阅读说明：本技术 抗旱相关蛋白IbSPB1及其编码基因与应用 (Drought-resistant related protein IbSPB1 and coding gene and application thereof ) 是由 翟红 刘庆昌 何绍贞 高少培 张欢 龙海东 白宜冬 李思语 于 2021-08-25 设计创作，主要内容包括：本发明公开了一种蛋白质IbSPB1在调控植物抗旱性中的应用。本发明首先公开了一种蛋白质在提高植物耐盐抗旱性中的应用；所述蛋白质为氨基酸序列如SEQ ID NO.1所示的蛋白质或SEQ ID NO.1所示的氨基酸序列的N端或/和C端连接蛋白标签得到的融合蛋白。本发明进一步公开了上述蛋白相关生物材料及其应用。本发明发现了IbSPB1蛋白及其编码基因,并将IbSPB1蛋白的编码基因导入甘薯中,得到IbSPB1的转基因甘薯植株。将转基因植株进行干旱胁迫处理,其抗旱性增强,在植物抗干旱的过程中起着重要的作用,在农业领域具有广阔的应用空间和市场前景。(The invention discloses an application of protein IbSPB1 in regulating and controlling plant drought resistance. The invention firstly discloses the application of protein in improving the salt and drought resistance of plants; the protein is a protein with an amino acid sequence shown in SEQ ID NO.1 or a fusion protein obtained by connecting protein labels at the N end or/and the C end of the amino acid sequence shown in SEQ ID NO. 1. The invention further discloses the protein-related biomaterial and application thereof. The invention discovers IbSPB1 protein and a coding gene thereof, and introduces the coding gene of the IbSPB1 protein into sweet potatoes to obtain a transgenic sweet potato plant of the IbSPB 1. The drought stress treatment is carried out on the transgenic plant, the drought resistance of the transgenic plant is enhanced, the transgenic plant plays an important role in the drought resistance process of the plant, and the transgenic plant has wide application space and market prospect in the agricultural field.)

1. Protein, characterized in that the protein is a1) or a2) or A3) as follows:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue on the amino acid sequence shown in A1), has more than 80% of identity with the protein shown in A1) and has the function of regulating and controlling plant drought resistance;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

2. The protein of claim 1, wherein said protein is derived from sweetpotato.

3. The biomaterial related to the protein of claim 1 or 2, wherein the biomaterial is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein of 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 containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;

B6) a transgenic plant tissue containing B1) the nucleic acid molecule, or a transgenic plant tissue containing B2) the expression cassette, or a transgenic plant tissue containing B3) the recombinant vector;

B7) a transgenic plant organ containing B1) the nucleic acid molecule, or a transgenic plant organ containing B2) the expression cassette, or a transgenic plant organ containing B3) the recombinant vector.

4. The biomaterial according to claim 3, characterized in that B1) the nucleic acid molecule is the gene of the following G1) or G2):

G1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

G2) the nucleotides of the coding strand are cDNA molecules or DNA molecules of SEQ ID No. 2.

5. A method for improving drought resistance of a plant, which comprises introducing a gene encoding the protein of claim 1 into a recipient plant to obtain a target plant having higher drought resistance than the recipient plant.

6. The method of claim 5, wherein the coding gene is G1) or G2) as follows:

G1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule shown as SEQ ID No. 2;

G2) the nucleotide sequence of the coding strand is a cDNA molecule or a DNA molecule as shown in SEQ ID No. 2.

7. The method of claim 5 or 6, wherein the recipient plant is a dicotyledonous plant.

8. The method according to claim 7, wherein the dicotyledonous plant is any one of:

1) tubular plants of the order florida;

2) a plant of the family Convolvulaceae;

3) ipomoea plants;

4) sweet potato.

9. Use of the protein of claim 1 or 2, and/or the biomaterial of claim 3 or 4, wherein the use is any one of the following:

p1) improving drought resistance of plants;

p2) preparing products for improving the drought resistance of plants;

p3) improving the rooting of plants under drought stress conditions;

p4) preparing a product for improving the rooting condition of plants under drought stress conditions;

p5) increasing the growth vigor of plants under drought stress conditions;

p6) preparing a product of the growth vigor of the plant under drought stress conditions;

p7) reduction of plant H under drought stress conditions₂O₂Content (c);

p8) preparation of plant H under conditions of reduced drought stress₂O₂The product of (a);

p9) plant breeding;

p10) sweet potato breeding.

