CN114574520A

CN114574520A - Application of TaSR45a gene in cultivating anti-gibberellic disease plants

Info

Publication number: CN114574520A
Application number: CN202210300458.3A
Authority: CN
Inventors: 何漪; 李刚; 张旭; 吴磊; 姜朋
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-06-03

Abstract

The invention discloses an application of TaSR45a gene from wheat in cultivating gibberellic disease resistant plants, belonging to the technical field of plant genetic engineering, wherein the nucleotide sequence of TaSR45a gene is shown as SEQ ID NO.1, and the coding amino acid sequence is shown as SEQ ID NO. 2; experiments show that the TaSR45a gene is overexpressed in Arabidopsis, wheat and other plants by utilizing an agrobacterium-mediated genetic transformation method, analysis and comparison prove that the gibberellic disease resistance of an overexpression TaSR45a transgenic plant is obviously improved, the gene is derived from wheat, and the overexpression does not influence the food safety, so that the gene is widely applied to cultivation of different gibberellic disease resistant transgenic plants.

Description

Application of TaSR45a gene in cultivating anti-gibberellic disease plants

Technical Field

The invention relates to application of a wheat TaSR45a gene in cultivation of a gibberellic disease-resistant transgenic plant, belonging to the technical field of plant genetic engineering.

Background

Wheat is one of the most important food crops in the world, and the demand for wheat is also greatly increased along with the increase of population. However, wheat production is facing significant challenges, and extensive outbreaks of various diseases seriously affect the yield and quality of wheat. According to statistics, the global wheat yield loss caused by fungal diseases is up to 15-20%, wherein the scab (Fusarium head light, FHB) is the cancer of wheat and the most serious harm is caused.

Wheat scab is a fungal disease caused by various fusarium, ears of wheat in a flowering period are easy to be infected by pathogenic bacteria to cause the withering of the ears, less fructification and grain emaciation, so that the yield of the wheat is reduced, the yield is generally reduced by 10-30%, and even the wheat is completely harvested in a pandemic. China is a country with the largest wheat scab damage area in the world, and due to the influences of factors such as climate warming, straw returning and the like, common wheat scab and repeated wheat scab regions are also expanded to Huang-Huai-Mai regions from the middle and lower reaches of Yangtze river. In recent years, the annual average incidence area of the scab in China is 7800 ten thousand mu, and the annual average incidence area accounts for about 20% of the wheat planting area. Gibberellic disease not only causes the reduction of wheat yield, but also produces toxins represented by Deoxynivalenol (DON), and seriously harms human and animal health. Wheat scab brings great threat to wheat safety production in China and seriously affects the healthy development of wheat industry in China. The excavation of the new wheat scab resistance gene can provide theoretical and practical basis for the cultivation of disease-resistant varieties, promote the cultivation and the application in production of the scab-resistant varieties and is beneficial to the guarantee of national food safety and food safety.

The variable shear is a ubiquitous post-transcriptional regulation mechanism in organisms, can process the same precursor mRNA to form different transcripts, and plays an important role in the growth and development of plants. Splicing of mRNA is accomplished primarily by the splicing complex. Serine/arginine-rich (SR) -rich proteins are the most widely studied class of splicing factors in recent years. The SR protein is used as an important component of a spliceosome to regulate constitutive and alternative splicing and influence the whole life process of plant growth and development, for example, ectopic expression of RSZ33 in Arabidopsis can influence the cell division process, and SCL33, SR34b and the like are influenced by external hormones to generate new transcripts, so that the adaptation of plants to new environments is regulated. SR protein is also involved in stress of plants, such as salt stress, high temperature stress and other stress reactions, but reports are less in plant disease resistance reactions. At present, the research on plant SR protein is still in the primary stage, and many functions and mechanisms related to the SR protein are not analyzed, especially the function of wheat in resisting scab is still unknown. The applicant selects a gene TaSR45a (Genbank accession: AK452238.1) related to scab resistance by screening a yeast library of a scab resistant variety Sumai No.3 in the early period, and the protein coded by the gene is a splicing factor specifically existing in plants, but the functional application of the protein is not clear.

Disclosure of Invention

Aiming at the problems, the invention provides application of a wheat TaSR45a gene in cultivating a scab-resistant plant.

The invention is realized by the following technical scheme:

firstly, the application provides the application of TaSR45a gene with the nucleotide sequence shown as SEQ ID NO.1 in cultivating gibberellic disease resistant transgenic plants, and the amino acid sequence coded by the TaSR45a gene is shown as SEQ ID NO. 2.

