阅读说明：本技术 小麦基因TaAn在提高植物DON耐受性和FHB抗性中的应用 (Application of wheat gene TaAn in improving DON tolerance and FHB resistance of plants ) 是由 廖玉才 刘刚 杨鹏 左东云 李和平 张静柏 黄涛 何伟杰 粘俊娜 易述远 于 2020-11-10 设计创作，主要内容包括：本发明属于植物基因工程技术领域,具体涉及利用小麦基因TaAn提高植物的DON耐受性和赤霉病抗性。通过筛选小麦悬浮细胞抑制差减杂交cDNA文库及RACE克隆基因全长的方法,从小麦品种郑麦9023中分离得到一个受DON诱导的基因TaAn。利用农杆菌介导的遗传转化方法,使该基因在拟南芥中超量表达,提高了转基因拟南芥对DON耐受能力和对赤霉病的抗性；利用农杆菌介导的遗传转化方法,使该基因在小麦中组成型表达,提高了转基因小麦对DON耐受能力,研究证实了该基因作为植物抗赤霉病功能的新用途。(The invention belongs to the technical field of plant genetic engineering, and particularly relates to a wheat gene TaAn Improve the DON tolerance and the gibberellic disease resistance of the plants. Separating a gene induced by DON from Zheng wheat 9023 of a small wheat variety by screening a wheat suspension cell inhibition differential hybrid cDNA library and a RACE clone gene full length method TaAn . The gene is overexpressed in arabidopsis thaliana by utilizing an agrobacterium-mediated genetic transformation method, so that the DON tolerance capacity and the gibberellic disease resistance of the transgenic arabidopsis thaliana are improved; the agrobacterium-mediated genetic transformation method is utilized to make the gene constitutively expressed in wheat, the DON tolerance capability of transgenic wheat is improved, and researches prove that the gene is used as a plantNew use of the compound for resisting head blight is provided.)

1. Wheat geneTaAnThe application of the polypeptide in improving the DON tolerance and the gibberellic disease resistance of plants.

2. Use according to claim 1, wherein said wheat geneTaAnThe nucleotide sequence of (A) is shown as SEQ ID NO.2, and the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO. 3.

3. Use according to claim 1 or 2, wherein the plant comprises arabidopsis thaliana and wheat.

4. An expression vector for improving the DON tolerance and the gibberellic disease resistance of plants, which is characterized in that the expression vector contains a nucleotide sequence shown in SEQ ID NO. 2.

Technical Field

The invention belongs to the technical field of wheat transgenosis, and particularly relates to a method for improving DON tolerance and FHB resistance of a plant by using a wheat gene TaAn. The gene is wheat TaAn gene, which is proved to be a new wheat gene with antibacterial peptide function and plays an important role in the defense reaction of plants. The TaAn gene is induced by DON and participates in the regulation and control of plant cell defense response. The tolerance of arabidopsis on DON and the resistance of FHB can be improved by over-expressing the gene; constitutive expression of the gene can improve the resistance of wheat to DON. The gene TaAn is expected to improve the scab resistance of plants, and provides a new and safe resistant germplasm resource for the breeding of transgenic wheat scab resistance.

Background

Wheat is one of the most important food crops in the world, wheat scab (FHB) caused by Fusarium is an important fungal disease worldwide, and pathogenic fungi mainly include Fusarium graminearum (Fusarium graminearum), Fusarium avenaceum (Fusarium avenaceum), Fusarium flavum (Fusarium culmorum) and Fusarium pyricularis (Fusarium poae), and the like, wherein Fusarium graminearum is the most important pathogenic bacterium worldwide. The large-scale gibberellic disease epidemic occurs every 4-5 years in the main wheat producing area. Since 2010, the prevalence frequency of the scab is on the trend of increasing year by year in China, and the scab is widely prevalent basically every 1-2 years, wherein 2012 is the most serious. From 2000 to 2018, the cultivated land area of wheat which is influenced by gibberellic disease every year in China is about 450 million hectares, which accounts for 20 percent of the total cultivated land area of the wheat, and the yield loss of the wheat is about 341 million tons every year. The gibberellic disease mainly infects the wheat head in the flowering phase and infects the surrounding spikelets through the vascular bundle of the spike stalk, so that the wheat head is withered, the kernel is abnormal in development and accompanied with red mildew stains. Fusarium graminearum produces various mycotoxins in the process of infecting wheat, such as Zearalenone (ZEA), Deoxynivalenol (DON), Nivalenol (Nivalenol, NIV), wherein DON is the main factor and seriously threatens the yield and quality of wheat. The chronic toxic effects of DON on animals are manifested by vomiting, loss of appetite, weight loss, abortion and immunosuppression.

The TaAn gene encodes 73 amino acids, has no functional annotation in NCBI database, and belongs to unknown functional protein of wheat. In previous studies, a 523bp EST fragment was selected from a suppression differential hybridization (SSH) library of wheat suspension cells, strongly induced by DON, and then cloned to the full length of the gene by 3 'RACE and 5' RACE, and named Taan. As no report is available on whether the TaAn gene in wheat can improve the FHB resistance of a plant, the TaAn gene is separated from wheat, after the TaAn gene is overexpressed in arabidopsis thaliana, the DON tolerance and the FHB resistance of arabidopsis thaliana can be improved, and the TaAn gene is constitutively expressed in wheat, so that the FHB resistance of the wheat can be improved, and the TaAn gene has very important significance for breeding new wheat disease-resistant varieties.

