阅读说明：本技术 一种调控玉米分蘖数的基因及其编码蛋白与应用 (Gene for regulating and controlling tillering number of corn, and encoded protein and application thereof ) 是由 林中伟 张旋 李艳 于 2019-11-05 设计创作，主要内容包括：本发明公开了一种调控玉米分蘖数的基因及其编码蛋白与应用。本发明提供了蛋白,为序列表中序列2所示的蛋白质或序列表中序列4所示的蛋白质；本发明克隆了一个可以调控玉米分蘖发育的基因tin1,在玉米中提高该基因的表达量可以使玉米分蘖数明显增多,同时还可以显著提高其雌穗数目,而对其他的农艺性状没有明显的影响。tin1基因的克隆,为今后的玉米分蘖数的精细改良提供了重要的理论基础和切实可行的新方法。因而,tin1基因在玉米分子设计育种中具有巨大的应用潜力。(The invention discloses a gene for regulating and controlling the tillering number of corn, and a coding protein and application thereof. The invention provides a protein which is a protein shown as a sequence 2 in a sequence table or a protein shown as a sequence 4 in the sequence table; the invention clones a gene tin1 which can regulate and control the tillering development of corn, and the improvement of the expression quantity of the gene in the corn can obviously increase the tillering number of the corn and can also obviously improve the number of the female ears without obviously influencing other agronomic traits. the cloning of the tin1 gene provides an important theoretical basis and a feasible new method for the fine improvement of the tillering number of corn in the future. Therefore, the tin1 gene has great application potential in corn molecule design and breeding.)

1. A protein is any one of the following (a1) - (a 5):

(a1) protein shown in a sequence 2 in a sequence table;

(a2) protein shown in a sequence 4 in a sequence table;

(a3) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a1) or (a 2);

(a4) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) or (a2) and is related to plant development;

(a5) a protein having 98% or more identity to (a1) or (a2) and involved in plant development.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is any one of the following (b1) - (b 6):

(b1) is a DNA molecule shown as a sequence 1 in a sequence table;

(b2) is a DNA molecule shown as a sequence 3 in a sequence table;

(b3) the coding region is a DNA molecule shown as a sequence 5 in a sequence table;

(b4) the coding region is a DNA molecule shown as a sequence 6 in a sequence table;

(b5) a DNA molecule having 95% or more identity to any one of (b1) to (b4) and encoding the protein;

(b6) a DNA molecule which hybridizes with the nucleotide sequence defined in any one of (b1) to (b4) under stringent conditions and encodes the protein of claim 1.

4. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.

5. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or 3 or the expression cassette, recombinant vector or recombinant microorganism of claim 4 in (c1) and/or (c2) as follows:

(c1) regulating and controlling the tillering number of the plant;

(c2) regulating and controlling the female spike number of the plant.

6. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or 3 or the expression cassette, recombinant vector or recombinant microorganism of claim 4 in (d1) and/or (d 2):

(d1) cultivating plants with increased tillering number;

(d2) and (5) cultivating plants with the increased number of female spikes.

7. A method of making a plant with increased tiller number and/or increased ear number comprising the steps of:

1) constructing a transgenic plant;

the method for constructing the transgenic plant comprises the following steps of 1) -A, 1) -B or 1) -C:

1) -a, increasing the content or activity of the protein of claim 1 in a recipient plant, resulting in a transgenic plant;

1) -B, increasing the expression level of a nucleic acid molecule encoding a protein according to claim 1 in a recipient plant, to obtain a transgenic plant;

1) -C, introducing a nucleic acid molecule encoding the protein of claim 1 into a plant of interest, resulting in a transgenic plant;

2) and (3) hybridizing the transgenic plant with a plant without tillering number, and selfing hybrid progeny to obtain a plant with increased tillering number and/or increased ear number.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a gene for regulating and controlling the tillering number of corn, and a coding protein and application thereof.