Further, the specific steps of the application include: the wheat TaSR45a gene with the nucleotide sequence shown in SEQ ID NO.1 is introduced into plant for expression to cultivate gibberellic disease resistant transgenic plant. The expression "introduced into a plant for expression" as described above means that the transgenic plant is obtained by using a technique which is conventional in the art, for example, the literature "Zhang X, Henriques R, Lin S, et al, Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method [ J ]. Nature Protocols,2006,1(2): 641.; hayta S, Smedley M A, Clarke M, et al, an Efficient Agrobacterium Transformation Protocol for Hexaploid and Tetraploid Wheat [ J ] Current Protocols,2021,1(3). "the disclosed transgenic method introduces a vector containing TaSR45a gene or an Agrobacterium strain containing the vector into a plant to cultivate transgenic plants resistant to gibberellic disease.

Further, the above plants include, but are not limited to, Arabidopsis, wheat, barley, to obtain transgenic material containing the gene for production.

Further, the plant is wheat, and the specific application steps are as follows;

1) preparation of a vector containing TaSR45a Gene

Performing PCR amplification by using TaSR45a gene as a template; then, recovering the PCR reaction product after electrophoresis, and connecting the product into a pEASY cloning vector to obtain a plasmid T-TaSR45 a;

the PCR amplification system comprises 2. mu.l of 2X Phanta Master Mix 25. mu.l, 2. mu.l of DNA template (SEQ ID NO.1) at a concentration of 0.5. mu.g/. mu.l, 1.5. mu.l of primer A at a concentration of 10. mu.M, 1.5. mu.l of primer B at a concentration of 10. mu.M, and 20. mu.l of sterile water; PCR reaction procedure: 1min at 95 ℃; at 95 ℃ for 10s, at 58 ℃ for 15s, at 72 ℃ for 30s, for 36 cycles; 5min at 72 ℃;

the DNA template nucleotide sequence of the TaSR45a gene is shown as SEQ ID NO.1, the nucleotide sequence of the primer A is shown as SEQ ID NO.3, and the nucleotide sequence of the primer B is shown as SEQ ID NO. 4;

2) construction of TaSR45a plant overexpression vector

2.1) PCR amplification Using T-TaSR45a as template

PCR amplification system 2 XPhanta Master Mix 25. mu.l, T-TaSR45a plasmid template 0.2. mu.l with concentration of 0.5. mu.g/. mu.l, primer C1.5. mu.l with concentration of 10. mu.M, primer D1.5. mu.l with concentration of 10. mu.M, and sterile water 21.8. mu.l; PCR reaction procedure: 1min at 95 ℃; at 95 ℃ for 10s, at 58 ℃ for 15s, at 72 ℃ for 30s, for 36 cycles; 5min at 72 ℃;

the nucleotide sequence of the primer C is shown as SEQ ID NO.7, and the nucleotide sequence of the primer D is shown as SEQ ID NO. 8;

2.2) digestion of the plant expression vector pLGY-02 with BamH I-HF

The enzyme cutting system is as follows: 25 mul of 2 XCutSmart Buffer, pLGY-021 mul of expression vector, 1 mul of BamH I, 23 mul of sterile water, and 30min of enzyme digestion at 37 ℃;

2.3) carrying out agarose gel electrophoresis separation on the PCR product obtained in the step 2.1) and the enzyme-digested vector obtained in the step 2.2), then carrying out gel recovery, and connecting the target gene fragment into the vector through homologous recombination to obtain a plant over-expression vector pLGY-TaSR45 a;

separating the PCR product and the carrier after enzyme digestion by agarose gel electrophoresis, recovering gel, connecting the target gene fragment into the carrier through homologous recombination (full-scale gold company, cargo number: CU101), and constructing to obtain a plant over-expression carrier pLGY-TaSR45 a;

3) agrobacterium-mediated genetic transformation

The plant over-expression vector pLGY-TaSR45a is transferred into agrobacterium EHA105, and the wheat immature embryo is genetically transformed to obtain the transgenic wheat plant with gibberellic disease resistance.

Furthermore, the plant is arabidopsis thaliana, and the specific application steps are as follows;

1) preparation of a vector containing TaSR45a Gene

the PCR amplification system comprises 2. mu.l of 2X Phanta Master Mix 25. mu.l, 2. mu.l of DNA template (SEQ ID NO.1) at a concentration of 0.5. mu.g/. mu.l, 1.5. mu.l of primer A at a concentration of 10. mu.M, 1.5. mu.l of primer B at a concentration of 10. mu.M, and 20. mu.l of sterile water; PCR reaction procedure: 1min at 95 ℃; at 95 ℃ for 10s, at 58 ℃ for 15s, at 72 ℃ for 50s, for 36 cycles; 5min at 72 ℃;

the nucleotide sequence of the DNA template is shown as SEQ ID NO.1, the nucleotide sequence of the primer A is shown as SEQ ID NO.3, and the nucleotide sequence of the primer B is shown as SEQ ID NO. 4;