10. Use according to claim 9, wherein the plant is any one of the following:

1) tubular plants of the order florida;

2) a plant of the family Convolvulaceae;

3) ipomoea plants;

4) sweet potato.

Technical Field

The invention relates to the technical field of biology, in particular to drought-resistant related protein IbSPB1, and a coding gene and application thereof.

Background

With the abnormal change of global climate, the destruction of ecological balance and the rapid increase of population, the shortage of water resources has become one of the most serious challenges facing the development of agricultural production nowadays. FAO investigation results show that drought stress is the most main reason for grain safety in developing countries, and influences on grain production far exceed natural disasters such as flooding, earthquake, typhoon, debris flow and the like. Due to the influences of factors such as abnormal global climate change, large annual change of rainfall, uneven rainfall spatial-temporal distribution and the like, the agricultural production in China is more and more seriously influenced by drought disasters, and the grain safety faces serious challenges.

Although sweet potatoes are drought-tolerant crops, the growth and development and yield of sweet potatoes are affected in the absence of water, as in other crops. By deeply researching the salt and drought resistance mechanism of plants, the salt and drought resistance gene resources are excavated, and the cultivation of new salt and drought resistant sweet potato varieties is one of the most economic and effective measures for utilizing saline-alkali soil and arid and semi-arid resources.

Therefore, the gene with the functions of drought resistance and salt tolerance is obtained by cloning and identifying through a genetic engineering means, and the gene is transferred into a plant body to improve the drought resistance and salt tolerance of the plant, so that the gene has important research and application values.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the drought resistance of plants.

In order to solve the technical problems, the invention firstly provides a protein, and the protein is A1) or A2) or A3) as follows:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue on the amino acid sequence shown in A1), has more than 80% of identity with the protein shown in A1) and has the function of regulating and controlling plant drought resistance;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

Further, the protein is derived from sweet potato.

Wherein SEQ ID No.1 consists of 333 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

Herein, the protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a protein of interest using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the protein of interest. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

Herein, the identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

Herein, the 80% or greater identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

The invention also provides a biological material related to the protein, wherein the biological material is any one of the following B1) to B7):

B1) nucleic acid molecules encoding the above proteins;

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 containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;

B6) a transgenic plant tissue containing B1) the nucleic acid molecule, or a transgenic plant tissue containing B2) the expression cassette, or a transgenic plant tissue containing B3) the recombinant vector;

B7) a transgenic plant organ containing B1) the nucleic acid molecule, or a transgenic plant organ containing B2) the expression cassette, or a transgenic plant organ containing B3) the recombinant vector.

Wherein, the nucleic acid molecule of B1) 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.

Further, the biological material B1) may be a gene of G1) or G2) as follows:

G1) the coding sequence of the coding chain is shown as cDNA molecule or DNA molecule shown in SEQ ID No. 2;

G2) the nucleotide sequence of the coding chain is shown as cDNA molecule or DNA molecule shown in SEQ ID No. 2.

Wherein, SEQ ID No.2 consists of 1002 nucleotides, the Open Reading Frame (ORF) thereof is from 1 st to 1002 th from the 5' end, and the encoded amino acid sequence is the protein shown as SEQ ID No. 1.

In the above-mentioned related biomaterials, the expression cassette described in B2) means a DNA capable of expressing the protein IbSPB1 in a host cell, and the DNA may include not only a promoter which promotes the transcription of the IbSPB1 gene but also a terminator which terminates the transcription of the IbSPB1 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 Physiol120: 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 jasmonic acid ester); 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 (Beachy et al (1985) EMBO J.4:3047-3053), used alone or in combination with other plant promoters. all references cited herein are incorporated in their entirety suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., Odell et al (1985) Nature 313: 810; Rober et al (1987) Gene,56: 125; Guerine et al (1991) mol.262. Gen.262; Dev.141: Sanfacon. 64; Gen. 141: 64, Gen. Fa., 5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

In the above-mentioned related biological materials, B3) said recombinant vector may contain a DNA molecule for encoding the protein IbSPB1 shown in SEQ ID No. 2.

