阅读说明：本技术 三萜相关蛋白质与其编码基因以及在提高植物三萜化合物含量中的应用 (Triterpene related protein, coding gene thereof and application of triterpene related protein in improving content of plant triterpene compound ) 是由 尹静 詹亚光 杨杰 肖佳雷 李影 张玉琦 常存 于 2019-09-20 设计创作，主要内容包括：本发明公开了三萜相关蛋白质与其编码基因以及在提高植物三萜化合物含量中的应用。本发明公开的三萜相关蛋白质为TRP1、TRP2或TRP3,TRP1为氨基酸序列是序列1的蛋白质；TRP2为氨基酸序列是序列3的蛋白质；TRP3为氨基酸序列是序列5的蛋白质。本发明的三萜相关蛋白及其编码基因以及TRP2和TRP3的组合、BpW和TRP1的组合均可以用来培育三萜化合物合成提高的植物,并进一步为植物中三萜化合物的高效合成调控和生物技术的利用提供了有效的基因资源,具有广泛的应用前景。(The invention discloses a triterpene related protein, a coding gene thereof and application of the triterpene related protein in improving the content of plant triterpene compounds. The triterpene related protein disclosed by the invention is TRP1, TRP2 or TRP3, and the TRP1 is a protein of which the amino acid sequence is a sequence 1; TRP2 is a protein of which the amino acid sequence is sequence 3; TRP3 is a protein whose amino acid sequence is sequence 5. The triterpene related protein and the coding gene thereof, the combination of TRP2 and TRP3 and the combination of BpW and TRP1 can be used for cultivating plants with improved triterpene compound synthesis, and further provide effective gene resources for efficient synthesis regulation and control of triterpene compounds in the plants and utilization of biotechnology, and have wide application prospects.)

1. A protein, designated TRP1, TRP2 or TRP 3;

the TRP1 is any one of A1) -A3) as follows:

A1) the amino acid sequence is the protein of sequence 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A1) or A2);

the TRP2 is any one of A4) -A6) as follows:

A4) the amino acid sequence is the protein of sequence 3;

A5) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table and has the same function;

A6) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A4) or A5);

the TRP3 is any one of A7) -A9) as follows:

A7) the amino acid sequence is the protein of sequence 5;

A8) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 5 in the sequence table and has the same function;

A9) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A7) or A8).

2. A biomaterial related to the protein of claim 1, which is biomaterial 1, biomaterial 2 or biomaterial 3;

the biomaterial 1 is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding TRP1 described in claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

the biological material 2 is any one of the following C1) to C7):

C1) a nucleic acid molecule encoding TRP2 described in claim 1;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector;

C5) a transgenic plant cell line comprising C1) the nucleic acid molecule or a transgenic plant cell line comprising C2) the expression cassette;

C6) transgenic plant tissue comprising the nucleic acid molecule of C1), or transgenic plant tissue comprising the expression cassette of C2);

C7) a transgenic plant organ comprising C1) said nucleic acid molecule, or a transgenic plant organ comprising C2) said expression cassette;

the biomaterial 3 is any one of the following D1) to D7):

D1) a nucleic acid molecule encoding TRP3 described in claim 1;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D3) a recombinant vector containing the nucleic acid molecule of D1) or a recombinant vector containing the expression cassette of D2);

D4) a recombinant microorganism containing D1) the nucleic acid molecule, or a recombinant microorganism containing D2) the expression cassette, or a recombinant microorganism containing D3) the recombinant vector;

D5) a transgenic plant cell line comprising D1) the nucleic acid molecule or a transgenic plant cell line comprising the expression cassette of D2);

D6) transgenic plant tissue comprising the nucleic acid molecule of D1) or transgenic plant tissue comprising the expression cassette of D2);

D7) a transgenic plant organ containing D1) the nucleic acid molecule or a transgenic plant organ containing D2) the expression cassette.

3. The biomaterial of claim 2, wherein: B1) the nucleic acid molecule encoding the TRP1 is any one of the following b11) -b 14):

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b12) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b13) a cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding the TRP1 of claim 1;

b14) a cDNA or DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in any one of b11) -b13) and encodes said TRP1 according to claim 1;

C1) the nucleic acid molecule encoding the TRP2 is any one of c11) -c14) as follows:

c11) the coding sequence is cDNA molecule or DNA molecule of sequence 4 in the sequence table;

c12) a cDNA molecule or a DNA molecule shown in a sequence 4 in a sequence table;

c13) a cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in c11) or c12) and encoding the TRP2 of claim 1;

c14) a cDNA or DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in any one of c11) -c13) and encodes said TRP2 according to claim 1;

D1) the nucleic acid molecule encoding the TRP3 is any one of d11) -d14) as follows:

d11) the coding sequence is cDNA molecule or DNA molecule of sequence 6 in the sequence table;

d12) a cDNA molecule or DNA molecule shown in a sequence 6 in a sequence table;

d13) a cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in d11) or d12) and encoding the TRP3 of claim 1;

d14) hybridizes under stringent conditions with a nucleotide sequence defined in any one of d11) -d13) and encodes a cDNA molecule or a DNA molecule of the TRP3 as claimed in claim 1.

4. A kit of parts, being any one of the following Y1-Y4:

y1, a kit of parts consisting of the protein named BpW, the TRP2 and the TRP3 in claim 1;

the BpW is any one of A10) -A12) as follows:

A10) the amino acid sequence is the protein of sequence 7;

A11) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 7 in the sequence table and has the same function;

A12) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A10) or A11);

y2, a kit consisting of a biomaterial named biomaterial 4, said biomaterial 2 and said biomaterial 3 in claim 2 or 3;

the biomaterial 4 is any one of the following E1) to E7):

E1) a nucleic acid molecule encoding said BpW;

E2) an expression cassette comprising the nucleic acid molecule of E1);

E3) a recombinant vector comprising the nucleic acid molecule of E1) or a recombinant vector comprising the expression cassette of E2);

E4) a recombinant microorganism containing E1) the nucleic acid molecule, or a recombinant microorganism containing E2) the expression cassette, or a recombinant microorganism containing E3) the recombinant vector;

E5) a transgenic plant cell line comprising the nucleic acid molecule of E1) or a transgenic plant cell line comprising the expression cassette of E2);

E6) transgenic plant tissue comprising the nucleic acid molecule of E1) or transgenic plant tissue comprising the expression cassette of E2);

E7) a transgenic plant organ containing the nucleic acid molecule of E1), or a transgenic plant organ containing the expression cassette of E2);

y3, a kit of parts consisting of the TRP1 and the BpW of claim 1;

y4, a kit of parts consisting of said biomaterial 1 and said biomaterial 4 as claimed in claim 2 or 3.

5. The kit of claim 4, wherein: E1) the nucleic acid molecule encoding the BpW is any one of e11) -e14) as follows:

e11) the coding sequence is cDNA molecule or DNA molecule of sequence 8 in the sequence table;

e12) a cDNA molecule or a DNA molecule shown in a sequence 8 in a sequence table;

e13) a cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in e11) or e12) and encoding BpW of claim 4;

e14) hybridizes under stringent conditions with a nucleotide sequence defined in any one of e11) -e13) and encodes a cDNA molecule or a DNA molecule of BpW as claimed in claim 4.

6. Use of the protein of claim 1, or a substance that modulates the content or activity of the protein of claim 1, or the biological material of claim 2 or 3, or the kit of parts of claim 4 or 5 for any of the following:

D1) regulating and controlling the content of the plant triterpenoid;

D2) preparing a product for regulating and controlling the content of the plant triterpenoid;

D3) increasing the content of plant triterpenoid;

D4) preparing a product for improving the content of the plant triterpenoid;

D5) cultivating plants with increased content of triterpenoid;

D6) preparing plant products with increased triterpene compound content.