2) construction of plant overexpression vector pCAMBI1301-TaSR45a

2.1) amplification of the target Gene TaSR45a

The PCR amplification system comprises 2 XPhanta Master Mix 25. mu.l, 0.2. mu.l of T-TaSR45a plasmid template with concentration of 0.5. mu.g/. mu.l, 1.5. mu.l of primer E with concentration of 10. mu.M, 1.5. mu.l of primer F with concentration of 10. mu.M, and 21.8. mu.l of sterile water; PCR reaction procedure: 1min at 95 ℃; at 95 ℃ for 10s, at 58 ℃ for 15s, at 72 ℃ for 30s, for 36 cycles; 5min at 72 ℃;

the nucleotide sequence of the primer E is shown as SEQ ID NO.5, and the nucleotide sequence of the primer F is shown as SEQ ID NO. 6;

2.2) NcoI-HF restriction of the plant expression vector pCAMBI1301

The enzyme cutting system is as follows: 2 x 25 mul of CutSmart Buffer, 13011 mul of expression vector pCAMBI, 1 mul of NcoI-HF, 23 mul of sterile water, and 30min of enzyme digestion at 37 ℃;

2.3) carrying out agarose gel electrophoresis separation on the PCR product obtained in the step 2.1) and the carrier subjected to enzyme digestion in the step 2.2), then carrying out gel recovery, and connecting the target gene fragment into a carrier pCAMBI1301 through homologous recombination to obtain a plant over-expression carrier pCAMBI1301-TaSR45 a;

3) agrobacterium-mediated genetic transformation of Arabidopsis

Transferring a plant over-expression vector pCAMBI1301-TaSR45a into agrobacterium GV3101, dip-dyeing arabidopsis flower buds by using an immersion flower method to the agrobacterium GV3101 containing the vector pCAMBI1301-TaSR45a, collecting T1 generation seeds after the grown siliques are mature, screening on a 1/2MS culture medium added with hygromycin (25mg/l), respectively harvesting T2 generation seeds, and then carrying out next round of screening, wherein the harvested seeds of a single plant are T3 generation, namely the gibberellic disease-resistant transgenic arabidopsis.

Vectors used in the present application include, but are not limited to, the vector pCAMBI1301 and the vector pLGY-02, both conventional commercially available vectors, as described in the references "Zhang Y, Zhou J, Wei F, et al.Nucleoredoxin Gene TaNRX1 Positively regulations Drou Tolerance in Transgenic Wheat (Triticum aestivum L.). Front Plant Sci.2021; 12:756338 Published 2021Nov 11 doi:10.3389/fpls 2021.756338 ". The Agrobacterium used includes, but is not limited to, conventional commercially available Agrobacterium strains such as Agrobacterium GV3101, Agrobacterium EHA105 and the like.

The invention proves the application of the plant specific splicing factor TaSR45a in resisting the gibberellic disease for the first time, and the over-expression TaSR45a gene can obviously improve the resistance of the plant to the gibberellic disease. In addition, the cloned gene is derived from wheat, so that the food safety is not influenced by the over-expression, and the gene can be widely applied to the cultivation of different gibberellic disease-resistant transgenic plants.

Drawings

FIG. 1 is a diagram of TaSR45a sequence agarose gel electrophoresis.

M is molecular weight standard DL 2000; lane 1 is TaSR45a fragment.

FIG. 2 is a schematic diagram showing the result of TaSR45a identification in transgenic Arabidopsis thaliana.

M is molecular weight standard DL 2000; lanes 1-4 are the results of the identification of TaSR45a overexpression Arabidopsis plants 1-4, and lane 5WT is wild type Arabidopsis.

FIG. 3: schematic diagram of the results of analysis of transgenic Arabidopsis gibberellic disease resistance phenotype;

a is wild plant, B is transgenic plant.

FIG. 4: schematic diagram of results of TaSR45a expression analysis in transgenic wheat.

And (3) qRT-PCR quantitative detection of TaSR45a gene in

transgenic lines

5, 6, 8, 9 and 10.

FIG. 5: the results of the phenotype analysis of resistance of transgenic wheat to gibberellic disease are shown schematically.

A is the phenotype of the transgenic plant line infected by the gibberellic disease for 15 days, B is the statistics of the number of diseased spikelets, and the resistance of the transgenic plant is obviously improved compared with that of the wild type gibberellic disease.