A plant expression vector can be used for constructing a recombinant vector containing the IbSPB1 coding gene expression cassette. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector and the like, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300-35S, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA 1391-Xb. When IbSPB1 is used to construct a recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CAMV)35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, 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 proper 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, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the related biological material, the recombinant microorganism B4) can be yeast, bacteria, algae and fungi.

In the above-mentioned related biological materials, B6) the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the above-mentioned related biological materials, B7) the transgenic plant organ may be a root, a stem, a leaf, a flower, a fruit and a seed of the transgenic plant.

In the related biological materials, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ may or may not include propagation material.

The invention also provides a method for improving the drought resistance of plants, which comprises the step of introducing the coding gene of the protein into a receptor plant to obtain a target plant with higher drought resistance than the receptor plant.

Further, the encoding gene in the above method is the following G1) or G2):

G1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule shown as SEQ ID No. 2;

G2) the nucleotide sequence of the coding strand is a cDNA molecule or a DNA molecule as shown in SEQ ID No. 2.

In the above method, the above-mentioned protein-encoding gene can be introduced into a target plant by a plant expression vector carrying the protein-encoding gene of the present invention. The plant expression vector carrying the protein-encoding gene of the present invention can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and culture the transformed plant cells or tissues into plants.

In a specific embodiment of the invention, the recombinant vector is a recombinant plasmid pCB-IbSPB 1. The recombinant plasmid pCB-IbSPB1 is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No.2 between the cleavage recognition sites of Kpn I and BamHI of the vector pCAMBIA1300-35S by using restriction enzymes Kpn I and BamH I, and keeping the other sequences of the vector pCAMBIA1300-35S unchanged.

In the recombinant vector pCB-IbSPB1, the promoter for promoting IbSPB1 gene transcription is the 35S promoter located in the vector pCAMBIA 1300-35S.

The invention also provides the application of the protein and/or the biological material, wherein the application is any one of the following:

p1) improving drought resistance of plants;

p2) preparing products for improving the drought resistance of plants;

p3) improving the rooting of plants under drought stress conditions;

p4) preparing a product for improving the rooting condition of plants under drought stress conditions;

p5) increasing the growth vigor of plants under drought stress conditions;

p6) preparing a product of the growth vigor of the plant under drought stress conditions;

p7) reduction of plant H under drought stress conditions₂O₂Content (c);

p8) preparation of plant H under conditions of reduced drought stress₂O₂The product of (a);

p9) plant breeding;

p10) sweet potato breeding.

Specifically, the breeding is drought-resistant breeding.

The above plant may be a dicot, which may be a tubular plant of the order florida. The tubular plant of the order florida may be a plant of the family Convolvulaceae. The Convolvulaceae plant can be Ipomoea plant. The Ipomoea plant may be Ipomoea batatas.

The growth vigor can be represented by the root length and the plant weight of the plant.

The invention provides IbSPB1 protein and a coding gene thereof, and the coding gene of the IbSPB1 protein is introduced into sweet potatoes to obtain a transgenic sweet potato plant of the IbSPB 1. The drought stress treatment is carried out on the transgenic plant, and compared with the contrast, the drought resistance of the transgenic plant is enhanced, which is embodied in that the growth vigor is enhanced and H is increased₂O₂The content is reduced. Therefore, the IbSPB1 gene and the protein coded by the gene play an important role in the process of plant drought resistance, have important application value in the research of improving plant drought resistance, and have wide application space and market prospect in the agricultural field.

Drawings

FIG. 1 shows the PCR detection result of transgenic sweetpotato plants; wherein M is marker; w is water; p is positive plasmid; WT is a wild type sweet potato plant; L1-L6 is a transgenic plant.

FIG. 2 shows the expression analysis of IbSPB1 in transgenic sweetpotato. Wherein WT is a wild type sweet potato plant; L1-L5 is a transgenic plant.