Disclosure of Invention

The invention aims to provide application of a TaAn gene of wheat in improvement of gibberellic disease resistance of plants. A gene TaAn induced by DON is separated from wheat by screening a wheat suspension cell suppression subtraction hybrid cDNA library and a RACE clone gene full length method, the DON tolerance capability and the gibberellic disease resistance of transgenic arabidopsis are further improved by overexpression of the gene in arabidopsis, the DON tolerance capability of transgenic wheat is improved by constitutive expression of the gene in wheat, and the novel application of the gene as a plant anti-gibberellic disease function is proved through research.

In order to achieve the purpose, the invention adopts the following technical scheme:

the nucleotide sequence of the wheat gene TaAn is shown as SEQ ID NO.2, and the nucleotide sequence of the encoded protein is shown as SEQ ID NO. 3.

The application of the wheat gene TaAn in improving the DON tolerance and the gibberellic disease resistance of plants: in the specific embodiment of the invention, PCR is utilized to amplify the coding region of wheat gene TaAn, EcoR I enzyme cutting sites and BamH I enzyme cutting sites are respectively added at two ends and are connected to an intermediate vector PTRAkc-VDM1-ERH to obtain a plant over-expression vector PTRAkc-35SS-TaAn, an agrobacterium-mediated genetic transformation method is utilized to transform arabidopsis thaliana to obtain transgenic arabidopsis thaliana with over-expression of the TaAn gene, and a spore solution of DON toxin and fusarium graminearum 5035 is inoculated to prove that the root length and fresh weight of the transgenic arabidopsis thaliana are obviously higher than those of a wild type T, T₂Generation and T₃The morbidity index of the generation transgenic line is obviously reduced, and the gibberellic disease resistance of transgenic arabidopsis is verified. The coding region of wheat gene Taan is amplified by PCR, Sma I enzyme cutting sites and Spe I enzyme cutting sites are respectively added at two ends of the coding region and are connected into an intermediate vector pCAMBIA3300 to obtain a plant constitutive expression vector pCAMBIA3300-Ubi-Taan, wheat is transformed by an agrobacterium-mediated genetic transformation method to obtain transgenic wheat with Taan gene constitutive expression, DON is inoculated in a seedling stage, and the result shows that the inhibition effect of the DON is obviously weakened compared with that of a wild type and a contrast in the root length of the transgenic wheat, and the DON toxin resistance of the transgenic wheat is verified. Wherein the plant over-expression vector PTRAkc-35SS-TaAn and the plant constitutive expression vector pCAMBIA3300-Ubi-TaAn comprise nucleotide sequences shown in SEQ ID NO. 2.

The invention has the advantages that:

the TaAn gene cloned by the invention can improve the tolerance of arabidopsis thaliana and wheat plants to mycotoxin DON, and obviously improve the resistance of transgenic arabidopsis thaliana to fusarium graminearum, a pathogen of scab.

The plant over-expression vector pTRAkc-35SS-TaAn used in the invention can efficiently express the TaAn gene in Arabidopsis, and the plant constitutive expression vector pCAMBIA3300-Ubi-TaAn can constitutively express the TaAn gene in wheat, so that the improvement of the gibberellic disease resistance and the DON toxin tolerance of wheat becomes possible.

Drawings

FIG. 1: the invention separates and clones TaAn gene and general technical route map of function identification.

FIG. 2: electrophorogram of full-length sequence clone of TaAn gene. Panel A is 3 'RACE product, panel B is 5' RACE product, and panel C is full-length cDNA of TaAn gene.

FIG. 3: the TaAn gene is inoculated to two wheat varieties of Zheng wheat 9023(Z9) and Sumai No.3 (S3) by DON, fusarium graminearum 5035 and Tri5^－Expression pattern after spore fluid. FIG. 3A is a graph showing the expression pattern of the TaAn gene after inoculation with DON toxin in wheat varieties Z9 and S3; FIG. 3, panel B, is a diagram of wheat varieties Z9 and S3 inoculated with different Fusarium graminearum strains 5035 and Tri5^－Expression pattern of the latter TaAn gene. Shows that the TaAn gene of the invention can be induced and expressed by DON and a toxigenic strain fusarium graminearum 5035, and a strain Tri5 with the deletion of the Tri5 gene^－Has no influence on the expression of TaAn gene.

FIG. 4: structural map of intermediate vector PTRAkc-VDM 1-ERH.

FIG. 5: the invention discloses a structural map of an arabidopsis thaliana overexpression vector PTRAkc-35 SS-TaAn.

FIG. 6: molecular identification and expression analysis of transgenic arabidopsis thaliana. A picture is PCR identification of transgenic arabidopsis, primers are TaAn-F2/TaAn-R2, and the size of a target product is 281 bp; b picture is expression quantity analysis of TaAn gene in transgenic Arabidopsis, the primer for amplifying TaAn gene is TaAn-F3/TaAn-R3, and the size of target fragment is 281 bp; the amplification primer of the reference gene Actin is Actin-F/Actin-R, and the size of the target fragment is 135 bp.

FIG. 7: t is₂Phenotype of transgenic Arabidopsis seedlings after 2 weeks of treatment at different concentrations of DON (0 ng/. mu.L, 5 ng/. mu.L, 10 ng/. mu.L and 15 ng/. mu.L).

FIG. 8: t is₃7 days and 10 days after inoculating fusarium graminearum 5035 spore liquid with arabidopsis thaliana.