Background

The development of the corn tillering is controlled by variable environmental factors and a complex gene regulation network. The genes related to the tillering development of the corn are cloned mainly three genes of tb1 (tetrosint branched 1), gt1(grass tillered 1) and tru1(tassels place upperers 1). Wherein the tb1 gene encodes a transcription factor of TCP family, which is involved in regulating the apical dominance of corn and influencing the branching ability of corn. In the cultivated corn, a Hopscitch type transposon with the length of about 4.9kb is inserted into a regulatory region with the upstream of about 60kb of tb1 gene, so that the expression level of the transposon is improved at the corn branch position, the corn branch is inhibited, and finally the cultivated corn loses the branching capability. gt1 and tru1 are located at the downstream of tb1 and are regulated by tb1, so that the tiller number of the corn is influenced to a certain extent. The tillering number of the corn plays an important role in the corn domestication and improvement process. Conventionally cultivated corn usually loses the habit of tillering and has only one strong main stem. However, in most cases, tillers with 2-4 ears still grow from specialty corn varieties such as sweet corn and pop corn. The reduction of tillering number in the conventional corn variety is beneficial to improving the planting density of the corn, thereby improving the yield. The proper increase of tillering number in special corn varieties (such as sweet corn, cracked corn, silage corn and the like) can obviously increase the single-plant ear number and biomass of the corn, thereby improving the yield to a certain extent. Therefore, the fine control of the tillering number of the corn has an important breeding target in corn breeding. Because of the gene of the tillering number cloned at present, the tillering number of the corn is influenced, and simultaneously, other agronomic characters and yield characters of the corn are also obviously influenced. Therefore, the method cannot be widely applied to the corn breeding process.

Disclosure of Invention

An object of the present invention is to provide a protein associated with plant tillering.

The protein provided by the invention is any one of the following (a1) - (a 5):

(a1) the protein shown in the sequence 2 in the sequence table is derived from a multi-tillering inbred line P51;

(a2) the protein shown in the sequence 4 in the sequence table is derived from a tillerless inbred line B37;

(a3) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a1) or (a 2);

(a4) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) or (a2) and is related to plant development;

(a5) a protein having 98% or more identity to (a1) or (a2) and involved in plant development.

In the present invention, the plant develops into plant tillers.

Nucleic acid molecules encoding the above proteins are also within the scope of the present invention.

The nucleic acid molecule is any one of the following (b1) to (b 6):

(b1) is a DNA molecule shown as a sequence 1 in a sequence table;

(b2) is a DNA molecule shown as a sequence 3 in a sequence table;

(b3) the coding region is a DNA molecule shown as a sequence 5 in a sequence table;

(b4) the coding region is a DNA molecule shown as a sequence 6 in a sequence table;

(b5) a DNA molecule having 95% or more identity to any one of (b1) to (b4) and encoding the protein;

(b6) a DNA molecule which hybridizes with any one of the nucleotide sequences defined in (b1) - (b4) under stringent conditions and encodes the protein.

Expression cassettes, recombinant vectors or recombinant microorganisms containing the above-described nucleic acid molecules are also within the scope of the present invention.

In the embodiment of the invention, the recombinant vector is pBCXUN-tin 1P51, which is formed by connecting a gene tin1P51 shown in a sequence 5 to a pBCXUN vector, and the recombinant vector expresses a protein tin1P51 shown in a sequence 2;

the recombinant vector pBCXUN-tin 1B37 is prepared by connecting a gene tin1B37 shown in a sequence 6 to a pBCXUN vector, and the recombinant vector expresses a protein tin1B37 shown in a sequence 4.

The use of the above-mentioned protein or the above-mentioned nucleic acid molecule or the above-mentioned expression cassette, recombinant vector or recombinant microorganism in (c1) and/or (c2) is also within the scope of the present invention:

(c1) regulating and controlling the tillering number of the plant;

(c2) regulating and controlling the female spike number of the plant.

The use of the above protein or the above nucleic acid molecule or the above expression cassette, recombinant vector or recombinant microorganism in (d1) and/or (d 2):

(d1) cultivating plants with increased tillering number;

(d2) and (5) cultivating plants with the increased number of female spikes.

It is another object of the present invention to provide a method for producing a plant with increased tiller number and/or increased ear number.

The method provided by the invention comprises the following steps:

1) constructing a transgenic plant;

the method for constructing the transgenic plant comprises the following steps of 1) -A, 1) -B or 1) -C:

1) -a, increasing the content or activity of the protein of the first interest in the recipient plant, resulting in a transgenic plant;

1) b, increasing the expression level of the nucleic acid molecule encoding the protein in the receptor plant to obtain a transgenic plant;

1) c, introducing the nucleic acid molecule for coding the protein into a target plant to obtain a transgenic plant;

2) and crossing the transgenic plant and a plant without tillering number, and selfing the filial generation to remove the target plant background to obtain the plant with increased tillering number and/or increased female ear number.

In the examples of the present invention, the target plant is B73.

The inventor finds that the expression level of tin1 at the tillerless inbred line B37 tillering bud is obviously lower than that of multi-tillering inbred line B37 tillering budCross-line P51. The tin1 gene is found not to affect the formation of lateral meristem through the phenotype analysis of near isogenic line,but finally influences the number of visible tillers of the corn by regulating and controlling the elongation of the tillering buds. In addition, the gene has no obvious influence on other agronomic traits while regulating and controlling the tillering number of the corn in a near-isogenic line. Through transgenic verification, the over-expression of the tin1 genes of two parent types is proved to increase the tillering number of the corn significantly (P-value)<0.01) and can also obviously increase the number of female ears (P-value) of the corn<0.01) so that the yield can be improved to some extent.