7. The following X1) or X2):

x1) a method for cultivating a plant having an increased content of a triterpene compound, which comprises allowing a plant of interest to express the protein of claim 1 in a recipient plant, or increasing the content of the protein of claim 1 in a recipient plant, or increasing the activity of the protein of claim 1 in a recipient plant, to obtain a plant having an increased content of a triterpene compound as compared with the recipient plant;

x2) a method for increasing the content of a triterpene compound in a plant, which comprises allowing a receptor plant to express the protein of claim 1, or increasing the content of the protein of claim 1 in the receptor plant, or increasing the activity of the protein of claim 1 in the receptor plant, to obtain a plant of interest having an increased content of a triterpene compound as compared with the receptor plant, thereby increasing the content of the triterpene compound in the plant;

x3) a method for breeding a plant having an increased content of a triterpene compound, which comprises allowing a plant having an increased content of a triterpene compound in a recipient plant to express BpW as defined in claim 4, TRP2 and TRP3 as defined in claim 1, or increasing the content of BpW as defined in claim 4, TRP2 and TRP3 as defined in claim 1 in a recipient plant, or increasing the activity of BpW as defined in claim 4, TRP2 and TRP3 in a recipient plant, to obtain a plant having an increased content of a triterpene compound as compared with the recipient plant;

x4) a method for increasing the content of a triterpene compound in a plant, comprising expressing in a recipient plant the BpW defined in claim 4, the TRP2 and the TRP3 defined in claim 1, or increasing the content of the BpW defined in claim 4, the TRP2 and the TRP3 defined in claim 1 in a recipient plant, or increasing the activity of the BpW defined in claim 4, the TRP2 and the TRP3 defined in claim 1 in a recipient plant, to obtain a plant of interest having an increased content of a triterpene compound as compared to the recipient plant, to achieve an increase in the content of a triterpene compound in a plant;

x5) a method for breeding a plant having an increased triterpene compound content, which comprises allowing BpW as described in claim 4 and TRP1 as described in claim 1 to exist in a recipient plant, or increasing BpW as described in claim 4 and TRP1 as described in claim 1 to exist in a recipient plant, or increasing BpW as described in claim 4 and TRP1 as described in claim 1 to exist in a recipient plant, to obtain a plant having an increased triterpene compound content as compared with the recipient plant;

x6) a method for increasing the content of a triterpene compound in a plant, comprising expressing BpW as defined in claim 4 and TRP1 as defined in claim 1 in a recipient plant, or increasing the content of BpW as defined in claim 4 and TRP1 as defined in claim 1 in a recipient plant, or increasing the activity of BpW as defined in claim 4 and TRP1 in a recipient plant to obtain a plant of interest having an increased content of a triterpene compound as compared with the recipient plant, thereby achieving an increase in the content of a triterpene compound in the plant.

8. The method of claim 7, wherein: x1) or X2) by introducing a gene encoding the TRP1, the TRP2 or the TRP3 in claim 1 into the recipient plant and expressing the gene;

x3) or X4) by introducing into said recipient plant a gene encoding said BpW of claim 4, said TRP2 and said TRP3 of claim 1 and expressing all of said genes encoding said BpW, said TRP2 and said TRP 3;

x5) or X6) by introducing into the recipient plant a gene encoding the BpW gene as claimed in claim 4 and a gene encoding the TRP1 gene as claimed in claim 1 and allowing both the gene encoding the BpW and the gene encoding the TRP1 to be expressed.

9. The use according to claim 4, or the method of claim 5 or 6, or the product of claim 7, wherein: the triterpene compound is betulin and/or betulinic acid;

and/or, the plant is M1) or M2) or M3):

m1) dicotyledonous or monocotyledonous plants;

m2) solanaceae or betulaceae plants;

m3) tobacco or white birch.

10. Amplifying a primer pair encoding the TRP1, the TRP2, or the TRP3 nucleic acid molecule of claim 1.

Technical Field

The invention relates to the field of biotechnology, triterpene related proteins, coding genes thereof and application of the triterpene related proteins in improving the content of plant triterpene compounds.

Background

Betula platyphylla (Betula platyphylla suk) contains an important secondary metabolite, namely betulin (TBP), wherein betulin, betulinic acid and oleanolic acid components are pioneer drugs with great development potential for resisting cancers, resisting Human Immunodeficiency Virus (HIV) and treating cardiovascular diseases and protecting liver.

Along with the increasing development of natural medicines, natural plant resources tend to be deficient, and how to effectively and reasonably utilize the natural plant resources is urgent; meanwhile, because the content of the secondary metabolites of the plants is low in a natural state, how to improve the yield of the secondary metabolites of the plants is a key link related to whether the development of natural medicines can be applied to actual production. However, secondary metabolic pathways are often extremely complex and there are often tedious interactions between different pathways. In order to change the content of certain secondary metabolites in plants or allow plants to synthesize new secondary metabolites according to the needs of people, besides the need of firstly clarifying their metabolic pathways, it is necessary to understand the expression regulation of enzymes in the metabolic pathways, the abundance of precursors, the sites of product synthesis, the mechanism of accumulation and transport after synthesis, etc. In order to make these natural substances better utilized by human beings, genetic modification of their metabolic pathways is an important research direction, and it is a new approach to improve the gene expression level of target pathway or inhibit the gene expression of other pathways by genetic engineering methods to promote the expression of secondary metabolites in plants or cells. Several secondary metabolites have been expressed in transgenic plants and cultured cells.

In higher plants, over 100 different triterpene backbones have been reported. These triterpene backbones are synthesized from the common precursor 2,3-oxidosqualene (2,3-oxidosqualene) via squalene cyclases (OSCs). The generation and modification stages of terpenes determine the structural diversity of terpenoids, and are the key point and difficulty in studying the secondary metabolism of plants such as terpenes.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the content of the plant triterpene compound.

In order to solve the technical problems, the invention firstly provides a protein (referred to as triterpene-related protein for short) which is derived from white birch (Betula platyphylla suk) and is related to the synthesis of triterpene compounds, and the name of the triterpene-related protein is TRP1, TRP2 or TRP 3;

the TRP1 is any one of A1) -A3) as follows:

A1) the amino acid sequence is the protein of sequence 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A1) or A2);

the TRP2 is any one of A4) -A6) as follows:

A4) the amino acid sequence is the protein of sequence 3;

A5) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table and has the same function;

A6) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A4) or A5);

the TRP3 is any one of A7) -A9) as follows:

A7) the amino acid sequence is the protein of sequence 5;

A8) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 5 in the sequence table and has the same function;

A9) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A7) or A8).

In order to facilitate the purification of the protein of A1), A4) or A7), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence No. 1 or No. 3 or No. 5 in the sequence listing is labeled as shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 1 and having the same function. A5) The protein of (4) is a protein having an identity of 75% or more or 75% or more to the amino acid sequence of the protein shown in SEQ ID No. 3 and having the same function. A8) The protein of (4) is a protein having an identity of 75% or more or 75% or more to the amino acid sequence of the protein shown in SEQ ID No. 5 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein in A2), A5) or A8) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. A5) The gene encoding the protein of (1) can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 4, and/or performing missense mutation of one or several base pairs, and/or attaching a coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. A8) The gene encoding the protein of (1) can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 6, and/or performing missense mutation of one or several base pairs, and/or attaching a coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in the sequence 2 encodes the protein shown in the sequence 1, the DNA molecule shown in the sequence 4 encodes the protein shown in the sequence 3, and the DNA molecule shown in the sequence 6 encodes the protein shown in the sequence 5.

The invention also provides a biological material related to the triterpene related protein, wherein the biological material is biological material 1, biological material 2 or biological material 3;

the biomaterial 1 is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the TRP 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

the biological material 2 is any one of the following C1) to C7):

C1) a nucleic acid molecule encoding the TRP 2;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector;

C5) a transgenic plant cell line comprising C1) the nucleic acid molecule or a transgenic plant cell line comprising C2) the expression cassette;

C6) transgenic plant tissue comprising the nucleic acid molecule of C1), or transgenic plant tissue comprising the expression cassette of C2);

C7) a transgenic plant organ comprising C1) said nucleic acid molecule, or a transgenic plant organ comprising C2) said expression cassette;

the biomaterial 3 is any one of the following D1) to D7):

D1) a nucleic acid molecule encoding the TRP 3;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D3) a recombinant vector containing the nucleic acid molecule of D1) or a recombinant vector containing the expression cassette of D2);

D4) a recombinant microorganism containing D1) the nucleic acid molecule, or a recombinant microorganism containing D2) the expression cassette, or a recombinant microorganism containing D3) the recombinant vector;

D5) a transgenic plant cell line comprising D1) the nucleic acid molecule or a transgenic plant cell line comprising the expression cassette of D2);

D6) transgenic plant tissue comprising the nucleic acid molecule of D1) or transgenic plant tissue comprising the expression cassette of D2);

D7) a transgenic plant organ containing D1) the nucleic acid molecule or a transgenic plant organ containing D2) the expression cassette.