Detailed Description

Unless otherwise specified, the examples are all conventional methods and experimental reagents used in the art. The wheat varieties Fielder and Sumai No.3 are common varieties, as disclosed in the documents "He Y, Wu L, Liu X, et al. TaUGT6, a novel UDP-glycosylation transfer enzyme genes industries the resistance to FHB and DON accumulation in wheat at J. Frontiers in Plant Science,2020:1549. Both Fielder and arabidopsis thaliana were from the collection of wheat genetic breeding team at the institute of food crops, academy of agricultural sciences, jiangsu province.

The examples relate to the formulation of the medium:

1/2MS culture Medium: 2.2g MS powder (Sigma-Aldrich, M5519), 30g sucrose, 8g agar powder, adding water to make up to 1L;

WLS-liq Medium: 10ml of 10 Xmacroelement, 1ml of 100 XFeEDTA, 1ml of 100 Xmicroelement, 1ml of 100 Xorganic component, 10g of glucose and 0.5g of MES, and adding water to fix the volume to 1L;

WLS-AS Medium: 10ml 10 Xmacroelements, 1ml 100 XFeEDTA, 1ml 100 Xmicroelements, 1ml 100 Xorganic components, 10g glucose, 0.5g MES、0.85mg AgNO₃、1.25mg CuSO₄·5H₂O, 100 mu M acetosyringone and 8g agar powder, and adding water to a constant volume of 1L;

WLS-Res medium: 100ml 10 Xmacroelements, 10ml 100 XFeEDTA, 10ml 100 Xmicroelements, 10ml 100 Xorganic components, 5ml 100mg/l 2,4-D, 0.5g glutamine, 0.1g hydrolysin, 0.75g MgCl₂·6H₂O, 40g of maltose, 1.95g of MES and 8g of agar powder, and adding water to a constant volume of 1L;

WLS-P5 Medium: 100ml 10 Xmacroelements, 10ml 100 XFeEDTA, 10ml 100 Xmicroelements, 10ml 100 Xorganic components, 5ml 100mg/l 2,4-D, 0.5g glutamine, 0.1g hydrolysin, 0.75g MgCl₂·6H₂O, 40g of maltose, 1.95g of MES, 5mg of glufosinate-butyl and 8g of agar powder, and adding water to a constant volume of 1L;

WLS-P10 Medium: 100ml 10 Xmacroelements, 10ml 100 XFeEDTA, 10ml 100 Xmicroelements, 10ml 100 Xorganic components, 5ml 100mg/l 2,4-D, 0.5g glutamine, 0.1g hydrolysin, 0.75g MgCl₂·6H₂O, 40g of maltose, 1.95g of MES, 10mg of glufosinate-butyl and 8g of agar powder, and adding water to a constant volume of 1L;

LSZ-P5 Medium: 100ml 10 Xmacroelements, 10ml 100 XFeEDTA, 10ml 100 Xmicroelements, 10ml 100 Xorganic components, 50ml 100mg/l zeatin, 0.5g MES, 20g sucrose, 2.5mg CuSO₄·5H₂O, 5mg of glufosinate-butyl and 8g of agar powder, and adding water to a constant volume of 1L;

wherein 10 × macroelements are 19.0g KNO3, 16.5g NH4NO3, 4.4g CaCl₂·2H₂O、3.7g MgSO₄·7H₂O and 1.7g KH₂PO4 is added into 1L water to obtain the product;

100 x trace elements of 2.23g MnSO₄·5H₂O、1.06g ZnSO₄·7H₂O、620mg H₃BO₃、83mg KI、25.0mg Na₂MoO₄·2H₂O、2.5mg CuSO₄·5H₂O and 2.5mg CoCl₂·6H₂O is obtained by diluting to a constant volume in 1L of water;

100 XFeEDTA 2.78g FeSO₄·7H₂O and 3.73g of EDTA are added into 1L of water in a constant volume mode; 100 Xorganic components are 10g inositol, 0.2g glycine, 100mg thiamine hydrochloride, 50mg pyridoxine hydrochloride and 50mg nicotinic acid, and the mixture is added to 1L water to obtain the final product.

Example 1 cloning of wheat TaSR45a

Ear RNA of wheat variety Sumai No.3 at heading stage was extracted using SV Total RNA Isolation System kit (Promega Co., Ltd., cat # Z3100), and cDNA was obtained using reverse transcription kit (TAKARA Co., cat # 6210A).