FIG. 3 shows drought resistance identification of transgenic IbSPB1 sweet potato plants and wild sweet potatoes; wherein WT is wild type sweet potato; l1, L2 and L3 are overexpression IbSPB1 transgenic sweet potato lines. Controls represent fresh weight and root length after 4 weeks of culture in normal medium, and drought represents fresh weight and root length after 4 weeks of culture in stress medium.

FIG. 4 is H of overexpression of IbSPB1 transgenic sweetpotato plants and wild type control₂O₂Measuring the content; wherein WT is wild type sweet potato; l1, L2 and L3 are overexpression IbSPB1 transgenic sweet potato lines. Controls represent 4 weeks of culture in normal medium and drought represents 4 weeks of culture in stress medium.

Detailed Description

Sweet potato variety lushu 3, Zhai hong, Shanli, Liu Qingchang, construction of hybrid cDNA library for inhibition of stem nematode induction and subtraction of sweet potato and analysis of expression sequence tags, report of agricultural biotechnology, 2010,18 (1): 141-148 ", the public is allowed to obtain from the sweet potato genetic breeding research laboratory of the university of agriculture of China after the author agrees, so as to repeat the experiment, and the method cannot be used for other purposes.

Chestnut flavor of sweet potato variety is disclosed in the literature "Yu B, ZHai H, Wang YP, Zang N, He SZ, Liu QC. efficient Agrobacterium tumefaciens-mediated transformation using organized genetic mutation cultures in sweet potato, Ipomoea batatas (L.) Lam.2007, Plant Cell, Tissue & Organ Culture, 90(3):265 and 273" (named Lizixiang in the literature), which can be obtained from the research room of sweet potato genetic breeding of the university of China after the agreement of the public authors to repeat the experiment and can not be used for other purposes.

pMD19-T vector was purchased from Beijing Zeping science and technology, Inc., under product catalog number A1360.

The vector pCAMBIA1300-35 was purchased from Wuhan transduction Biolabs, Inc., and has catalog number VT 4004.

Agrobacterium tumefaciens EHA105 was purchased from Beijing Bylendi Biotechnology, Inc.

Escherichia coli DH5a (from Beijing Quanjin Biotechnology Co., Ltd., catalog No. CD201-01)

The following examples were processed using SPSS19.0 statistical software, with P < 0.05 (x) indicating a significant difference, P < 0.01 (x) indicating a very significant difference, and P < 0.001 (x) indicating a very significant difference.

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 examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 obtaining of IbSPB1 protein related to improving drought resistance of sweet Potato and its encoding Gene

1.1 cloning of IbspB1 Gene cDNA of Ipomoea batatas

Experimental materials: sweet potato variety Lushu No. 3

1.1.1 extraction of Total RNA from sweet Potato

Grinding 0.1g of young and tender leaves of sweet potato into powder in liquid nitrogen, adding into a 2mL centrifuge tube, and extracting the total RNA of the sweet potato by using a TIANGEN RNAprep pure plant total RNA extraction kit (catalog number: DP432), wherein the kit comprises: lysis solution RL, deproteinization solution RW1, rinsing solution RW, RNase-Free ddH₂O, RNase-Free adsorption column CR3, RNase-Free filtration column CS, DNase I, buffer RDD, RNase-Free centrifuge tube, RNase-Free collection tube. Collecting 1 μ L, performing 1.2% agarose gel electrophoresis to detect its integrity, diluting 2 μ L to 500 μ L, and detecting its quality (OD) with ultraviolet spectrophotometer_260nm) And purity (OD)_260nm/OD_280nm) The extracted Luma 3 total RNA is detected by non-denaturing gel agarose gel electrophoresis, the 28S and 18S bands are clear, the brightness ratio of the two is 1.5-2: 1, the total RNA is not degraded, the obtained mRNA meets the experimental requirements, and the method can be used for cloning the IbSBP1 protein cDNA full length.

1.1.2 full-Length cloning of IbSPB1 Gene cDNA

Primers were designed for full-length cloning of the IbSPB1 cDNA.