In the above biological material, the nucleic acid molecule encoding the TRP1 described in B1) may be any one of the following B11) to B14):

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b12) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b13) a cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding the TRP 1;

b14) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in any one of b11) -b13) under stringent conditions and codes for the TRP 1;

C1) the nucleic acid molecule encoding the TRP2 may be any one of c11) -c14) as follows:

c11) the coding sequence is cDNA molecule or DNA molecule of sequence 4 in the sequence table;

c12) a cDNA molecule or a DNA molecule shown in a sequence 4 in a sequence table;

c13) a cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in c11) or c12) and encoding said TRP 2;

c14) a cDNA molecule or a DNA molecule which hybridizes with a nucleotide sequence defined in any one of c11) -c13) under stringent conditions and codes for the TRP 2;

D1) the nucleic acid molecule encoding the TRP3 may be any one of d11) -d14) as follows:

d11) the coding sequence is cDNA molecule or DNA molecule of sequence 6 in the sequence table;

d12) a cDNA molecule or DNA molecule shown in a sequence 6 in a sequence table;

d13) a cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined by d11) or d12) and encoding said TRP 3;

d14) hybridizes with any one of the nucleotide sequences defined by d11) -d13) under strict conditions and encodes the cDNA molecule or the DNA molecule of the TRP 3.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc. The triterpene related protein is named as TRP1, TRP2 or TRP 3;

the nucleotide sequence encoding TRP1, TRP2 or TRP3 of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of TRP1, TRP2 or TRP3 isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode TRP1, TRP2 or TRP3 protein and have the function of TRP1, TRP2 or TRP3 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 or sequence 3 or sequence 5 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above biomaterial, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above-mentioned biological material, the expression cassette containing a nucleic acid molecule encoding a TRP1 protein (TRP1 gene expression cassette) described in B2) refers to a DNA capable of expressing the TRP1 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of the TRP1 gene but also a terminator that terminates transcription of the TRP1 gene. Further, the expression cassette may also include an enhancer sequence. C2) The expression cassette containing a nucleic acid molecule encoding a TRP2 protein (TRP2 gene expression cassette) refers to a DNA capable of expressing a TRP2 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of the TRP2 gene but also a terminator that terminates transcription of the TRP2 gene. Further, the expression cassette may also include an enhancer sequence. D2) The expression cassette containing a nucleic acid molecule encoding a TRP3 protein (TRP3 gene expression cassette) refers to a DNA capable of expressing a TRP3 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of the TRP3 gene but also a terminator that terminates transcription of the TRP3 gene. Further, the expression cassette may also include an enhancer sequence.

Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beach et al (1985) EMBO J. 4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) plant cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic acids sRs.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The existing expression vector can be used for constructing a recombinant vector containing the TRP1 gene expression cassette or the TRP2 gene expression cassette or the TRP3 gene expression cassette. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Corp.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may be specifically pCAMBIA1303 vector.

B3) The recombinant vector can be pCAMBIA1303-TRP 1. The pCAMBIA1303-TRP1 is a recombinant vector obtained by inserting the TRP1 gene shown in the 1 st to 1530 th positions of the sequence 2 between multiple cloning sites of the pCAMBIA1303 vector by using NcoI. The pCAMBIA1303-TRP1 can express a fusion protein formed by the TRP1 protein shown in a sequence 1, GUS and GFP, and the expression of the fusion protein is driven by a 35S promoter.

C3) The recombinant vector can be pCAMBIA1303-TRP 2. The pCAMBIA1303-TRP2 is a recombinant vector obtained by inserting TRP2 gene shown in 1 st to 1185 th positions of sequence 4 between multiple cloning sites of pCAMBIA1303 vector by using NcoI. The pCAMBIA1303-TRP2 can express a fusion protein formed by the TRP2 protein shown in a sequence 3, GUS and GFP, and the expression of the fusion protein is driven by a 35S promoter.

D3) The recombinant vector can be pCAMBIA1303-TRP 3. The pCAMBIA1303-TRP3 is a recombinant vector obtained by inserting the TRP3 gene shown in the 1 st to 1536 th positions of the sequence 6 between multiple cloning sites of the pCAMBIA1303 vector using NcoI. The pCAMBIA1303-TRP3 can express a fusion protein formed by the TRP3 protein shown in a sequence 5, GUS and GFP, and the expression of the fusion protein is driven by a 35S promoter.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. Wherein, the bacterium can be Agrobacterium, such as Agrobacterium LBA 4404.

In the above biological material, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

The invention also provides a kit, which is any one of the following Y1-Y4:

y1, a kit consisting of the protein designated BpW, the TRP2 and the TRP 3;

the BpW is any one of A10) -A12) as follows:

A10) the amino acid sequence is the protein of sequence 7;

A11) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 7 in the sequence table and has the same function;

A12) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A10) or A11);

y2, a kit consisting of a biomaterial named biomaterial 4, said biomaterial 2 and said biomaterial 3;

the biomaterial 4 is any one of the following E1) to E7):

E1) a nucleic acid molecule encoding said BpW;

E2) an expression cassette comprising the nucleic acid molecule of E1);

E3) a recombinant vector comprising the nucleic acid molecule of E1) or a recombinant vector comprising the expression cassette of E2);

E4) a recombinant microorganism containing E1) the nucleic acid molecule, or a recombinant microorganism containing E2) the expression cassette, or a recombinant microorganism containing E3) the recombinant vector;

E5) a transgenic plant cell line comprising the nucleic acid molecule of E1) or a transgenic plant cell line comprising the expression cassette of E2);

E6) transgenic plant tissue comprising the nucleic acid molecule of E1) or transgenic plant tissue comprising the expression cassette of E2);

E7) a transgenic plant organ containing the nucleic acid molecule of E1), or a transgenic plant organ containing the expression cassette of E2);

y3, a kit consisting of the TRP1 and the BpW;

y4, a kit consisting of said biomaterial 1 and said biomaterial 4.

The above-mentioned kit has any of the following functions:

F1) regulating and controlling the content of the plant triterpenoid;

F2) preparing a product for regulating and controlling the content of the plant triterpenoid;

F3) increasing the content of plant triterpenoid;

F4) preparing a product for improving the content of the plant triterpenoid;

F5) cultivating plants with increased content of triterpenoid;

F6) preparing plant products with increased triterpene compound content.

In the above kit, the nucleic acid molecule encoding BpW of E1) may be any one of E11) to E14) as follows:

e11) the coding sequence is cDNA molecule or DNA molecule of sequence 8 in the sequence table;

e12) a cDNA molecule or a DNA molecule shown in a sequence 8 in a sequence table;

e13) a cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in e11) or e12) and encoding said BpW;

e14) hybridizes under stringent conditions with a nucleotide sequence defined in any one of e11) -e13) and encodes the BpW cDNA or DNA molecule.

E3) The recombinant vector can be pCAMBIA 1303-BpW. The pCAMBIA1303-BpW is a recombinant vector obtained by inserting BpW gene shown in 1 st to 2265 th positions of sequence 8 between multiple cloning sites of pCAMBIA1303 vector by using NcoI. The pCAMBIA1303-BpW can express a fusion protein formed by BpW protein shown in a sequence 7, GUS and GFP, and the expression of the fusion protein is driven by a 35S promoter.