The cDNA sequence of TaSR45a gene obtained through the public website http:// plants. ensemble. org/is shown in SEQ ID NO. 1. The 20 nucleotides after the initiation codon of TaSR45a gene were selected as forward primer 5'-ATGTCGTATTCTAGGTACAG-3' (SEQ ID NO.3), the 20 nucleotides before the termination codon was selected as reverse primer 5'-GGTGCTTGCTGAGGTTGAGC-3' (SEQ ID NO.4), primers for amplifying TaSR45a gene were synthesized, and the above cDNA was used as template for PCR amplification of full-length cDNA.

PCR reaction System (Novozan, cat # P511):

master Mix 25. mu.l, cDNA template 2. mu.l (0.5. mu.g/. mu.l), primers SEQ ID NO.3 and SEQ ID NO.4 each 1.5. mu.l (10. mu.M), sterile water 20. mu.l. The PCR reaction program is set to 95 ℃ for 1 min; at 95 ℃ for 10s, at 58 ℃ for 15s, at 72 ℃ for 30s, for 36 cycles; 5min at 72 ℃.

The PCR product was electrophoresed in a 1% agarose gel, and the result of electrophoresis is shown in FIG. 1, lane 1 is TaSR45a, which is a target fragment. The target fragment was recovered by gel (Nonanza, cat # DC301), ligated into pEASY cloning vector (Takara, cat # CB111), and subjected to sequencing verification by Biotechnology engineering (Shanghai) GmbH, and the plasmid with the correct sequencing was named T-TaSR45 a.

Example 2 application of TaSR45a in improving gibberellic disease resistance in Arabidopsis thaliana

Construction of TaSR45a Arabidopsis thaliana overexpression vector

A homologous recombination primer design website (https:// www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools) is utilized to select a plant expression vector pCAMBI1301 (purchased from CAMBIA corporation) with 15bp nucleotides at an NcoI site of an enzyme cutting site, 21 nucleotides after an initiation codon and 21 nucleotides before a termination codon of a TaSR45a gene, reverse complementary sequences are respectively utilized to design primers, and a target fragment is subjected to PCR amplification by taking the T-TaSR45a obtained in example 1 as a template, wherein the forward primer is 5'-GGACTCTTGACCATGGTATCGTATTCTAGGTACAGG-3' (SEQ ID NO.5) and the reverse primer is 5'-CTCAGATCTACCATGGGGTGCTTGCTGAGGTTGAGC-3' (SEQ ID NO. 6).

PCR reaction System (Novozan, cat # P511):

master Mix 25. mu.l, T-TaSR45a plasmid 0.2. mu.l (0.5. mu.g/. mu.l), primers SEQ ID NO.5 and SEQ ID NO.6 each 1.5. mu.l (10. mu.M), sterile water 21.8. mu.l. The PCR reaction program is set to 95 ℃ for 1 min; 36 cycles of 95 ℃ for 10s, 58 ℃ for 15s, and 72 ℃ for 30 s; 5min at 72 ℃.

The plant expression vector pCAMBI1301 was digested with NcoI-HF (NEB, cat # R3193). The enzyme cutting system is as follows: 2 XCutSmart Buffer 25. mu.l, expression vector pCAMBI 13011. mu.g, NcoI-HF 1. mu.l, sterile water 23. mu.l, 37 ℃ for 30 min. And separating the PCR product and the enzyme-cut carrier by agarose gel electrophoresis and then recovering the gel. The target gene fragment is connected into a vector pCAMBI1301 through homologous recombination (all-open gold company, cargo number: CU101) to construct and obtain a plant over-expression vector pCAMBI1301-TaSR45 a.

2. Agrobacterium-mediated genetic transformation of Arabidopsis

The plant overexpression vector pCAMBI1301-TaSR45a was transferred into Agrobacterium GV3101 (Shanghai Diego, cat # AE1001) and verified by PCR. Agrobacterium GV3101 containing a plant expression vector was used to impregnate Arabidopsis flower buds using the general Arabidopsis transformation method "floral dip" (Zhang X, Henriques R, Lin S, et al, Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method [ J ]. Nature Protocols,2006,1(2): 641.). After the siliques grown from the siliques are mature, collecting T1 generation seeds, screening on a 1/2MS culture medium added with hygromycin (the final concentration is 25mg/l), respectively harvesting T2 generation seeds, then carrying out next round of screening on a 1/2MS culture medium added with hygromycin (the final concentration is 25mg/l), transplanting green seedlings on the culture dish, and obtaining T3 generation seeds from a single plant, namely, obtaining a homozygous transgenic line.

3. Identification of transgenic Arabidopsis thaliana

For the transgenic plant T3, DNA extracted from leaves is amplified by PCR and detected by 1% agarose gel electrophoresis, the detection result is shown in figure 2, the amplification products are respectively spotted on lanes 1-4 shown in figure 2, and the lanes 1-4 are respectively named as 1-4 transgenic strains. FIG. 2 shows the successful acquisition of 4 Arabidopsis plants independently transformed with TaSR45a gene.