The primer sequences are as follows:

primer 1: 5'-ATGGCTTTTCCTCAGCAG-3'

Primer 2: 5'-TTACATATAAACCTCTATACCTATAT-3'

The total RNA extracted in the step 1 is reversely transcribed into template DNA by QuantScript RT Kit (TIANGEN, Beijing), and PCR amplification is carried out by using high-fidelity LA enzyme. Detecting the PCR amplification product by agarose gel electrophoresis to obtain an amplification fragment with the length of 1002 bp.

After sequencing, the PCR product has a nucleotide sequence shown in SEQ ID No.2, a gene shown in the sequence is named IbSPB1 gene, a coding region of the gene is nucleotides 1 to 1002 from the 5' end of the SEQ ID No.2, a protein coded by the gene is named IbSPB1 protein or protein IbSPB1, an amino acid sequence is SEQ ID No.1 and consists of 333 amino acid residues.

1.2 construction of plant expression vectors

According to the coding sequence of the cDNA of IbsPB1 gene of sweet potato, designing and amplifying a primer sequence of the complete coding sequence, respectively introducing Kpn I and BamH I enzyme cutting sites into forward and reverse primers (primer 3 and primer 4), wherein the primer sequences are as follows:

primer 3: 5' -CGGGGTACCCCGATGGCTTTTCCTCAGCAGA-3' (the underlined part is the Kpn I cleavage site sequence),

primer 4: 5' -CGCGGATCCGCGTTACATATAAACCTCTATACCTATAT-3' (the underlined part is the BamH I cleavage site).

Using artificially synthesized SEQ ID No.2 as a template, and carrying out PCR amplification to obtain a product for later use.

The vector pCAMBIA1300-35S (Wuhan transduction biology laboratory Co., Ltd., product catalog number VT4004) was digested with Kpn I and BamH I, the large fragment of the vector was recovered, at the same time, the PCR product was digested with Kpn I and BamH I, the intermediate fragment of about 1.0kb was recovered, and the recovered large fragment of the vector was ligated with the intermediate fragment of about 1.0kb to obtain the desired plasmid. The target plasmid is transformed into escherichia coli DH5a (purchased from Beijing Quanyujin biotechnology limited, product catalog number is CD201-01), cultured for 20h at 37 ℃, subjected to PCR analysis and enzyme digestion identification of the recombinant vector, and subjected to sequencing verification. Sequencing results show that a sequence shown by 1 st to 1002 th sites from 5' ends of SEQ ID No.2 is inserted between Kpn I and BamH I recognition sites of the vector pCAMBIA1300-35S, other nucleotide sequences of the vector pCAMBIA1300-35S are kept unchanged, the construction of the recombinant vector is correct, and the recombinant vector is named as pCB-IbSPB 1. In pCB-IbSPB1, the promoter that initiates transcription of the IbSPB1 gene is the 35S promoter located in the vector pCAMBIA 1300-35S.

1.3 plant expression vector transformation of Agrobacterium

(1) 200 mu L of agrobacterium tumefaciens EHA105 competent cells are taken out from a low-temperature refrigerator at minus 80 ℃, placed on ice for thawing, and added with 1 mu g of the plant expression vector pCB-IbSBP1 obtained in the step 1, and mixed evenly.

(2) Freezing with liquid nitrogen for 1min, and incubating at 37 deg.C for 5 min.

(3) Adding 800 μ L LB liquid culture medium, and culturing at 28 deg.C for 2-6 h.

(4) mu.L of the resulting suspension was applied to LB solid medium (containing 100. mu.g/mL rifampicin (Rif) and 25. mu.g/mL kanamycin (Kan)), and the applied solution was spread uniformly, followed by sealing the petri dish. The plates were inverted and incubated at 28 ℃ for 2 d.

(5) Taking a single colony which is positive in PCR identification, inoculating the single colony into LB liquid culture medium containing Rif of 100 mu g/mL and Kan of 25 mu g/mL, culturing at 28 ℃ for 30h to logarithmic phase, taking a proper amount of agrobacterium, and diluting the agrobacterium with the liquid MS culture medium by 30 times for later use to obtain agrobacterium liquid (agrobacterium liquid containing target genes) introduced into pCAMBIA1300-IbSPB 1.