FIG. 9: the invention constructs an intermediate vector structure map of a wheat constitutive expression vector Pcambia 3300-Ubi-Nos.

FIG. 10: the invention constructs a structural map of a wheat constitutive expression vector Pcambia 3300-Ubi-TaAn.

FIG. 11: t is₂And generating a PCR identification result of the transgenic wheat plant.

FIG. 12: t is₂Phenotype of transgenic wheat seedlings after 1 week of treatment with different concentrations of DON (0mM, 10mM, 20 mM).

Detailed Description

Example 1: isolation and cloning of the full-Length sequence of the TaAn Gene

523bp EST fragments selected from a wheat suspension cell suppression subtraction hybrid library (SSH) were strongly induced by DON (previous work of the inventors, not published). Based on the sequence, the fragment-specific primers TaAn-F1/TaAn-F2 and TaAn-R1/TaAn-R2 were designed, combined with 3 '-RACE and 5' -RACE universal primers (see Table 1), and the total volume of the PCR reaction system was 50. mu.L, 1. mu.L (about 100ng) of cDNA template, 5. mu.L of 10 XKOD plus buffer enzyme reaction buffer, 5. mu.L of 2.5mM dNTP, 1.5. mu.L of 10. mu.M primer, 1. mu.L of KOD enzyme, and 50. mu.L of double distilled water according to the procedures described in the RACE kit (available from Clontech). The reaction procedure is as follows: denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, denaturation at 60 deg.C for 30s, and elongation at 72 deg.C for 5min in 35 cycles. The PCR amplification result is shown in FIG. 2, the 3 'RACE obtains a 422bp fragment (FIG. 2A), the 5' RACE obtains a 468bp fragment (FIG. 2B), the 523bp fragment is obtained after the two are spliced, a primer TaAn-F3/TaAn-R3 (Table 1) is designed to amplify the full length of TaAn and the fragment is sequenced to obtain the 523bp fragment (FIG. 2C, SEQ ID NO. 1).

TABLE 1 primers used for Gene cloning

Description of table 1: english letters F and R after the name of the primer represent a forward primer and a reverse primer, respectively.

Example 2: detecting expression pattern of TaAn gene in wheat variety

Selecting two wheat varieties, Zheng wheat 9023 (for FHB infection, national scrutiny variety, national scrutiny wheat 2003027, abbreviated as Z9, see Cheng et al, Tissue-specific and genetic-index expressionon of a fusion protein associating a Fusarium-specific antibody and a fusion enzyme promoter presenting a mutant Fusarium pathogen and a fusion enzyme promoter, plant Biotechnology journal.2015.doi:10.1111/pbi.12289) and Sumai No.3 (resistant to FHB, bred by the Oncoinaceae in 1970, highly resistant to gibberellic disease, S3 for short, see Cheng et al, Tissue-specific and pathogen-induced expression of a fusion protein associating a Fusarium-specific antibody and a fusion enzyme promoter, plant Biotechnology 2015.12289), as well as a fusion protein promoter expressing a polypeptide promoter, plant biochemical, 12252. A. a. After pregermination, the seeds were placed in a refrigerator at 4 ℃ for vernalization (pregermination and vernalization are conventional methods in the art) for 2 weeks and transplanted into a greenhouse. Selecting wheat ear with consistent growth state at early stage of wheat flowering, cutting off wheat stem of wheat ear with scissors, injecting into 10 μ L, 5 × 10 with microsyringe⁵Inoculating one marked middle spikelet to each strain, covering a transparent plastic bag, keeping the humidity for 3 days, then tearing off the bag, sampling the spikelets inoculated with the spore liquid for 24h, 48h, 72h and 96h respectively, and sampling the spikelets inoculated with the DON toxin for 4h, 12h, 24h and 48 h. The samples were first frozen in liquid nitrogen and then stored in a freezer at-80 ℃. Total RNA was extracted using Trizol (available from Invitrogen) reagent (experimental procedures were performed according to Trizol reagent instructions). Using reverse transcriptase SuperScript^TMIII (from Invitrogen) was reverse transcribed to synthesize cDNA (according to Invitrogen's reverse transcriptase reagent instructions) under the following reaction conditions: 5min at 65 ℃, 60min at 50 ℃ and 10min at 70 ℃. Using the cDNA synthesized by the above reverse transcription as a template, a primer pair TaAn-F3/TaAn-R3 (see Table 1) gene was subjected to specific PCR amplification. Meanwhile, the primer Actin-F/Actin-R is used for carrying out specific amplification (the length of an amplification product is 135bp) on the wheat Actin gene (AB181991) to be used as an internal reference gene for carrying out quantitative analysis. The reaction conditions are as follows: 5min at 95 ℃; 95 ℃ 10sec, 60 ℃ 5sec, 72 ℃ 34sec, 45 cycles. Real-time quantitative analysis of fluorescence detection during the reaction (real-time quantitative analysis of fluorescence detection is a common method in the art, see Li et al, Resistance to FusIum head light and cutting light in leather associated with activity of a cytochrome P450 gene, phytopathology 2010.100: 183-191). The results showed (FIG. 3A) that the TaAn gene was strongly induced by the DON toxin, and induced expression 305-fold and 440-fold in Z9 and S3, respectively, 4h after inoculation, reached a high peak of induction at 17040-fold in Z9, followed by a gradual decrease in expression, and reached a high peak at 2729-fold in S324 h, followed by a gradual decrease in expression, but the induction efficiency was still high. The fold induction in Z9 and S3 was 7354 fold and 2729 fold, respectively, at 24h, and 5793 fold and 1715 fold, respectively, at 48 h. Thus, DON can rapidly activate the TaAn gene, the expression level is increased sharply after activation, the expression level in Z9 is 56 times of that in 4h for 12h, and the expression level is 24 times and 19 times of that in 4h although the expression level is slowly reduced at 24h and 48 h. As shown in FIG. 3B, the TaAn gene was also induced by Fusarium graminearum 5035 toxigenic fungi (a highly pathogenic strain of Fusarium graminearum isolated and stored in Wuhan 1999 in the laboratory of the inventors, Xu et al, division of the heart synthase Chs1 from Fusarium particulate matter in an alternative structure of cells and recovered virus, fungal Genetics and biology 2010.47:205-^－(A mutant strain of the deletion of the Tri5 gene of Fusarium graminearum constructed and maintained by the inventors' laboratory, see Xu et al, partition of the component synthase Chs1 from Fusarium particulate results in an alternative structure of cell walls and reduced vision. fungal Genetics and biology.2010.47: 205-. After fusarium graminearum 5035 starts producing toxin in the ear (36h), the TaAn gene starts to be induced and expressed, and the induced expression of the gene is only related to DON and has no relation with the wheat variety and FHB resistance (namely genetic background) of the wheat variety.