PCR reaction System (Novozan, cat # P511):

master Mix 25. mu.l, Arabidopsis DNA template 2. mu.l (0.5. mu.g/. mu.l), primers SEQ ID NO.5 and SEQ ID NO.6 each 1.5. mu.l (10. mu.M), sterile water 20. mu.l. The PCR reaction program is set to 95 ℃ for 1 min; at 95 ℃ for 10s, at 58 ℃ for 15s, at 72 ℃ for 30s, for 36 cycles; 5min at 72 ℃.

4. Transgenic Arabidopsis gibberellic disease resistance identification

Wild type and the above T3 transgenic Arabidopsis lines were sown and leaf infestation (Li G, Zhou J, Jia H,2019.et al. mutation of a histidine-rich caldium-binding-protein gene in wheat head tolerance to fusarium head light) [ J]Nature Genetics,2019,51: 1106-: selecting plants with consistent growth state, respectively taking leaves with consistent size, pricking wounds with gun head in the middle of the leaves with consistent size, and dripping 10 μ l of the solution containing 1 × 10⁵The fusarium graminearum spore liquid is subjected to wet culture in a culture dish for 5 days, and then the gibberellic disease resistance expression of wild plants and transgenic plants is observed, the result is shown in figure 3, lanes 1-4 are identification results of 4 randomly selected TaSR45a overexpression arabidopsis plants (which are sequentially numbered as 1-4 by the applicant), and in lane 5, WT is wild arabidopsis. FIG. 3 shows that the withered yellow range of transgenic Arabidopsis plant leaves (shown as B in FIG. 3) is obviously smaller than that of wild plants (shown as A in FIG. 3), which indicates that after TaSR45a gene is transferred, Arabidopsis thaliana is subjected to transformationThe resistance phenotype is obviously improved after the infection of the gibberellin.

Example 3 application of TaSR45a in improving gibberellic disease resistance in wheat

Construction of plant overexpression vector of TaSR45a

A website (https:// www.takarabio.com/learning-centers/cloning/primer-design-an d-other-tools) is designed by utilizing homologous recombination primers, 16bp nucleotides at the BamH I site of a restriction enzyme site of a plant expression vector pCAMBI1301, 20 nucleotides after an initiation codon of a TaSR45a gene and reverse complementary sequences 20 nucleotides before a termination codon are selected to respectively design primers, the T-TaSR45a obtained in the embodiment is used as a template to carry out PCR amplification on a target fragment, the forward primer is 5'-GGCCTACTAGGGATCCATGTCGTATTCTAGGTACAG-3' (SEQ ID NO.7), and the reverse primer is 5'-CTCTGAGCTCGGATCCGGTGCTTGCTGAGGTTGAGC-3' (SEQ ID NO. 8).

The PCR amplification system comprises 2 XPhanta Master Mix 25. mu.l, T-TaSR45a 0.2.2. mu.l with concentration of 0.5. mu.g/. mu.l, primer SEQ ID NO. 71.5. mu.l with concentration of 10. mu.M, primer SEQ ID NO. 81.5. mu.l with concentration of 10. mu.M, and sterile water 21.8. mu.l; PCR reaction procedure: 1min at 95 ℃; 36 cycles of 95 ℃ for 10s, 58 ℃ for 15s, and 72 ℃ for 30 s; 5min at 72 ℃.

The plant expression vector pLGY-02 (present at the institute of crops, college of agricultural sciences, Shandong province) was digested with BamH I-HF (NEB, cat # R3136) in the following manner: 2 XCutSmart Buffer 25. mu.l, expression vector pLGY-021. mu.g, BamH I1. mu.l, sterile water 23. mu.l, enzyme digestion 30min at 37 ℃.

And separating the PCR product and the enzyme-cut carrier by agarose gel electrophoresis and then recovering the gel. The target gene fragment is connected into a vector through homologous recombination (whole formula gold company, cargo number: CU101) to construct and obtain a plant over-expression vector pLGY-TaSR45 a.

2. Agrobacterium-mediated genetic transformation

The plant overexpression vector pLGY-TaSR45a was transformed into Agrobacterium EHA105 (Shanghai Weidi Biometrics, cat # AC1012) and verified by PCR. A general immature embryo-based genetic transformation method was used: stripping fertilized wheat variety Fielder young embryos for 14 days in a sterile environment, putting the young embryos into a WLS-liq culture medium, centrifuging for 10min, adding agrobacterium containing a target gene for 5min, placing young embryo scutellum upwards in a WLS-AS culture medium, carrying out dark culture, cutting off embryonic axis after 3 days, transferring to a WLS-Res culture medium, carrying out dark culture for 5 days, transferring to a WLS-P5 culture medium, carrying out dark culture for 3 weeks, transferring to a WLS-P10 culture medium, carrying out LSZ-P5 culture, carrying out continuous illumination culture and differentiation to obtain transgenic wheat plants, randomly selecting the wheat plants, and sequentially numbering the wheat plants by the applicant AS 5-9.