Example 2 genetic transformation and regeneration of sweetpotato

2.1 explant preparation

Peeling the sterilized stem tip of chestnut under microscope, cutting stem tip differentiated tissue cell with size of 0.1mm with scalpel, inoculating on MS solid culture medium containing 2.0 mg/L2, 4-D, and culturing under dark culture condition. After the cells produced embryoid bodies, small pieces of embryoid bodies were uniformly placed in an MS liquid medium containing 2.0mg/L of 2,4-D, shake-cultured, and subcultured once in 7 days (see Yu et al, 2007 for a specific method).

2.2, introducing the coding sequence of IbSPB1 cDNA into the chestnut flavor of a sweet potato variety by an agrobacterium-mediated method. The specific method comprises the following steps:

adding the prepared agrobacterium liquid containing the target gene into the sweet potato variety chestnut fragrant embryonic suspension cell mass, standing for 5-8mins, uniformly placing the suspension cell mass on an MS solid culture medium containing 2.0 mg/L2, 4-D and 30mg/L AS, carrying out co-culture, carrying out dark culture at the temperature of 27 +/-1 ℃ for 3 days. Subsequently, the embryogenic cell mass was cultured in MS liquid medium containing 2.0 mg/L2, 4-D for delayed culture. 7-10 days later, putting the cell mass on a solid MS culture medium which is paved with filter paper and contains 2.0 mg/L2, 4-D and 0.25mg/L hygromycin for screening culture at the temperature of 27 +/-1 ℃ for dark culture for 10 days; transferring the callus with good growth state to MS solid culture medium paved with a layer of filter paper and containing 2.0 mg/L2, 4-D and 0.5mg/L hygromycin for selective culture at 27 +/-1 ℃, carrying out dark culture, and replacing the culture medium every 2 weeks; after 8 weeks, surviving resistant calli were carefully transferred to 1.0mg/L ABA solid MS medium for induction at 27. + -. 1 ℃ for 13h daily with 3000lux light. After 2-4 weeks, the green somatic embryos were transferred to solid MS medium at 27. + -. 1 ℃ for 13h daily with 3000lux light. 4-8w later, growing into a complete regeneration plant to obtain a pseudotransgenic plant.

The identification of the transgenic plants uses a method combining PCR detection and qRT-PCR detection.

A. PCR detection

Extracting genome DNA of a pseudotransgenic plant and a wild sweet potato plant (chestnut fragrance of a sweet potato variety) by using a CTAB method. PCR detection is carried out by a conventional method, and the used primers of the IbSPB1 gene are as follows:

primer 5: 5'-TTCTTCACTTCATTGCCATCC-3'

Primer 6: 5'-GGAAGGTGGCTCCTACAAA-3' are provided.

To a 0.2ml Eppendorf centrifuge tube were added 2. mu.l of 10 XPCR buffer, 1. mu.l of 4dNTP (10mol/L), 1. mu.l of each primer (10. mu. mol/L), 2. mu.l of template DNA (50ng/ul), 1ul of Taq DNA polymerase, and H₂O to a total volume of 20. mu.l. The reaction program comprises 36 cycles of denaturation at 94 ℃ for 4mins, renaturation at 57 ℃ for 1.5mins and extension at 72 ℃ for 1min for 30s. The pCB-IbSPB1 vector plasmid is used as a positive control, water and wild type sweet potato plants are used as negative controls, and then electrophoresis detection is carried out.

The results are shown in FIG. 1, from which it can be seen that the quasi-transgenic plant L1-L6 and the positive control amplified a target band of about 1000bp, indicating that the IbSPB1 gene has been integrated into the genome of sweetpotato, and proving that these plants of quasi-transgenic plant L1-L6 are transgenic plants, hereinafter also referred to as transgenic sweetpotato positive plants; the target band was not amplified from water and wild type sweet potato plants.

B、qRT-PCR

Extracting RNA of the positive plant of the transgenic sweet potato, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR by taking the wild sweet potato plant as a control.