Example 3: construction of TaAn gene overexpression vector and transformation of Arabidopsis thaliana

In order to better analyze the function of the TaAn gene, the TaAn gene is over-expressed in Arabidopsis thaliana, and the function of the gene is researched from the phenotype of transgenic Arabidopsis thaliana.

Super meterThe vector construction method comprises the following steps: taking cDNA obtained by reverse transcription of total RNA after treatment of Zheng wheat 9023(Z9) DON for 12h as a template, amplifying a cDNA fragment (shown as a sequence shown in a sequence table SEQ ID NO: 2) containing a complete coding region of a TaAn gene by using a primer TaAn-F2/TaAn-R2 (shown in a table 1), adding EcoR I and BamH I enzyme cutting sites at two ends of the cDNA fragment respectively, and carrying out reaction under the following conditions: pre-denaturation at 94 ℃ for 5 min; 30sec at 94 ℃, 30sec at 58 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min. After electrophoresis of the PCR product, a target fragment of about 234bp was excised, and a DNA fragment recovery kit (purchased from Axgen) was used to add 3 volumes of DE-A buffer (kit-of-products) to the gel, dissolve the gel in a 75 ℃ water bath for 6min, after the gel was completely dissolved, add 1.5 volumes of DE-B buffer (kit-of-products) to the gel, transfer the gel to a recovery column, centrifuging at 12000rpm for 1min, discarding the effluent, adding 500 μ L washing buffer 1 to the recovery column, centrifuging at 12000rpm for 1min, adding 700 μ L washing buffer 2 to the recovery column, centrifuging at 12000rpm for 1min, washing with washing buffer 2 again, adding 25 μ L of precipitation buffer, standing for 2min, centrifuging at 12000rpm for 2min to obtain purified PCR product, namely TaAn gene, digesting the purified PCR product by EcoR I and BamH I respectively, and precipitating and recovering by absolute ethyl alcohol. Meanwhile, the vector PTRAkc-VDM1-ERH (shown in figure 4, modified by molecular biotechnology laboratory of wheat crops of Huazhong university of agriculture) is respectively cut by EcoR I and BamH I, the carrier skeleton is recovered by cutting gel about 7500bp, and finally the cut target fragment is cut by T₄DNA ligase (NEB) was ligated to the vector backbone to construct an overexpression vector, which was transformed into E.coli DH 5. alpha. to obtain recombinant vector PTRAkc-35SS-TaAn by screening positive clones (FIG. 5).

Genetic transformation and screening identification of transgenic plants:

genetic transformation of Taan Gene Arabidopsis thaliana

The overexpression vector PTRAkc-35SS-TaAn is transferred into Arabidopsis thaliana by Agrobacterium-mediated Arabidopsis thaliana genetic transformation method, which is reported by Zhang Xiuren et al (Zhang Xiuren et al, Agrobacterium-mediated transformation of Arabidopsis thaliana genetic transformation method)the flow di p method, Nature protocols, 1:641-646, 2006). The specific operation steps are as follows: when Arabidopsis thaliana was grown to about 3cm of terminal inflorescence, the terminal inflorescence was removed to stimulate the growth of the lateral inflorescence. And (4) carrying out transformation when the lateral inflorescence grows to 6-10 cm. Before transformation, the soil is allowed to absorb sufficient water, and pollinated flowers and young siliques are removed. And carrying out streak culture on the agrobacterium tumefaciens until a single colony appears, picking the single colony to 5mL of YEB culture medium, carrying out shake culture at 28 ℃ and 200r/min for 40h, and carrying out shake culture according to the ratio of 1: inoculating 100 proportion into 50mL YEB culture medium, shaking and culturing at 28 deg.C and 200r/min to OD₆₆₀And (4) when the concentration reaches about 0.8, centrifuging at 6000r/min for 15min to collect thalli, and resuspending thalli precipitates by using an equal-volume transformation heavy suspension. The part above the rosette leaves of Arabidopsis plants was immersed in the resuspension for 15 sec. The transformed plants were placed flat in a tray with wetted filter paper, covered with a preservative film to maintain humidity, and after 1h, treated again in the same manner. Keeping the plants in the dark for 24h, taking the plants upright, and culturing the plants at 22 ℃ under 8h of light. And harvesting seeds after the plants normally bloom and seed, drying and storing at-20 ℃ for later use. Putting the harvested seeds into a centrifugal tube, treating the seeds with 70% alcohol for 1min, then treating the seeds with 0.15% hypochlorous acid solution for 2min, and washing the seeds with sterile water for a plurality of times; the seeds were sown on 1/2MS medium (pH 5.7, containing Kan 50mg/mL) to screen positive transformants, about 1000 seeds per dish were vernalized at 4 ℃ for 3-4 d, and then cultured at 22 ℃ under 8h of light. The plant which can grow normally is transplanted into nutrient soil after 5 or 6 leaves grow.