The present invention also provides any one of the following uses of the TRP1, the TRP2 or the TRP3, or a substance modulating the content or activity of the TRP1, the TRP2 or the TRP3, or the biological material 1, the biological material 2 or the biological material 3, or the kit:

D1) regulating and controlling the content of the plant triterpenoid;

D2) preparing a product for regulating and controlling the content of the plant triterpenoid;

D3) increasing the content of plant triterpenoid;

D4) preparing a product for improving the content of the plant triterpenoid;

D5) cultivating plants with increased content of triterpenoid;

D6) preparing plant products with increased triterpene compound content.

The present invention also provides the process described in any one of the following X1) -X6):

x1) a method for breeding a plant having an increased content of a triterpene compound, which comprises expressing said TRP1, said TRP2 or said TRP3 in a recipient plant, or increasing the content of said TRP1, said TRP2 or said TRP3 in a recipient plant, or increasing the activity of said TRP1, said TRP2 or said TRP3 in a recipient plant, to obtain a plant having an increased content of a triterpene compound as compared with said recipient plant;

x2) a method for increasing the content of a triterpene compound in a plant, comprising expressing said TRP1, said TRP2 or said TRP3 in a recipient plant, or increasing the content of said TRP1, said TRP2 or said TRP3 in a recipient plant, or increasing the activity of said TRP1, said TRP2 or said TRP3 in a recipient plant, to obtain a desired plant having an increased content of a triterpene compound as compared to said recipient plant, thereby increasing the content of a triterpene compound in a plant;

x3) a method for breeding a plant with increased content of triterpene compounds, which comprises expressing said BpW, said TRP2 and said TRP3 in a recipient plant, or increasing the content of said BpW, said TRP2 and said TRP3 in a recipient plant, or increasing the activity of said BpW, said TRP2 and said TRP3 in a recipient plant, to obtain a plant with increased content of triterpene compounds compared with said recipient plant;

x4) a method for increasing the content of a triterpene compound in a plant, comprising expressing said BpW, said TRP2 and said TRP3 in a recipient plant, or increasing the content of said BpW, said TRP2 and said TRP3 in a recipient plant, or increasing the activity of said BpW, said TRP2 and said TRP3 in a recipient plant, to obtain a desired plant having an increased content of a triterpene compound as compared to said recipient plant, to achieve an increased content of a triterpene compound in a plant;

x5) a method for breeding plants with increased triterpene compound content, which comprises increasing the content of said BpW and said TRP1 in a recipient plant, or increasing the content of said BpW and said TRP1 in a recipient plant, or increasing the activity of said BpW and said TRP1 in a recipient plant, to obtain a target plant with increased triterpene compound content compared with said recipient plant;

x6) a method for increasing the content of a triterpene compound in a plant, comprising expressing said BpW and said TRP1 in a recipient plant, or increasing the content of said BpW and said TRP1 in a recipient plant, or increasing the activity of said BpW and said TRP1 in a recipient plant, to obtain a plant of interest having an increased content of a triterpene compound as compared to said recipient plant, thereby achieving an increase in the content of a triterpene compound in a plant.

In the above method, X1) or X2) the method may be carried out by introducing the coding gene for the TRP1, the TRP2 or the TRP3 into the recipient plant and allowing the coding gene to be expressed;

x3) or X4) by introducing the genes encoding the TRP2 and the TRP3 into the recipient plant and expressing all of the BpW, the TRP2 and the TRP3 genes;

x5) or X6) by introducing the gene encoding BpW and the gene encoding TRP1 into the recipient plant and allowing both the gene encoding BpW and the gene encoding TRP1 to be expressed.

In the above method, the gene encoding TRP1 may be B1) the nucleic acid molecule. The gene encoding TRP2 may be C1). The gene encoding TRP3 may be D1) the nucleic acid molecule. The gene encoding BpW may be E1) the nucleic acid molecule.

In the above method, the TRP1 encoding gene, the TRP2 encoding gene, the TRP3 encoding gene and the BpW encoding gene may be modified as follows and then introduced into a recipient plant to achieve a better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the amino acid sequence of the TRP1, the TRP2, the TRP3, or the gene encoding the TRP BpW of the present invention may be changed according to the codon preferred by a recipient plant to conform to the plant preference; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding TRP1 can be introduced into a recipient plant using a recombinant expression vector containing the gene encoding TRP 1. The recombinant expression vector can be specifically the pCAMBIA1303-TRP 1.

The gene encoding TRP2 can be introduced into a recipient plant using a recombinant expression vector containing the gene encoding TRP 2. The recombinant expression vector can be specifically the pCAMBIA1303-TRP 2.

The gene encoding TRP3 can be introduced into a recipient plant using a recombinant expression vector containing the gene encoding TRP 3. The recombinant expression vector can be specifically the pCAMBIA1303-TRP 3.

The gene encoding BpW can be introduced into a recipient plant using a recombinant expression vector containing the gene encoding BpW. The recombinant expression vector can be specifically the pCAMBIA 1303-BpW.

The recombinant expression vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant molecular Biology (2nd Edition)).

The plant of interest is understood to comprise not only the first generation plant in which the protein of interest or the gene encoding it has been altered, but also the progeny thereof. For the plant of interest, the gene may be propagated in the species, or transferred into other varieties of the same species, including commercial varieties in particular, using conventional breeding techniques. The plant of interest includes seeds, callus, whole plants and cells.

In the present invention, the triterpene compound may be betulin and/or betulinic acid.

The plant may be M1) or M2) or M3):

m1) dicotyledonous or monocotyledonous plants;

m2) solanaceae or betulaceae plants;

m3) tobacco or white birch.

The primer pair for amplifying the nucleic acid molecule coding the TRP1, the TRP2 or the TRP3 also belongs to the protection scope of the invention.

Experiments prove that the triterpene related proteins (TRP1, TRP2 and TRP3) and coding genes thereof can improve the content of triterpene compounds in plants, and the combination of BpW, TRP2 and TRP3 and the combination of BpW and TRP1 can further promote the synthesis of the triterpene compounds: in the plants which are independently and transiently expressed with TRP2 and TRP3, the contents of betulin and betulinic acid are both obviously higher than those of wild plants, the contents of the betulin in the plants which are independently and transiently expressed with TRP2 and TRP3 are respectively 1.50 times and 1.46 times of those of the wild plants, and the contents of the betulinic acid are respectively 12.40 times and 11.85 times of those of the wild plants; when the genes BpW, TRP2 and TRP3 are expressed in the plant at the same time, the contents of betulin and betulinic acid are obviously higher than that of the wild plant, and are respectively 1.85 times and 13.62 times of that of the wild plant; BpW and TRP1 are used for co-infecting plants, the betulinic acid content is obviously higher than that of the plants which are separately infected by BpW and TRP1, and is 10.27 times of that of wild plants, and is 11.65 times and 10.97 times of that of the plants which are separately infected by BpW and TRP 1; BpW when the plant is infected together with TRP1, the betulin content is 1.16 times of that of wild plant, 1.34 times and 1.40 times of that of BpW and TRP1 respectively; in a plant with the TRP1 transferred gene, the relative expression quantity of the BpSS, BpSE and BpW genes is obviously improved, wherein the BpW gene has the most obvious effect of up-regulation, and the relative expression quantity of the BpDS and BpY genes is obviously down-regulated. The three triterpene related proteins (TRP1, TRP2 and TRP3) and coding genes thereof, the combination of TRP2 and TRP3 and the combination of BpW and TRP1 can be used for cultivating plants with improved triterpene compound synthesis, and further provide effective gene resources for efficient synthesis control and biotechnology utilization of triterpene compounds in plants, thereby having wide application prospect.

Drawings

FIG. 1 shows the HPLC detection of the betulin and betulinic acid content in tobacco with transient expression. (a) The betulin content in tobacco is instantly expressed by TRP1, TRP2 and TRP 3; (b) the betulinic acid content in tobacco with over-expression of TRP1, TRP2 and TRP 3. WT represents wild type Nicotiana benthamiana, BpW represents BpW-expressing tobacco, TRP1 represents TRP 1-expressing tobacco, TRP2 represents TRP 2-expressing tobacco, TRP3 represents TRP 3-expressing tobacco, BpW + TRP1 represents TRP1 and BpW-co-expressing tobacco, BpW + TRP2+ TRP3 represents BpW, TRP2 and TRP 3-co-expressing tobacco.