Taking sweet potato Actin (Actin) gene as an internal reference, the primer sequence is as follows:

IbActin-F：5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′

IbActin-R：5′-TGGAAAATTAGAAGCACTTCCTGTGAAC-3′

the sequence of the IbSPB1 primer is as follows:

primer 7: 5'-ACTTCTTGCAGCCACGATCA-3'

Primer 8: 5'-ACGCCAATCGCCCATTATCA-3'

The qRT-PCR result is shown in figure 2, and the result shows that the IbSPB1 gene is expressed in different degrees in transgenic plants. Transgenic sweet potato plant strains L1, L2 and L3 are selected for tissue culture and propagation, so that overexpression IbSPB1 transgenic sweet potato plant strains L1, L2 and L3 are obtained, and subsequent tests are carried out.

Example 3 drought resistance identification of transgenic plants

3.1 phenotypic characterization

The overexpressed IbSPB1 transgenic sweet potato lines L1, L2, L3 and wild type sweet potato (sweet potato variety chestnut) of example 2 were cultured on MS medium (normal medium) and MS medium containing polyethylene glycol (concentration 20%), respectively (solid medium obtained by adding polyethylene glycol to the MS medium so that the content of polyethylene glycol is 20%, stress medium), 3 plants per line per medium. The culture conditions are 27 +/-1 ℃, 13h and 3000lux of light are carried out every day, and after the culture is carried out for 4 weeks, the growth state and the rooting condition are observed and measured.

The fresh weight (fresh weight of whole plant including root and aerial parts) and root length (length of main root) of each plant before and after the stress treatment were measured separately.

As shown in FIG. 3, the wild type sweetpotato (WT) was less grown on the MS medium with polyethylene glycol (20%) and was difficult to root; the growth states and rooting conditions of the 3 overexpression transgenic sweet potato strains are better than those of wild type control in different degrees, and the result shows that the overexpression IbSPB1 improves the drought resistance of the transgenic sweet potato plants.

3.2、H₂O₂Determination of content

Hydrogen peroxide (H)₂O₂) Kit (sumac gming biology, catalog No.: H2O2-2-Y) to determine the H of sweet potato plants₂O₂And (4) content.

Plants overexpressing IbSPB1 transgenic sweet potato lines L1, L2 and L3 and wild type sweet potato (sweet potato variety chestnut) were subcultured on MS solid medium (stress medium) and MS solid medium (normal medium) containing polyethylene glycol (20% concentration), respectively, 3 plants per line. The culture conditions are 27 + -1 deg.C, 13H per day, 3000lux light, culturing for 4 weeks, and taking leaf for H₂O₂Content determination was repeated 3 times.

Plants overexpressing IbSPB1 transgenic sweetpotato lines L1, L2, L3 and wild-type control H₂O₂The results of the assay are shown in FIG. 4, which shows the H of 3 transgenic lines on MS medium containing polyethylene glycol (20% concentration)₂O₂The content is obviously lower than that of wild sweet potato plants.

The transgenic sweet potato plant H₂O₂The content determination result shows that the drought resistance of the transgenic sweet potato plant over expressing IbSPB1 is obviously improved compared with the wild sweet potato plant, which indicates that the protein IbSPB1 and the coding gene thereof can be used for regulating and controlling the stress tolerance of plants, particularly improving the drought resistance of the plants.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

<120> drought-resistant related protein IbSPB1, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 333

<212> PRT

<213> sweet potato (Ipomoea batatas)