Molecular identification and expression quantity analysis of transgenic arabidopsis positive plant

Extraction of transgenic T₀DNA was extracted from fresh leaves of the plant (using a plant genomic DNA extraction kit, purchased from Tiangen Biochemical technology (Beijing) Ltd.) and PCR positive test was carried out using the target gene primer TaAn-F2/TaAn-R2, the results of which are shown in panel A of FIG. 6.

Analysis of expression level of TaAn gene in transgenic Arabidopsis thaliana plant

Total RNA of transgenic positive plants An-1 and An-2 was extracted using Trizol (purchased from Invitrogen corporation) reagent (the experimental procedure was performed according to Trizol reagent instructions). Using reverse transcriptase SuperScript^TMIII (from Invitrogen) was reverse transcribed to synthesize cDNA (according to Invitrogen's reverse transcriptase reagent instructions) under the following reaction conditions: 5min at 65 ℃, 60min at 50 ℃ and 10min at 70 ℃. Using the cDNA synthesized by the above reverse transcription as a template, a primer pair TaAn-F3/TaAn-R3 (see Table 1) gene was subjected to specific PCR amplification. Meanwhile, a primer Actin-F/Actin-R is used for carrying out specific amplification (the length of an amplification product is 135bp) on the wheat Actin gene (AB181991) so as to be used as an internal reference gene for carrying out semi-quantitative analysis. The reaction conditions are as follows: 5min at 95 ℃; 95 ℃ 10sec, 60 ℃ 5sec, 72 ℃ 34sec, 28 cycles. The concentration of the PCR product was detected by electrophoresis. The results showed (see panel B of FIG. 6) that the TaAn gene was abundantly expressed in transgenic Arabidopsis thaliana.

Example 4: taan overexpression transgenic Arabidopsis T₂/T₃Growth conditions of the generation lines under DON stress

This example selects over-expressed T of TaAn transgenic Gene₂The generational lines (numbered An-1) were subjected to DON stress tests at different concentrations. The method comprises the following specific steps: the seeds of the overexpression transgenic strain (An-1) are disinfected by a conventional method (firstly treated by 70% alcohol for 1min, then treated by 0.15% hypochlorous acid solution for 2min, washed by sterile water for several times), germinated on 1/2MS culture medium containing 50mg/L kanamycin, wild type Arabidopsis (non-transgenic, abbreviated as WT) is sowed on 1/2MS culture medium without kanamycin, vernalized at 4 ℃ for 2 days, placed at 20 ℃, and after 7 days of growth, seeds which are well germinated and have consistent growth vigor are selected and transferred to 1/2MS culture medium containing four DONs with different concentrations of 0 ng/muL, 5 ng/muL, 10 ng/muL and 15 ng/muL. After 2 weeks of growth, the root length and fresh weight of the transgenic and wild-type plants were measured and the test was repeated 3 times with 20 seedlings per line. The test results show (FIG. 7) that under the conditions of two concentrations of DON of 0 ng/. mu.L and 5 ng/. mu.L, the root length and the fresh weight of wild type and transgenic plants are not obviously different, but DON can inhibit the growth of Arabidopsis. At concentrations of 10 ng/. mu.L and 15 ng/. mu.L DON, growth of wild type and transgenic plants was inhibited, but root length and fresh weight of transgenic plants were significantly higher than those of wild type, and the difference was greatest at a concentration of 15 ng/. mu.L DON. Therefore, a DON concentration of 15 ng/. mu.L was selected for subsequent DONAnd (4) stress test.

Different transgenic lines (numbered An-1 and An-2) T₂/T₃Strain lines were tested under stress at a concentration of 15 ng/. mu.L DON. The method comprises the following specific steps: overexpression of transgenic lines (An-1 and An-2) T₂/T₃The seeds were sterilized (1 min with 70% alcohol, 2min with 0.15% hypochlorous acid, 5 washes with sterile water), germinated on 1/2MS medium containing 50mg/L kanamycin, wild type Arabidopsis (WT) was sown on 1/2MS medium without kanamycin, vernalized at 4 ℃ for 2 days, placed at 20 ℃, and after 7 days of growth, seedlings that germinated well and grew consistently were selected and transferred to 1/2MS medium containing 15 ng/. mu.L DON. After 2 weeks of growth, the root length and fresh weight of transgenic and wild type arabidopsis thaliana were measured and the experiment was repeated 3 times with 20 seedlings per line. The results of the experiments show (Table 2) that different transgenic lines (An-1 and An-2) T₂/T₃The root length and fresh weight of the material are higher than those of a wild type material under the DON concentration of 15 ng/muL, which shows that the DON tolerance of transgenic arabidopsis can be improved by the overexpression of the TaAn gene, and specific results are shown in Table 2.