FIG. 2 shows the HPLC results of WT (wild type), TRP2 and TRP3 transiently expressed tobacco. The left side is the detection result of betulin, and the lowest graph is the standard substance; the right side is the detection result of betulinic acid, and the lowest graph is the standard.

FIG. 3 is the PCR identification picture of transgenic birch and the detection result of target gene expression. a is a PCR identification electrophoretogram of the transgenic white birch; b is the result of detecting the expression level of TRP1 gene; c is the expression detection result of the gene related to the triterpene synthesis pathway in wild type Nicotiana benthamiana and TRP1-5 strain.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

IS medium: 1L of culture medium contains 370mg/L MgSO₄·7H₂O；170mg/L KNO₃；710mg/L Ca(NO₃)₂·4H₂O；140mg/L KC1；680mg/L NH₄NO₃；80mg/LKH₂PO₄；27.8mg/L FeSO₄·7H₂O；37.3mg/L Na₂-EDTA；8mg/L MnSO₄·4H₂O；0.8mg/L KI；9mg/L ZnSO₄·7H₂O；0.25mg/L CuSO₄·5H₂O；3.2mg/L H₃BO₃(ii) a 100mg/L inositol; 0.8mg/L nicotinic acid (VB)₃) (ii) a 0.1mg/L thiamine hydrochloride (VB)₁) (ii) a 0.1mg/L pyridoxine hydrochloride (VB)₆) (ii) a10 mg/L urea; 1mg/L fumaric acid; 10mg/L tyrosine; 1mg/L ascorbic acid, and the balance of water.

Example 1 triterpene-related proteins TRP1, TRP2 and TRP3 can increase the content of triterpene compounds in plants

This example provides proteins (denoted as triterpene-related proteins) derived from white birch (Betula platyphylla Suk) and related to the synthesis of triterpene compounds, which are designated as TRP1, TRP2, TRP3, TRP1, TRP2 and TRP3, and the amino acid sequences of the three proteins are represented as sequences 1, 3 and 5 in the sequence table, respectively, in white birch, DNA molecules encoding the three proteins are represented as DNAs represented as sequences 2, 4 and 6 in the sequence table, and the three DNA molecules are designated as TRP1 gene, TRP2 gene and TRP3 gene, respectively.

Construction of recombinant expression vector

Extracting total RNA of the white birch tissue culture seedling, carrying out reverse transcription to synthesize cDNA, amplifying a TRP1 gene shown in a sequence 2 by using TRP1-F and TRP1-R, amplifying a TRP2 gene shown in a sequence 4 by using TRP2-F and TRP2-R, amplifying a TRP3 gene shown in a sequence 6 by using TRP3-F and TRP3-R, wherein the primer sequences are as follows:

TRP1-F：5′-GATCATGGAGGTCTGGCTCA-3′；

TRP1-R：5′-CAACATGGTGGCATGGACTGT-3′；

TRP2-F：5′-CTGGGTTCCATGTCTTATACTTG-3′；

TRP2-R：5′-ACCACCCCACAGACTGGATTATG-3′；

TRP3-F：5′-CAGCTCTACCATGGAAGCCCTA-3′；

TRP3-R：5′-CCTTTTCCCTTCAACTGCCC-3′。

a recombinant vector was obtained by inserting the TRP1 gene shown in the 1 st to 1530 th positions of the sequence 2 between multiple cloning sites in the pCAMBIA1303 vector using NcoI, and the recombinant vector having the TRP1 gene with the correct sequence was designated as pCAMBIA1303-TRP1, and pCAMBIA1303-TRP1 was able to express a fusion protein of the TRP1 protein shown in the sequence 1, GUS and GFP, and the expression of this fusion protein was driven by 35S promoter.

A recombinant vector was obtained by inserting TRP2 gene represented by 1-1185 th position of the sequence 4 between multiple cloning sites of pCAMBIA1303 vector using NcoI, and the recombinant vector having the correct sequence containing TRP2 gene was designated as pCAMBIA1303-TRP2, pCAMBIA1303-TRP2 was able to express a fusion protein of TRP2 protein represented by the sequence 3 with GUS and GFP, and the expression of this fusion protein was driven by 35S promoter.

A recombinant vector was obtained using the TRP3 gene shown in 1 st to 1536 th positions of sequence 6 among multiple cloning sites of the pCAMBIA1303 vector with NcoI, and the recombinant vector containing the TRP3 gene with the correct sequence was designated as pCAMBIA1303-TRP3, pCAMBIA1303-TRP3 was able to express a fusion protein of the TRP3 protein shown in sequence 5 with GUS and GFP, and the expression of this fusion protein was driven by 35S promoter.

The recombinant vector is obtained by using BpW genes shown in 1 st to 2265 th sites of a sequence 8 in a sequence list of multiple cloning sites of a pCAMBIA1303 vector through NcoI, the recombinant vector with a correct sequence containing BpW genes is marked as pCAMBIA1303-BpW, pCAMBIA1303-BpW can express a fusion protein formed by BpW protein shown in a sequence 7 in the sequence list, GUS and GFP, and the expression of the fusion protein is driven by a 35S promoter.

Wherein, the DNA molecule shown in the sequence 8 in the sequence table codes BpW protein shown in the sequence 7 in the sequence table.

BpW the gene is derived from white birch.

Secondly, the TRP1, TRP2 and TRP3 can improve the content of triterpenoid in Nicotiana benthamiana

1. Transient expression of Nicotiana benthamiana

Respectively introducing pCAMBIA1303-TRP1, pCAMBIA1303-TRP2, pCAMBIA1303-TRP3, pCAMBIA1303-BpW and an origin vector pCAMBIA1303 into Agrobacterium LBA4404, and respectively marking the obtained recombinant bacteria as LBA4404-pCAMBIA1303-TRP1, LBA4404-pCAMBIA1303-TRP2, LBA4404-pCAMBIA1303-TRP3, LBA4404-pCAMBIA1303-BpW and LBA4404-pCAMBIA 1303-1303.

(1) Single colonies of Agrobacterium, pCAMBIA1303-TRP1, pCAMBIA1303-TRP2, pCAMBIA1303-TRP3, pCAMBIA1303-BpW and LBA4404-pCAMBIA1303 were picked up, inoculated into LB liquid medium containing rifampicin (100mg/L) and kanamycin (50mg/L), shake-cultured at 28 ℃ for 2 days, and centrifuged at 4000g for 10min to collect the cells.

(2) The collected cells were resuspended in 20mL of a permeant (10mM MES, 10mM MgCl)₂100. mu.M acetosyringone, pH 5.7) to a final concentration OD₆₀₀And (4) incubating at room temperature for 2h at 1.0 to obtain a resuspension of each strain for later use.

(3) After the step (2) is finished, respectively infiltrating the lower epidermis of 3-week-old lamina of Nicotiana benthamiana (Nicotiana benthamiana) into each strain heavy suspension obtained in the step (2) by using a 1mL needleless injector, culturing the Nicotiana benthamiana plants permeated with bacterial liquid in a tissue culture room at 25 ℃ under long-day conditions (16h light and 8h dark), respectively marking the transient expression tobacco obtained by LBA4404-pCAMBIA1303-TRP1, LBA4404-pCAMBIA1303-TRP2, LBA4404-pCAMBIA1303-TRP3 and LBA4404-pCAMBIA1303-BpW as TRP1 expression tobacco, LBA 2 expression tobacco, TRP3 expression tobacco and BpW expression tobacco, respectively marking the transient expression tobacco after LBA4404-pCAMBIA1303-TRP2 and LBA4404-pCAMBIA1303-TRP3 are infected, and jointly marking the transient expression tobacco suspension obtained by the transient expression of the LBA4404-pCAMBIA1303-TRP3 and the transient expression tobacco suspension obtained by the same volume as the tobacco after the LBA 4404-TRP 865-TRP 4 expression, The instantaneous expression tobacco obtained by infecting Nicotiana benthamiana after equal-volume mixing of bacterial suspensions of LBA4404-pCAMBIA1303-BpW is marked as TRP1 and BpW co-expression tobacco, the instantaneous expression tobacco obtained by LBA4404-pCAMBIA1303 is no-load control tobacco, and the material is taken after 3 days after bacterial liquid infiltration to detect the content of the triterpene compound.