<400> 1

Met Ala Phe Pro Gln Gln Lys His Phe Gln Gln Pro Pro Pro Gln Pro

1 5 10 15

His Gln Gln Ser Lys Ala Phe Arg Asp Leu Tyr Asn Val Glu Gly Gln

20 25 30

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

35 40 45

Ser Gln His Pro Pro Tyr Ile Pro Pro Phe Gln Val Ala Gly Leu Ala

50 55 60

Pro Gly Thr Val Glu Glu Ser Gly Gln Asp Leu Gln Trp Asn Tyr Gly

65 70 75 80

Leu Glu Pro Lys Lys Lys Arg Pro Lys Glu Gln Asp Phe Leu Glu Asn

85 90 95

Asn Ser Pro Ile Ser Ser Leu Asp Phe Leu Gln Pro Arg Ser Val Ser

100 105 110

Thr Gly Leu Gly Leu Ser Leu Asp Asn Gly Arg Leu Ala Ser Ser Gly

115 120 125

Asp Ser Ser Phe Leu Gly Leu Ser Gly Asp Asp Ile Glu Arg Glu Leu

130 135 140

Gln Arg Gln Asp Ala Glu Ile Asp Arg Tyr Ile Lys Val Gln Gly Asp

145 150 155 160

Arg Leu Arg Gln Ala Ile Leu Glu Lys Val Gln Ala Asn Gln Leu His

165 170 175

Thr Ile Ser Tyr Phe Glu Glu Lys Val Ile Gln Lys Leu Arg Glu Arg

180 185 190

Glu Ala Glu Val Glu Asn Ile Asn Lys Lys Asn Val Asp Leu Glu Met

195 200 205

Gln Met Glu Gln Leu Ala Leu Glu Ala Asn Ala Trp Gln Gln Arg Ala

210 215 220

Lys Tyr Asn Glu Ser Leu Ile Asn Thr Leu Lys Phe Asn Leu Gln Gln

225 230 235 240

Val Tyr Ala Gln Ser Lys Asp Ser Lys Glu Gly Cys Gly Asp Ser Glu

245 250 255

Val Asp Asp Thr Ala Ser Cys Cys Asn Gly Arg Ala Ile Asp Phe His

260 265 270

Leu Leu Cys Arg Asp Gly Asn Glu Val Lys Lys Leu Met Thr Cys Lys

275 280 285

Val Cys Arg Val Asn Thr Val Cys Met Leu Leu Leu Pro Cys Lys His

290 295 300

Leu Cys Leu Cys Lys Glu Cys Glu Ser Lys His Ser Thr Cys Pro Leu

305 310 315 320

Cys Gln Ser Thr Lys Tyr Ile Gly Ile Glu Val Tyr Met

325 330

<210> 2

<211> 1002

<212> DNA

<213> sweet potato (Ipomoea batatas)

<400> 2

atggcttttc ctcagcagaa acacttccag caaccgccgc ctcaaccaca ccaacaatcc 60

aaagctttca gagatttata taacgtggag ggtcagattt cacagcctgt ggcttacttc 120

aacggtccta atcttcccga tcagtctcag catcctcctt atattcctcc ttttcaagtg 180

gctggattag ctcctggtac tgtggaagaa agtgggcagg atttgcagtg gaattatggg 240

ttggagccga agaagaagag gccaaaggag caagattttc tggagaataa ttctccgata 300

tcttctctag acttcttgca gccacgatca gtgtctactg gcctcggatt gtcccttgat 360

aatgggcgat tggcgtcatc tggggactcc tcctttctgg gtctttctgg ggatgacatt 420

gaacgcgagc tgcagagaca ggatgctgag attgataggt acatcaaagt tcagggtgac 480

cgtttgaggc aagctatttt agagaaggtt caagcgaatc aactacatac tatatcctat 540

tttgaagaaa aggtcattca aaagctacgc gagagagagg ctgaggttga aaacatcaac 600

aagaaaaatg tcgaccttga gatgcaaatg gaacaattag ctctggaagc caatgcttgg 660

caacagcgag ccaaatacaa tgaaagcctg attaacacac tcaaattcaa cttacaacag 720

gtttatgctc aaagcaaaga tagtaaggaa ggatgtggtg acagtgaggt ggatgataca 780

gcatcttgtt gtaatgggcg tgccattgat tttcacctgc tttgcaggga tggcaatgaa 840

gtgaagaagt tgatgacttg taaggtttgt agagtcaaca cagtatgcat gctactgtta 900

ccatgtaagc atctctgtct gtgtaaagaa tgtgaaagta agcatagtac ttgcccattg 960

tgtcagtcta caaagtatat aggtatagag gtttatatgt aa 1002