TABLE 2 transgenic lines (An-1 and An-2) T₂/T₃The material was treated for 2 weeks at 15 ng/. mu.L DON concentration for root length and fresh weight

a indicates significant difference at 0.05 level

b indicates significant difference at 0.01 level

Example 5: taan overexpression transgenic Arabidopsis T₂FHB resistance analysis of Generation lines

This example selects different transgenic lines (numbered An-1 and An-2) T₂/T₃Inoculating fusarium graminearum 5035 spore liquid with the concentration of 5 multiplied by 10⁵After one/mL, plants were analyzed for disease. The method comprises the following specific steps: overexpression of transgenic lines (An-1 and An-2) T₂/T₃Seed disinfection (70% ethanol treatment for 1min, 0.15% hypochlorous acid treatment for 2 min)Washed several times with sterile water), germinated on 1/2MS medium containing 50mg/L kanamycin, and wild type Arabidopsis (WT) was sown on 1/2MS medium not containing kanamycin, vernalized at 4 ℃ for 2 days, placed at 20 ℃, grown for 7 days, and then transplanted into a small pot. The soil used for the test is carbon soil: vermiculite: perlite is 2: 1: 1 (weight ratio). Growing for about 35 days at 20 ℃ under short illumination (8-hour illumination/16 darkness), wherein the arabidopsis flowers vigorously, selecting plants with consistent growth states, carrying out spray inoculation on fusarium graminearum 5035 spores, and uniformly spraying once on each plant. The specific inoculation method is as follows: 0.001% Silwet L-77 is added into the spore liquid with the adjusted concentration, a small sprayer with a good spraying effect is selected and uniformly sprayed to the inflorescence part of the arabidopsis thaliana, and the sprayer is shaken at any time to enable the spore liquid to be uniformly suspended, so that the precipitation of the spore liquid is avoided; covering the arabidopsis thaliana subjected to spray inoculation with a large plastic cover for moisturizing, keeping the arabidopsis thaliana in the dark for 2 days, and fully germinating spores with the relative humidity of 100 percent by spraying water every day for moisturizing; after 2 days, the cultivation is carried out by illumination, and water spraying and moisture preservation are continued; the disease was investigated on day 7 and 10 after inoculation. Each time 20 strains were inoculated, three replicates.

Arabidopsis thaliana survey is mainly used for counting The incidence of inflorescences (F), old horns (OS) and new horns (NS), and The final incidence index (FAD) is The sum of The three, namely FAD value F + NS + OS specific scoring criteria are shown in Table 3(Urban et al, Arabidopsis Suppeptible to The central early bright future genes Fusarium graminearum and Fusarium culmorum, The Plant Journal, 32: 961-.

TABLE 3 investigation statistical method of inoculating fusarium graminearum 5035 to arabidopsis thaliana

The results show that the FAD values of transgenic plants were lower than those of wild type at both day 7 and day 10 (table 4). Wherein, T₂The FAD values of generation transgenic line An-1 at 7 th and 10 th days were 6.59 and 8.41, which were reduced by 41% and 38% compared with WT, respectively,while the FAD values of An-2 at day 7 and day 10 were 5.10 and 5.91, which were 55% and 57%, respectively, lower than WT, and T₃The FAD values of the transgenic lines were also significantly reduced (fig. 8). Therefore, the results show that the overexpression of the TaAn gene in Arabidopsis can improve the FHB resistance of Arabidopsis plants.

TABLE 4 investigation of the disease index (FAD) after inoculation of Arabidopsis with Fusarium graminearum 5035

b indicates significant differences at the 0.01 level.

Example 6: construction of TaAn gene constitutive expression vector and transformation of wheat

Construction of Pcambia3300-Ubi-TaAn plant expression vector

In order to better analyze the function of the TaAn gene, the TaAn gene is expressed in wheat constitutively, and the function of the gene is researched from the phenotype of transgenic wheat. The construction method of the wheat constitutive expression vector comprises the following steps: using cDNA obtained by reverse transcription of total RNA after treatment of Zheng wheat 9023(Z9) DON for 12h as a template, using a primer TaAn-F2/TaAn-R2 (see table 1) to amplify a cDNA fragment (see a sequence shown in a sequence table SEQ ID NO: 2) containing a complete coding region of a TaAn gene, adding Sma I and Spe I enzyme cutting sites at two ends of the cDNA fragment respectively, and carrying out reaction under the following conditions: pre-denaturation at 94 ℃ for 5 min; 30sec at 94 ℃, 30sec at 58 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 1 min. After electrophoresis of the PCR product, a target fragment of about 234bp was excised, and a DNA fragment recovery kit (purchased from Axgen) was used to add 3 volumes of DE-A buffer (kit-in-band) to the gel, dissolve the gel in a 75 ℃ water bath for 6min to completely dissolve the gel, add 1.5 volumes of DE-B buffer (kit-in-band) to the gel, transfer the gel to a recovery column, centrifuge the gel for 1min at 12000rpm, discard the effluent, add 500. mu.L of washing buffer 1, centrifuge the gel for 1min at 12000rpm to the recovery column, add 700. mu.L of washing buffer 2, centrifuge the gel again at 12000rpm for 1min, wash the gel once again with washing buffer 2, add 25. mu.L of elusion buffer, stand the gel for 2min, and centrifuge the gel for 2min at 12000rpm to obtain pure DNAAnd (3) digesting the purified PCR product, namely the TaAn gene, by using Sma I and Spe I respectively, and precipitating and recovering the purified PCR product by using absolute ethyl alcohol. Simultaneously, the vector pCAMBIA3300-Ubi-Nos (shown in figure 9, modified by molecular biotechnology laboratory of wheat crop of Huazhong agricultural university) is respectively cut by Sma I and Spe I, then the vector skeleton is recovered by cutting gel about 10688bp, and finally the cut target fragment is cut by T₄The DNA ligase (NEB) is connected to a vector skeleton to construct an over-expression vector, then escherichia coli DH5 alpha is transformed, and a plant constitutive expression vector pCAMBIA3300-Ubi-TaAn (figure 10) is obtained by screening positive clones.