2. Detecting the content of triterpene compounds in the instantly expressed Nicotiana benthamiana

Detecting the contents of betulin and betulinic acid in each of the transiently expressed tobaccos obtained in step 1 by high performance liquid chromatography, and using non-transformed wild type Nicotiana benthamiana as a control. The method comprises the following specific steps: taking tobacco leaves, drying at 60 ℃ to constant weight, grinding, precisely weighing 0.3g of tissue dry sample, adding 25mL of hydrochloric acid-absolute ethanol solution (obtained by mixing hydrochloric acid aqueous solution with the hydrogen chloride concentration of 36-38% (mass percentage) and ethanol according to the volume ratio of 2: 8), refluxing at 90 ℃ for 3h, cooling, shaking up, filtering, and collecting filtrate. Precisely measuring 15mL of filtrate and 15mL of distilled water, mixing, placing on a water bath at 80 ℃ to evaporate ethanol, adding 20mL of diethyl ether into the residual liquid for extraction, collecting the upper layer diethyl ether extract, extracting the residual liquid for 2 times by using 20mL of diethyl ether, combining the three upper layer diethyl ether extracts, evaporating to dryness at the low temperature of 40 ℃, dissolving by using 1mL of methanol, filtering by using a 0.45-micrometer organic filter membrane to obtain a liquid to be detected, detecting betulin and betulinic acid in the liquid to be detected by using high performance liquid chromatography, respectively using betulin (BWB 50068, a Branch of Beijing century Olympic Biotechnology Co., Ltd., BWB50067) and betulinic acid (BWB 50067, a Branch of Beijing century Olympic technology Co., Ltd.) as standard products, and carrying out quantitative analysis by using a standard curve method (external standard method) according to retention time of the standard products.

High Performance Liquid Chromatography (HPLC) detection conditions: a chromatographic system of 600-; the mobile phase is acetonitrile: water 9: 1 (v/v); the column temperature is 25 ℃; sensitivity 16 AUFS; the flow rate is 1.0 mL/min; detecting wavelength 210nm, and injecting 20 μ L (white)The betulin regression equation is: y 10000000x +63763, R²0.9995; betulinic acid regression equation: y is 9 × 10⁶X +68020R2 ═ 0.9994), the results are shown in fig. 1, fig. 2 and table 1.

TABLE 1 content of betulin and Betulinic acid (mg/g dry weight) in tobacco with different genes transiently expressed

Tobacco	Betulin	Betulinic acid
			Wild type Benshi tobacco	2.16	0.0363
TRP2 expressing tobacco	3.23	0.45
			TRP3 expressing tobacco	3.15	0.43
Tobacco co-expressing BpW, TRP2 and TRP3	3.99	0.494
			TRP1 expressing tobacco	1.78	0.034
BpW expressing tobacco	1.86	0.032
			Tobacco co-expressing TRP1 and BpW	2.50	0.373

The results show that the betulin and betulinic acid content of the unloaded control tobacco and the wild type Nicotiana benthamiana are not obviously different.

The contents of betulin and betulinic acid in tobacco transiently expressed by TRP2 and TRP3 are both significantly higher than that in wild type Nicotiana benthamiana, the contents of betulin in tobacco transiently expressed by TRP2 and TRP3 are respectively 1.50 times and 1.46 times of that in wild type Nicotiana benthamiana, and the contents of betulinic acid are respectively 12.40 times and 11.85 times of that in wild type Nicotiana benthamiana. When the BpW, TRP2 and TRP3 genes are simultaneously expressed in tobacco, the contents of betulin and betulinic acid are obviously higher than that of wild type Nicotiana benthamiana, and are respectively 1.85 times and 13.62 times of that of the wild type Nicotiana benthamiana. The above results indicate that both TRP2 and TRP3 genes play an important role in the biosynthesis of betulin and betulinic acid, and the combination of BpW, TRP2 and TRP3 genes can further increase the content of betulin and betulinic acid.

In tobacco transiently expressed with TRP1, betulin and betulinic acid content were not increased compared with wild type Nicotiana benthamiana. BpW, when the tobacco is infected with TRP1 together, the betulinic acid content is obviously higher than that of the tobacco which is separately infected with BpW and TRP1 respectively, and is 10.27 times of that of wild type Nicotiana benthamiana, and is 11.65 times and 10.97 times of that of the tobacco which is separately infected with BpW and TRP1 respectively, which shows that the TRP1 can promote the biosynthesis of the betulinic acid when the gene BpW is over-expressed. BpW and TRP1 are used for co-infecting tobacco, the betulin content is obviously higher than that of BpW and TRP1 respectively and is 1.16 times of wild type Nicotiana benthamiana, and is 1.34 times and 1.40 times of BpW and TRP1 respectively and independently infecting tobacco. It is demonstrated that TRP1 and BpW genes may act synergistically in the biosynthesis of betulin and betulinic acid.

Thirdly, TRP1, TRP2 and TRP3 can regulate and control the expression of triterpene synthetic pathway related genes in white birch

1. Preparation of transgenic white birch

And (2) respectively carrying out genetic transformation on the recombinant bacteria LBA4404-pCAMBIA1303-TRP1, LBA4404-pCAMBIA1303-TRP2, LBA4404-pCAMBIA1303-TRP3 and LBA4404-pCAMBIA1303 in the second step to prepare the transgenic white birch, wherein the steps are as follows:

sterile seedlings (WPM culture medium +0.5mg/L IBA + sucrose 20g/L + agar 5.3g/L, pH 6.5) are induced from excellent white birch seeds in the northeast forestry university seed orchard, and materials with strong stems and large leaves are selected for experiments.

Genetic transformation of white birch tissue is carried out by a leaf disc method, and an explant comprising leaves, stem segments and petioles of white birch tissue culture seedlings is taken. The explants were precultured for 3 days in IS medium containing 6-BA (5mg/L) to give precultured explants. Activating the recombinant bacteria, selecting single bacterial colony of the recombinant bacteria, placing the single bacterial colony in LB liquid culture medium containing rifampicin (100mg/L) and kanamycin (50mg/L), and shake culturing at 28 ℃ until bacterial liquid OD₆₀₀The bacterial solution was centrifuged at 4,000r/min for 5min and the cells were collected, about 0.6-0.8. Resuspending the thallus with sterile water of the same volume to obtain a recombinant bacterial suspension, immersing the pre-cultured explant leaves into the recombinant bacterial suspension for 5min, taking out the material, and sucking excess liquid with sterile filter paper to obtain the infected explant. The infected explants were cultured in the dark at 25 ℃ for 3 days in IS medium containing 5 mg/L6-BA and 0.6mg/L NAA. After dark culture IS finished, washing the leaf surface clean by sterile water containing 500mg/L of cefuroxime axetil for a plurality of times, sucking the water on the leaf surface by filter paper, then putting the leaf surface clean in an IS culture medium containing 500mg/L of cefuroxime axetil, 10mg/L of hygromycin, 5mg/L of 6-BA and 0.6mg/L of NAA, and continuously carrying out bacteria removal for a plurality of days until the callus grows out. Transferring the callus to IS culture medium containing 6-BA (1mg/L), cefamycin (300mg/L) and hygromycin (10mg/L), and inducing to obtain transgenic white birch. The culture conditions are as follows: the temperature is 24-26 ℃, the illumination intensity is 2,000lx, the illumination time is 16h/d, and the humidity is 40-50%.

The transgenic plant obtained from LBA4404-pCAMBIA1303-TRP1 was designated as TRP1 gene white birch, the transgenic plant obtained from LBA4404-pCAMBIA1303-TRP2 was designated as TRP2 gene white birch, the transgenic plant obtained from LBA4404-pCAMBIA1303-TRP3 was designated as TRP3 gene white birch, and the transgenic plant obtained from LBA4404-pCAMBIA1303 was designated as no-load control white birch.