The above transformation methods are conventional in the art, and are disclosed in the literature "Ishida Y, Tsunashima M, Hiei Y, et al.Wheat (Triticum aestivum L.) transformation using the methods of distribution [ M ]. Agrobacterium protocols.Springer, New York, NY,2015: 189-.

3. Identification of overexpressed transgenic wheat plant molecules

Quantitative expression analysis primers for TaSR45a were designed by primer5 software, with the forward primer being 5'-CCCATATGAAAGAAGGCAAT-3' (SEQ ID NO.9) and the reverse primer being 5'-CGGGGCGAGCAAGACATGCGC-3' (SEQ ID NO. 10). And (3) respectively extracting the RNA of the transgenic wheat plant and the RNA of the wild plant obtained in the step (2), carrying out qRT-PCR amplification by using the designed primer after reverse transcription, and analyzing the gene expression difference of the over-expressed plant and the wild plant.

The qRT-PCR reaction system is as follows: 2 XTB

Premix Ex Taq^TMII (takara, cat # RR820) 5. mu.l, 0.3. mu.l (10. mu.M) of each of the primer SEQ ID NO.9 and the primer SEQ ID NO.10, 1. mu.l (50ng) of the template, and 3.4. mu.l of sterile water. The qRT-PCR reaction program was: pre-denaturation at 95 ℃ for 30s for 1 cycle; 5s at 95 ℃,20 s at 60 ℃, 10s at 72 ℃ and 45 cycles; the melting curve analysis was 95 ℃ for 10s,65 ℃ for 10s, and 95 ℃ for 5 s.

The detection results are shown in fig. 4, and 5-9 in fig. 4 are qRT-PCR quantitative detection results of TaSR45a gene of transgenic wheat plants (over-expressed plants) randomly selected and self-named by the applicant, and it can be seen that the expression level of TaSR45a in the over-expressed plants is obviously higher than that of wild plants.

4. Transgenic wheat scab resistance identification

And (3) performing gibberellic disease resistance identification on the transgenic wheat plant (overexpression plant) obtained in the step (2), wherein the identification method comprises the following steps: selecting one upper spikelet of the initial-flowering ear to inoculate 10 μ l of 1 × 10⁵The fusarium graminearum spore liquid is bagged and moisturized for 3 days, the number of the small scab is counted every 2 days after inoculation for 5 days, the counting result is shown in fig. 5, A in fig. 5 is a phenotype photo of a transgenic plant line infected by gibberellic disease for 15 days, and B in fig. 5 is the counting result of the number of the small scab, and the gibberellic disease resistance level of TaSR45a overexpression wheat plants is obviously improved.

Sequence listing

<110> agricultural science and academy of Jiangsu province

Application of <120> TaSR45a gene in cultivation of anti-gibberellic disease plants

<130> ywx0-01-20220324-01-NO0020

<141> 2022-03-24

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 975

<212> DNA

<213> wheat (Triticum aestivum)

<400> 1

atgtcgtatt ctaggtacag gagtcgttca aggagcgtgg actcgagtga tgttgagaac 60

cctgggaaca atctcttcgt gactggttta tcatctcgtc taactgatcg agatctggag 120

aagcatttct ctacagaggg agaggtgatt gatgcaagta tagtacttga tccatggaca 180

agggaatcac ggggatttgg gtttgttacc atggctactc ttaaggaggc agaacgctgc 240

atcaaatatc tcgaccgttc agtgctggaa ggtcgtgtca ttactgttga gaaggcaaag 300

cgaagacgag gtcgaacccc aacaccagga aggtatctgg gcgccaaatc atcgcgtgga 360

aggaggtatt cccgaagcag gtcacctgtt cggagagacc gttacagctc acgctactcg 420

tctgaccgag aacgctctta ttctccttat cgcagaggac gatcatacgc tcgtcgtcac 480

atgcgtagat catactcccc atatgaaaga aggcaatcct actctcccta tggcagaagg 540

agatcatact ctccttccaa caggtcggag tctccctgcg acaggcgaag gtcctactca 600

ccctacgaca ggcgcatgtc ttgctcgccc cgccatggtc atcgccaccg ttcaagatct 660

ccataccgct acagaaggcg gaggtcgcgc tcccatgacc gttctgtttc agcatactac 720

agcaggcgct attctccaag gagcagaaga cggagctact ctcgcagcat atcgccacgc 780

aggagctact cccgcagctg ctctccggcg tcggaagaat caaggagctg ttctccgcgg 840

aaaggacgcg ccgaaagcaa ggcatcacgc agcaggtcac ctgggaagag gcgttccaga 900

gaaagctatg ctcacagccg cagttcatgc tcgaggtccg tctccaggga acgctcaacc 960

tcagcaagca cctga 975

<210> 2

<211> 324

<212> PRT

<213> wheat (Triticum aestivum)