Agrobacterium genetic transformation and screening identification of transgenic wheat

Transformation of Agrobacterium EHA105 by Heat shock method

The agrobacterium tumefaciens strain preserved at the temperature of minus 80 ℃ is taken to be in a sensitive state at room temperature for a moment until part of the agrobacterium tumefaciens strain is melted, and the agrobacterium tumefaciens strain is inserted into ice when the agrobacterium tumefaciens strain is in an ice-water mixture state. 0.01-1 μ g plasmid DNA is added per 100 μ L competence, the tube bottom is lightly beaten by hand and mixed well, and then placed on ice for 5min, liquid nitrogen for 5min, water bath for 5min at 37 ℃ and ice bath for 5min in sequence. Adding 700 μ L LB liquid medium without antibiotics, and culturing at 28 deg.C for 2-3h under shaking. Centrifuging at 6000r/min for 1min, leaving 100. mu.L of supernatant, gently blowing and beating the resuspended pellet, coating the pellet on an LB solid plate containing corresponding antibiotics (kanamycin 25. mu.g/mL, rifampicin 50. mu.g/mL), and performing inverted culture at 28 ℃ for 2-3 d.

PCR identification of Agrobacterium

Single colonies growing on the plates were picked and inoculated on LB plates (kanamycin 25. mu.g/mL, rifampicin 50. mu.g/mL) and streaked for scale-up culture at 28 ℃ for 1-2 days. The purified single colony was picked up with sterilized toothpick in 50. mu.L ddH₂And O, mixing, and decocting in boiling water for 15 min. After centrifugation at 12000r/min for 1min, the cells were placed on ice, and 10. mu.L of the supernatant was used as a template. The PCR reaction system is as follows: 2.5. mu.L of 10 XPCR Buffer, 2. mu.L of 1.25mmol/L dNTPs, 0.5. mu.L of each 10. mu.mol/L primer, 0.3. mu.L of Easy Taq enzyme (5U/. mu.L), 10. mu.L of template, and complement ddH₂O to 25. mu.L. The PCR reaction program is: 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃ (varied according to the primers), and 30s at 72 ℃ for 35 cycles; 72 ℃ for 10min and 10 ℃ for 10 min.

Wheat agrobacterium genetic transformation system

Selecting tender green wheat grains with white fluff on bodies after 10-14d of wheat flowers, pouring the wheat grains into a triangular flask in a sterile environment, disinfecting the wheat grains with 70% ethanol for 30s, disinfecting the wheat grains with 0.1% mercury bichloride for 6-8min, washing the wheat grains with sterile water for 5 times, and washing the wheat grains with sterile water for 2min each time, wherein the triangular flask needs to be continuously shaken in the whole process to ensure that the seeds are fully contacted with liquid. Stripping a semi-transparent young embryo with the size of about 0.8-1.2mm with a dissecting needle under aseptic condition, inoculating 60-80 embryos on an induction culture medium with an upward scutellum, culturing in dark at 25 ℃ for 7-9 days, and inducing callus.

Inoculating and culturing agrobacterium tumefaciens one day before transformation, and mixing the stored agrobacterium tumefaciens liquid containing the target plasmid according to the ratio of 1: 50 in a ratio of 2mmol/L MgSO₄100mg/L Rif, 100mg/L Carb and 25mg/L Kana, shaking and culturing overnight at 28 ℃ and 220r/min at constant temperature in a dark environment until the OD600 of the bacterial suspension is 1.0, and then using for transformation (the culture time is about 17-20h, and the specific time depends on the strain). The cells were collected by centrifugation at 4500 Xg for 10min at 4 ℃ and then the supernatant was discarded, and the cells were resuspended in a staining solution until the OD600 became 0.8-0.9, 200. mu.M AS was added thereto, and the cells were cultured in a dark environment at 28 ℃ and 220r/min in a constant temperature shaking table for 1-3 hours, and then 100mg/L of L-ascorbic acid and 0.015% Silwet L-77 were added thereto to prepare an Agrobacterium staining solution. Selecting the callus with good growth state, placing in a triangular flask with a volume of 50mL, drying for 30min without sealing, adding agrobacterium-mediated dyeing liquid, immersing the callus, performing ultrasonic treatment at 20MHz for 30s, performing shaking dark culture at a constant temperature of 100r/min at 28 ℃ for 30 min. After completion of the padding, the padding solution was removed and the calli were transferred to a petri dish with whatman No.1 filter paper (200. mu.L dd H had been added)₂O), piling in stacks, wherein each stack has the diameter of about 2cm and the height of 1cm, and airing the filter paper in a super clean bench. Co-cultivation was carried out at 23 ℃ in the dark for 3 d.