The gene tin1 for regulating and controlling the tillering development of the corn is over-expressed in the corn, so that the tillering number of the corn can be obviously increased by improving the expression quantity of the gene, the number of the female ears can be obviously increased, and other agronomic traits are not obviously influenced. the cloning of the tin1 gene provides an important theoretical basis and a feasible new method for the fine improvement of the tillering number of corn in the future. Therefore, the tin1 gene has great application potential in corn molecule design and breeding.

Drawings

FIG. 1 is a photograph of a phenotype of a parental population and a strategy for constructing a definitive population; FIG. 1A shows the phenotype (upper) and grain phenotype (lower) of the single stalk parent B37 and the multi-tillering parent P51 at the mature stage; FIG. 1B shows a strategy for QTL positioning population construction.

FIG. 2 shows comparison of tillering phenotype of plants of tin1 near isogenic line; FIG. 2a is a comparison of the phenotype of the seedling stage of plants of the near isogenic line carrying the B37 type tin1 allele (NIL-B37) and the P51 type tin1 allele (NIL-P51); FIG. 2B is a comparison of the phenotype of the plants of the near isogenic line carrying the allele of tin1 of type B37 and the allele of tin1 of type P51 at maturity; FIG. 2c is the tillering shoot phenotype at the seedling stage of a near isogenic line plant carrying the B37 type tin1 allele; FIG. 2d is the tillering shoot phenotype at the seedling stage of a near isogenic line plant carrying the P51 type tin1 allele; FIG. 2e is the tillering shoot phenotype of the juxtametochore plant with the B37 type tin1 allele; FIG. 2f tillering shoot phenotype of plants of the near isogenic line with P51 type tin1 allele at the elongation stage; FIG. 2g shows the tillering shoot phenotype of a plant of the near isogenic line with the B37 type tin1 allele at the mature stage; FIG. 2h shows the tillering shoot phenotype of the plant in the mature stage of the near isogenic line with P51 type tin1 allele; FIG. 2i shows the difference between the tillering gradient of plants of the near isogenic line with the B37 type tin1 allele and the P51 type tin1 allele; FIG. 2j is the statistical difference in tillering number phenotype at maturity for the near isogenic line with the B37 type tin1 allele and the P51 type tin1 allele.

FIG. 3 shows the phenotypic comparison of other agronomic traits for the line tin1 NIL.

FIG. 4 shows the positioning and cloning of tin1 site; FIG. 4a shows the initial positioning result of tin1 locus; FIG. 4b shows the fine positioning of tin1 locus; FIG. 4c shows candidate genes within the fine localization interval of the tin1 locus.

FIG. 5 shows a comparison of expression patterns of tin1 in a tin1 near isogenic line; note: "" indicates P-value < 0.01.

FIG. 6 shows the difference in expression level of transgenic over-expressed lines and the tillering behavior of their progeny; FIG. 6a shows the difference in tiller number between transgenic overexpression line OEtin1B37-1 and control CK at the pollen scattering stage; FIG. 6b stem base detail of control CK of transgenic over-expressed plants; FIG. 6c Stem base of transgenic overexpression line OEtin1B37-1A detail view; FIG. 6d turn The mature phenotype of the gene over-expression plant OEtin1B37-1 and the control CK is different; FIG. 6e tin1 difference in expression levels in 4 transgenic overexpressing families versus control CK; FIG. 6f tillering number phenotype statistical analysis of 4 transgenic over-expressed families versus control CK; FIG. 6g shows the phenotypic statistical analysis of the number of ears of transgenic over-expressed plants OEtin1B37-1 and control CK; "x" represents 0.001 significant levels.

FIG. 7 shows the tillering behavior of transgenic over-expression lines at mature stage; FIG. 7a shows the difference in tillering number between transgenic over-expressed plants OEtin1P51 and control CK at the mature stage; FIG. 7B shows the difference in tillering number between transgenic over-expressed plants OEtin1B37-2 and control CK at the mature stage; FIG. 7c shows the difference in tillering number between transgenic over-expressed plants OEtin1B37-3 and control CK at the mature stage; each figure is divided into upper and lower groups, and the upper is enlarged below.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Maize B37 (named B37 in the literature), B73 maize (named B73 in the literature), and maize P51 (named P51 in the literature) are all described in: bukowski R, Guo X, Lu Y, et al.construction of third-generation Zea Mays hash map [ J ]. Gigascience,2017,7(4): gix 134.