<400> 2

Met Ser Tyr Ser Arg Tyr Arg Ser Arg Ser Arg Ser Val Asp Ser Ser

1 5 10 15

Asp Val Glu Asn Pro Gly Asn Asn Leu Phe Val Thr Gly Leu Ser Ser

20 25 30

Arg Leu Thr Asp Arg Asp Leu Glu Lys His Phe Ser Thr Glu Gly Glu

35 40 45

Val Ile Asp Ala Ser Ile Val Leu Asp Pro Trp Thr Arg Glu Ser Arg

50 55 60

Gly Phe Gly Phe Val Thr Met Ala Thr Leu Lys Glu Ala Glu Arg Cys

65 70 75 80

Ile Lys Tyr Leu Asp Arg Ser Val Leu Glu Gly Arg Val Ile Thr Val

85 90 95

Glu Lys Ala Lys Arg Arg Arg Gly Arg Thr Pro Thr Pro Gly Arg Tyr

100 105 110

Leu Gly Ala Lys Ser Ser Arg Gly Arg Arg Tyr Ser Arg Ser Arg Ser

115 120 125

Pro Val Arg Arg Asp Arg Tyr Ser Ser Arg Tyr Ser Ser Asp Arg Glu

130 135 140

Arg Ser Tyr Ser Pro Tyr Arg Arg Gly Arg Ser Tyr Ala Arg Arg His

145 150 155 160

Met Arg Arg Ser Tyr Ser Pro Tyr Glu Arg Arg Gln Ser Tyr Ser Pro

165 170 175

Tyr Gly Arg Arg Arg Ser Tyr Ser Pro Ser Asn Arg Ser Glu Ser Pro

180 185 190

Cys Asp Arg Arg Arg Ser Tyr Ser Pro Tyr Asp Arg Arg Met Ser Cys

195 200 205

Ser Pro Arg His Gly His Arg His Arg Ser Arg Ser Pro Tyr Arg Tyr

210 215 220

Arg Arg Arg Arg Ser Arg Ser His Asp Arg Ser Val Ser Ala Tyr Tyr

225 230 235 240

Ser Arg Arg Tyr Ser Pro Arg Ser Arg Arg Arg Ser Tyr Ser Arg Ser

245 250 255

Ile Ser Pro Arg Arg Ser Tyr Ser Arg Ser Cys Ser Pro Ala Ser Glu

260 265 270

Glu Ser Arg Ser Cys Ser Pro Arg Lys Gly Arg Ala Glu Ser Lys Ala

275 280 285

Ser Arg Ser Arg Ser Pro Gly Lys Arg Arg Ser Arg Glu Ser Tyr Ala

290 295 300

His Ser Arg Ser Ser Cys Ser Arg Ser Val Ser Arg Glu Arg Ser Thr

305 310 315 320

Ser Ala Ser Thr

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtcgtatt ctaggtacag 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggtgcttgct gaggttgagc 20

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggactcttga ccatggtatc gtattctagg tacagg 36

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctcagatcta ccatggggtg cttgctgagg ttgagc 36

<210> 7

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggcctactag ggatccatgt cgtattctag gtacag 36

<210> 8

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctctgagctc ggatccggtg cttgctgagg ttgagc 36

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cccatatgaa agaaggcaat 20

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cggggcgagc aagacatgcg c 21

Claims

1. An application of TaSR45a gene in cultivating anti-gibberellic disease plants, wherein the nucleotide sequence of TaSR45a gene is shown in SEQ ID NO. 1.

2. The use of claim 1, wherein the TaSR45a gene is introduced into cells, tissues or individual plants of host plants to develop transgenic plants resistant to gibberellic disease.

3. The use according to claim 1, wherein the plant comprises at least one of Arabidopsis, wheat, barley.

4. The use of claim 2, wherein said introduction into a host plant is carried out by introducing a vector containing TaSR45a gene or an Agrobacterium strain containing said vector into cells, tissues or plant individuals of a host plant to produce transgenic plants resistant to gibberellic disease.