After co-cultivation, the adherent calli were transferred to recovery medium, subcultured once with fresh recovery medium after dark cultivation at 25 ℃ for 10d, dark cultivation at 25 ℃ for 10 d. The screening process of materials in the PMI screening system comprises the following steps: transferring the callus after recovery culture to a differentiation and screening medium (PI) with mannose of 10g/L, and screening for 2 weeks under the condition of illumination at 25 ℃; transferring the callus with the differentiated green dots to a regeneration screening culture medium (PII) with mannose of 5g/L, and screening for 2 weeks under the illumination condition of 25 ℃; the differentiated green shoots were transferred to rooting medium (PIII) containing 15g/L mannose and cultured at 25 ℃ under light for 3 weeks. Transferring the screened resistant seedlings into a strong seedling culture medium (MST1) without a screening agent, rooting and strengthening the seedlings for 2-3 weeks under the illumination condition of 25 ℃, vernalizing the resistant seedlings for 2 weeks at 4 ℃ after the robust roots grow out, then transplanting the resistant seedlings into a greenhouse, and in a PMI system, screening the materials with better performance without using strong seedlings, and transplanting the materials after direct vernalization.

Screening and identification of transgenic positive wheat plants

Extraction of T₂DNA was extracted from fresh leaves of transgenic wheat (using a plant genomic DNA extraction kit, purchased from Tiangen Biochemical technology (Beijing) Ltd.) and PCR positive test was performed with the target gene primer Taan-WF/Taan-WR, and the results are shown in FIG. 11. Lane 1 is a water control, lane 2 is untransformed X76 wheat (negative control), and lanes 3 and 4 are positive plants transformed with the TaAn gene.

TABLE 5 primers used for screening and identification of transgenic wheat

Description of table 5: the English letter W after the name of the primer represents wheat, and F and R represent a forward primer and a reverse primer respectively.

Example 7: taan constitutive expression of transgenic T₂Growth condition of wheat generation under DON stress

This example selects constitutively expressed T transformed with TaAn Gene₂The generation wheat line (numbered An-1) was subjected to DON stress tests at different concentrations. The method comprises the following specific steps: sterilizing wild wheat (non-transgenic, WT) and constitutive expression transgenic line (An-1) seeds by conventional method (treating with 70% ethanol for 1min, treating with 0.15% hypochlorous acid solution for 2min, cleaning with sterile water for several times), germinating on 1/2MS culture medium, sowing on 1/2MS culture medium containing DON (0mM, 10mM, 20mM), vernalizing at 4 deg.C for 3 days, standing at 20 deg.C, growing for 7 days, and measuring transgenic plantThe plants and wild type plants were root-long and the test was repeated 3 times, 20 seedlings per line. The test results show (figure 12) that under the conditions of DON at two concentrations of 10mM and 20mM, the root length of wild wheat X76 is significantly inhibited by DON, but the root length of transgenic wheat An-1 is significantly reduced in DON inhibition compared with wild wheat and a control. The results of the experiments (Table 6) show that different transgenic lines (An-1 and An-2) T₂The material has higher root length than wild type under DON concentrations of 10mM and 20mM, which shows that the constitutive expression of TaAn gene can improve the DON tolerance of transgenic wheat, and the specific results are shown in Table 2.

TABLE 6 wheat transgenic lines (An-1 and An-2) T₂The material was treated at DON concentrations of 10mM, 20mM for 1 week of root length

a indicates significant difference at 0.05 level

b indicates significant differences at the 0.01 level.

Sequence listing

<110> university of agriculture in Huazhong

Application of <120> wheat gene TaAn in improving DON tolerance and FHB resistance of plants

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 523

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagaaacaca tcaagcacac actgacacac gatcagtagc agagaagacc tgagttttct 60

ctaagcttgg agagcagagg aaagaaagaa agaaagagag ggacggggaa attttagtgg 120

tgtagaagct tagcagagta taaatggggt tttgctgtgg gtgttcggat gtcaaggtgc 180

tccccaagaa caactccttg gcttcctcgc cctcgccgtc cgctaaagat tccagcgatg 240

gcgccaagaa gaagcagcct caagccgtaa agaaggaagg gaaagagaag aaaaggagca 300

accttgaccg ggccgccatg gcgtcgcccc gcctcccctt ccattctcga cccggcctaa 360

tgtaatagag tttgcagatt ttcagaacaa tagtgcatac aacatacaat cctgtagctt 420

ctcttattag cttacgcccg ggtttgcctc ggttgttccg ctgaagattt catttgtact 480

gtgtccacgt gtactgtttt attcatggac ggtcggttga tcc 523

<210> 2

<211> 222

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggggtttt gctgtgggtg ttcggatgtc aaggtgctcc ccaagaacaa ctccttggct 60

tcctcgccct cgccgtccgc taaagattcc agcgatggcg ccaagaagaa gcagcctcaa 120

gccgtaaaga aggaagggaa agagaagaaa aggagcaacc ttgaccgggc cgccatggcg 180

tcgccccgcc tccccttcca ttctcgaccc ggcctaatgt aa 222

<210> 3

<211> 73

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Gly Phe Cys Cys Gly Cys Ser Asp Val Lys Val Leu Pro Lys Asn

1 5 10 15

Asn Ser Leu Ala Ser Ser Pro Ser Pro Ser Ala Lys Asp Ser Ser Asp

20 25 30

Gly Ala Lys Lys Lys Gln Pro Gln Ala Val Lys Lys Glu Gly Lys Glu

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Lys Lys Arg Ser Asn Leu Asp Arg Ala Ala Met Ala Ser Pro Arg Leu

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Pro Phe His Ser Arg Pro Gly Leu Met

65 70