2. PCR identification of transgenic white birch

And (3) carrying out PCR amplification identification on each transgenic white birch obtained in the step (1) by using a primer GUS-F/R, taking a non-transgenic wild type white birch plant and a no-load control white birch as negative controls, and taking a pCAMBIA1303 plasmid as a positive control. The reaction was carried out using 2 XT 5Direct PCR Kit (Plant) Kit from Biotechnology Ltd of Beijing Optimalaceae: taking 1-2mm leaf blades, putting the leaf blades into a centrifugal tube, adding 50 mu L of lysine Buffer A, heating at 95 ℃ for 5-10min, standing, taking 1 mu L as a template, taking GUS-F/R as a specific primer, and carrying out PCR amplification conditions: 3min at 98 ℃; 30 cycles of 98 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 45 s; and identifying positive plants at 72 ℃ for 5 min. The primers GUS-F/R are as follows:

GUS-F：ATTACGGCAAAGTGTGGGT，GUS-R：TGACGCACAGTTCATAGAG。

the results (a in fig. 3) show that transgenic birch as well as the positive control show the purposive band, demonstrating that the fragment comprising TRP1, TRP2, TRP3 gene has integrated into the plant genome. Neither wild type birch plants nor empty-load control birch plants contained TRP1 gene, TRP2 gene or TRP3 gene.

3. Detecting the expression level of target gene in transgenic white birch

Quantitative PCR using real-time fluorescence (Premix Ex Taq^TMTaKaRa) method detects the relative expression level of TRP1 gene in 5-strain TrP1 transgenic white birch seedlings (TRP1-1, TRP1-5, TRP1-6, TRP1-7 and TRP1-8), and wild white birch (WT) without transgene and idle-load control white birch are used as control. The primers are TRP1-F and TRP1-R, Tubulin gene (Tubulin gene) is used as internal reference, primers of TU-F and TU-R are used as internal reference, and primers of the primers are used as external referenceAs in table 2.

The results (b in fig. 3) show that there was no significant difference in the relative expression amount of TRP1 gene in wild type white birch and empty control white birch; compared with wild white birch, the expression levels of TRP1 genes in transgenic strains TRP1-1, TRP1-5, TRP1-6, TRP1-7 and TRP1-8 are remarkably up-regulated and are respectively 14.52, 17.47, 14.86, 4.74 and 7.64 times of that of the wild white birch, wherein the expression level of TRP1 in the TRP1-5 strain is the highest.

4. Detecting expression quantity of triterpene synthesis pathway related gene in transgenic white birch

Quantitative PCR using real-time fluorescence (Premix Ex Taq^TMTaKaRa) method detects relative expression quantity of triterpene synthesis pathway related genes in transgenic plant TRP1-5, and wild type birch (WT) without transgene and idle control birch are used as control. Tubulin gene (Tubulin gene) is used as an internal reference, and the internal reference primers are TU-F and TU-R. The primers used are shown in Table 2.

TABLE 2 fluorescent quantitative PCR primer sequences

The results (c in fig. 3) show that there was no significant difference in the relative expression of each gene in wild type white birch and no-load control white birch; compared with wild white birch, the relative expression of the BpSS, BpSE and BpW genes is obviously improved, wherein the up-regulation effect of BpW gene is most obvious, and the relative expression of the BpDS and BpY genes is obviously reduced. The betula alba TRP1 gene can promote the up-regulation expression of betulin and betulinic acid synthesis genes.

<110> northeast university of forestry

<120> triterpene-related protein, coding gene thereof and application thereof in increasing content of plant triterpene compound

<160> 8

<170> PatentIn version 3.5

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cggctctggt gcattttgga caaagcggct aaagacaaca tctcggtgga cttgcaggac 540

ttgttgctac gcttgacgtt cgataatata tgcggcctca cgttcggtaa agacccggaa 600

acgctctctc cggagctatc cgataacccg tttgccatgg cctttgacac cgccaccgaa 660

accactctcc aacggcttct ctacccgggt ttcctttgga gattggagaa gttattgggt 720

atcggagcgg agaggagatt gaaagaaagc cttcaagtcg tcgaaaacta catgaacgac 780

gccatcacgg cgcgcaagga atctccgtca gatgatttac tctctcgctt catgaacaaa 840

cgtgacgtcg acggcaaaac cttcacagtc tccgtcctcc aacgaatcgc tctcaacttc 900

gtcctcgccg gccgagacac gtcgtcggtg gcgctgagct ggttcttctg gctcgtcatg 960

cacaacccgg aggtcgagga caagatcatc gaggagatat caaccgtcct gaacgacaca 1020

cgtggcgccg accacttgaa atgggtccaa gagccgttgg agttcgacga ggccgaccgg 1080

ttggtctacc tgaaagccgc acttgccgaa acgctgcgtt tgttcccgtc cgtgccggag 1140

gacttcaagt acgtcgtctc cgacgatgtt ttaccggacg gcactttcgt accggccggt 1200

tcgacggtga cgtattcgat atattctgtt gggaggatga agagtatatg gggcgaggac 1260

tgcatggagt ttaaaccgga gcggtggctc tcggccgagg gagaccgctt cgaaccgccc 1320

aaagatgggt acaagttcgt ggccttcaat gctggaccga ggacttgtct tgggaaggac 1380

ttggcttacc tgcaaatgaa gtctgtagct tcggctgtgc ttttgaggta ccggatatcg 1440

caggttcccg gtcaccgggt ggagcagaag atgtctctga cgctgttcat gaagaatggg 1500

ctccacgtgt acgtgcaacc acgtcagctc tctgtgtga 1539

<210> 7

<211> 755

<212> PRT

<213> white birch (Betula platyphylla suk.)

<400> 7

Met Trp Lys Leu Lys Ile Ala Glu Gly Gly Pro Gly Leu Val Ser Gly

1 5 10 15

Asn Asp Phe Ile Gly Arg Gln His Trp Glu Phe Asp Pro Asp Ala Gly

20 25 30

Thr Pro Gln Glu Arg Ala Glu Val Glu Lys Val Arg Glu Glu Phe Thr

35 40 45

Lys Asn Arg Phe Gln Met Lys Gln Ser Ala Asp Leu Leu Met Arg Met

50 55 60

Gln Leu Arg Lys Glu Asn Pro Cys Gln Pro Ile Pro Pro Pro Val Lys

65 70 75 80

Val Lys Glu Thr Glu Val Ile Thr Glu Glu Ala Val Ile Thr Thr Leu

85 90 95

Arg Arg Ser Leu Ser Phe Tyr Ser Ser Ile Gln Ala His Asp Gly His

100 105 110

Trp Pro Gly Glu Ser Ala Gly Pro Leu Phe Phe Leu Gln Pro Phe Val

115 120 125

Met Ala Leu Tyr Ile Thr Gly Asp Leu Asn Thr Ile Phe Ser Pro Ala

130 135 140

His Gln Lys Glu Ile Ile Arg Tyr Leu Tyr Asn His Gln Asn Glu Asp

145 150 155 160

Gly Gly Trp Gly Phe His Ile Glu Gly His Ser Thr Met Phe Gly Ser

165 170 175

Ala Leu Ser Tyr Ile Ala Leu Arg Ile Leu Gly Glu Gly Leu Glu Asp

180 185 190

Gly Glu Asp Gly Ala Met Ala Lys Ser Arg Lys Trp Ile Leu Asp His

195 200 205

Gly Gly Leu Val Ala Ile Pro Ser Trp Gly Lys Phe Trp Ala Thr Val

210 215 220

Leu Gly Leu Tyr Glu Trp Ser Gly Cys Asn Pro Leu Pro Pro Glu Phe

225 230 235 240

Trp Leu Leu Pro Asp Ile Phe Pro Ile His Pro Gly Lys Met Leu Cys

245 250 255

Tyr Cys Arg Leu Val Tyr Met Pro Met Ser Tyr Leu Tyr Gly Lys Arg

260 265 270

Phe Val Gly Pro Ile Thr Gly Leu Ile Gln Ser Leu Arg Gln Glu Leu

275 280 285

Tyr Asn Glu Pro Tyr His Gln Ile Asn Trp Asn Lys Ala Arg Ser Thr

290 295 300

Val Ala Lys Glu Asp Leu Tyr Tyr Pro His Pro Leu Ile Gln Asp Leu

305 310 315 320

Leu Trp Gly Phe Leu His His Val Ala Glu Pro Val Leu Thr His Trp

325 330 335

Pro Phe Ser Met Leu Arg Glu Lys Ala Leu Lys Ala Ala Ile Gly His

340 345 350

Val His Tyr Glu Asp Glu Asn Ser Lys Tyr Leu Cys Ile Gly Ser Val

355 360 365

Glu Lys Val Leu Cys Leu Ile Ala Cys Trp Ala Glu Asp Pro Asn Gly

370 375 380

Glu Ala Tyr Lys Leu His Leu Gly Arg Ile Pro Asp Asn Tyr Trp Val

385 390 395 400

Ala Glu Asp Gly Leu Lys Ile Gln Ser Phe Gly Cys Gln Met Trp Asp

405 410 415

Ala Gly Phe Ala Ile Gln Ala Ile Leu Ser Cys Asn Leu Asn Glu Glu

420 425 430

Tyr Trp Pro Thr Leu Arg Lys Ala His Glu Phe Val Lys Ala Ser Gln

435 440 445

Val Pro Glu Asn Pro Ser Gly Asp Phe Lys Ala Met Tyr Arg His Ile

450 455 460

Asn Lys Gly Ala Trp Thr Phe Ser Met Gln Asp His Gly Trp Gln Val

465 470 475 480

Ser Asp Cys Thr Ala Glu Gly Leu Lys Val Ala Ile Leu Phe Ser Gln

485 490 495

Met Pro Pro Asp Leu Val Gly Glu Lys Ile Glu Lys Glu Arg Leu Tyr

500 505 510

Asp Ala Val Asn Val Ile Leu Ser Leu Gln Ser Ser Asn Gly Gly Phe

515 520 525

Pro Ala Trp Glu Ser Gln Arg Ala Tyr Gly Trp Leu Glu Lys Phe Asn

530 535 540

Pro Thr Glu Phe Phe Glu Asp Thr Leu Ile Glu Arg Glu Tyr Val Glu

545 550 555 560

Cys Thr Ser Ser Ala Val His Gly Leu Ala Leu Phe Arg Lys Phe Tyr

565 570 575

Pro Arg His Arg Arg Thr Glu Ile Asp Ser Ser Ile Tyr Arg Gly Ile

580 585 590

Gln Tyr Ile Glu Asp Val Gln Glu Pro Asp Gly Ser Trp Tyr Gly His

595 600 605

Trp Gly Ile Cys Tyr Thr Tyr Gly Thr Trp Phe Ala Val Gly Ala Leu

610 615 620

Ala Ala Cys Gly Arg Asn Tyr Lys Asn Cys Pro Ala Leu Arg Lys Ser

625 630 635 640

Cys Glu Phe Leu Leu Ser Lys Gln Leu Pro Asn Gly Gly Trp Gly Glu

645 650 655

Ser Tyr Leu Ser Ser Gln Asn Lys Val Trp Thr Asn Ile Glu Gly Asn

660 665 670

Arg Ala Asn Leu Val Gln Thr Ala Trp Ala Leu Leu Ser Leu Ile Asp

675 680 685

Ala Gly Gln Ala Glu Ile Asp Pro Thr Pro Ile His Arg Gly Val Arg

690 695 700

Val Leu Ile Asn Ser Gln Met Glu Asp Gly Asp Phe Pro Gln Gln Glu

705 710 715 720

Ile Thr Gly Val Phe Met Arg Asn Cys Thr Leu Asn Tyr Ser Ser Tyr

725 730 735

Arg Asn Ile Phe Pro Ile Trp Ala Leu Gly Glu Tyr Arg Arg Arg Val

740 745 750

Leu Phe Ala

755

<210> 8

<211> 2268

<212> DNA

<213> white birch (Betula platyphylla suk.)

<400> 8

atgtggaagt tgaagatagc ggaaggaggg ccagggctgg tgagcggaaa tgatttcatc 60

gggcggcaac actgggaatt cgacccggat gccggcactc cccaagagcg tgctgaagtt 120

gaaaaggtcc gcgaggagtt caccaaaaat cggtttcaga tgaaacaaag cgctgatctt 180

ttgatgagga tgcagcttag gaaggagaac ccatgccaac caattccacc accagtgaaa 240

gtgaaagaaa cagaggtgat aacagaggaa gcagtgatta ctacactgag aagatcacta 300

agcttttatt cctccattca agctcatgat ggccactggc ctggtgaatc tgctggcccc 360

ttgtttttcc ttcaaccctt tgtaatggca ttatacatca ctggagatct caatactatt 420

ttttccccag cacaccagaa ggaaattatt cgatacttgt ataatcatca gaacgaagat 480

ggaggctggg ggttccatat agagggtcac agcacaatgt ttgggtcagc tttgagctac 540

attgccttga gaatacttgg agagggactt gaagatggtg aagatggggc tatggctaaa 600

agccggaaat ggattcttga ccatggtggt ttagtggcta ttccttcatg gggaaagttt 660

tgggccacgg tactgggact gtatgagtgg tcaggctgca atccactgcc cccagagttc 720

tggcttcttc ctgatatctt tcccatacat ccaggtaaaa tgttatgcta ctgtcgcttg 780

gtttacatgc caatgtctta tttatatggg aagaggtttg ttggtccaat cactggattg 840

attcaatcac ttagacaaga gttatataac gagccttacc atcaaattaa ctggaataaa 900

gcccggagta cagttgcaaa ggaggatctc tactatccgc atcccctcat acaagatctg 960

ctatggggat ttcttcacca tgtagccgag cctgtcctga cgcattggcc cttttcaatg 1020

ctgagagaga aggcactcaa agctgcaatt ggtcatgtac attatgagga cgagaacagc 1080

aaataccttt gcattggaag cgttgaaaag gtattatgtt tgattgcatg ttgggctgaa 1140

gatccaaatg gggaggcata caagcttcat ctaggaagga ttccagacaa ctattgggtt 1200

gctgaagatg gcttaaaaat tcagagtttc ggctgtcaga tgtgggatgc gggttttgct 1260

attcaagcaa ttctctcttg caatttaaac gaagagtatt ggccaacact tcgtaaagca 1320

catgagtttg taaaggcttc acaggtccca gaaaaccctt ctggggactt caaagccatg 1380

taccgccaca taaacaaagg agcatggaca ttctcgatgc aggaccatgg atggcaagtc 1440

tctgactgca ccgctgaagg gctgaaggtt gcaatcttgt tctcgcaaat gcctccggac 1500

cttgtcgggg aaaaaattga gaaagagcgg ttatatgatg ctgtgaatgt cattctttct 1560

ctacaaagta gcaatggtgg tttcccagca tgggagtctc aaagagcata tggttggttg 1620

gagaagttca accccacgga attctttgaa gataccctta ttgagcgaga gtacgtagag 1680

tgcacttcat ctgcagttca tggtctggca ctctttagga agttctatcc ccggcaccgg 1740

aggacggaga tagatagtag catttacagg ggaattcaat acattgaaga cgtgcaagaa 1800

cctgatggat catggtatgg tcattggggg atttgctaca cctacggtac atggtttgct 1860

gtaggggcac tggcagcctg tggaagaaac tacaaaaatt gtcctgcatt gcgcaaatct 1920

tgtgaatttt tgctatcaaa gcagctacct aatggtggat ggggagaaag ttacctatca 1980

agccaaaaca aggtgtggac gaatatagaa ggcaaccgtg caaatttagt ccaaacagca 2040

tgggccttgt tatccctcat tgatgctggg caggccgaga tagatccaac gccaattcat 2100

cgtggagtaa gagtattgat caattcacag atggaagatg gtgactttcc tcaacaggaa 2160

atcactggag tatttatgcg aaactgcaca ctaaactact catcatatag aaacattttt 2220

ccaatatggg ctcttggaga atatcggagg cgagttctat ttgcatga 2268