Application of TaGLO gene in regulating wheat scab resistance

Abstract

The invention belongs to the field of agricultural biology, and particularly relates to application of a TaGLO gene to regulation and control of wheat scab resistance. The invention finds that the gene TaGLO from the wheat scab resistance variety 'Bainong 4299' can interact with the 'Bainong 4299' scab resistance protein His to improve the plant disease resistance, and the cDNA sequence of the TaGLO gene is as shown in SEQ ID NO: 1. The invention also finds that after the TaGLO gene is silenced in the plant, the disease resistance of the plant is increased. Therefore, the TaGLO gene can be used for analyzing a TaHis gibberellic disease resistance mechanism and improving the gibberellic disease resistance of wheat, and has a wide application space in theoretical analysis of plant resistance, breeding and cultivation.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biology and specifically relates to TagLO Application of genes in regulating wheat fusarium head blight resistance. Background Art

[0002] Wheat is one of the most important food sources for mankind. Fusarium head blight is a semi-saprophytic fungal disease that mainly occurs in crops such as wheat, barley and corn, and wheat fusarium head blight (FHB) is one of the most harmful fungal diseases. Wheat fusarium head blight is a global disease caused by Fusarium graminearum. During the wheat flower filling period, Fusarium graminearum infects the ear and continues to spread along the ear axis, causing the death of the entire ear. The diseased grains contain fungal toxins such as deoxynivalenol (DON), which not only endangers the health of humans and animals, but also seriously affects the edible and feeding value. Therefore, research on wheat resistance to fusarium head blight has attracted great attention from disease-resistant genetic breeders.

[0003] Cultivating and planting Fusarium head blight-resistant varieties is the most economical and environmentally friendly way to control the damage of wheat Fusarium head blight (LiG, Zhou J, Jia H, et al. Mutation of a histidine-rich calcium-binding-proteingene in wheat confers resistance to Fusariumhead blight. Nature genetics, 2019, 51(7): 1106-1112). The scarcity of wheat Fusarium head blight resistance genes and the unclear verification of resistance mechanisms restrict the progress of wheat Fusarium head blight resistance breeding. Therefore, the cloning and functional research of disease resistance genes is of great significance for the study of the molecular mechanism of wheat disease resistance and molecular disease resistance breeding. Summary of the invention

[0004] The present invention discovered a gene from the wheat scab resistant variety "Bainong 4299" TagLO It can interact with the "Bainong 4299" Fusarium scaly resistance protein His to improve plant disease resistance. The present invention also found that the silenced TagLO After the gene is added, the plant's disease resistance increases. TagLO Genes can be used to analyze TaHis The mechanism of resistance to ergot and improving wheat's resistance to ergot have broad application space in plant resistance theory analysis, breeding and cultivation.

[0005] The present invention specifically adopts the following technical solutions: The present invention first provides a gene related to wheat scab resistance—— TagLOGene, which is derived from common wheat ( Triticum aestivum L.), TagLO The cDNA sequence of the gene is shown in SEQ ID NO: 1; The present invention also provides the TagLO The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0006] The present invention further provides a TagLO The recombinant vector of the gene. The starting vector of the recombinant vector is the pMD18-T vector for cloning, the pSL038-1 vector for VIGS and the pTCK303 vector for transgenic expression.

[0007] The present invention further provides a cell line or a recombinant bacterium containing the recombinant vector.

[0008] The present invention provides the TagLO The application of the gene, the protein, the recombinant vector, the cell line or the recombinant bacteria in regulating wheat head blight resistance can be specifically achieved by inhibiting TagLO Gene expression or reduction of TaGLO protein content can improve wheat fusarium head blight resistance. TagLO Gene expression can be silenced using RNAi TagLO Gene.

[0009] The invention provides a method for cultivating wheat resistant to scab, comprising inhibiting TagLO Steps to reduce gene expression or TaGLO protein content. For example, RNAi method can be used to silence the wheat TagLO Genes, including: (1) Construction of RNAi silencing vector; The RNAi silencing vector process is as follows: wheat cDNA is used as a template, GLO-F (RNAi) and GLO-R (RNAi) primers are used for amplification, and the amplified product is cloned into a plant expression vector, such as PTCK303 vector, to obtain the RNAi silencing vector; the sequences of GLO-F (RNAi) and GLO-R (RNAi) are as follows: GLO-F(RNAi):5'-GGGGTACCACTAGTTGGAAGAGGTCGTGAAGGGT-3'; GLO-R(RNAi): 5'-CGGGATCCGAGCTCAATTTGTCGGCCTCGGTGAC-3'.

[0010] (2) The RNAi silencing vector was transformed into wheat through Agrobacterium-mediated genetic transformation, and positive transgenic plants were obtained by screening and identification.

[0011] The beneficial effects of the present invention are: First, the FHB resistance gene His reported by previous researchers was constructed into the yeast two-hybrid bait vector pGBKT7 to obtain the bait vector His-pGBKT7. After screening the wheat yeast two-hybrid library by yeast two-hybrid technology, it was identified that wheat TaGLO interacted with His. Specific primers were further designed for PCR amplification to obtain TagLO Full-length coding region complete sequence.

[0012] The present invention utilizes BSMV-VIGS technology to TagLO Gene transient silencing is shown TagLO The resistance of wheat to fusarium head blight was significantly improved after gene silencing. TagLO Gene overexpression and RNAi silencing vectors, obtained through Agrobacterium-mediated transgenic technology TagLO Transgenic wheat with overexpressed and silenced genes, analysis of resistance to Fusarium head blight showed silencing TagLO Gene can significantly improve wheat fusarium resistance, while overexpression TagLO The gene reduces resistance to wheat fusarium wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Indicates VIGS silencing of wheat Bainong 607 TagLO The phenotype of Fusarium head blight resistance response 14 days after Fusarium head blight inoculation. Left: control wheat plants; Right: silent TagLO Wheat plants.

[0014] Figure 2 Represents wheat TagLO Detection of gene expression. BSMV:00: indicates VIGS empty-load control plants; BSMV-TaGLO1, BSMV-TaGLO2, BSMV-TaGLO3: indicates BSMV: TaGLO gene silencing-treated plants.

[0015] Figure 3 Expressing silence TagLO The phenotype of the Fusarium head blight resistance response of transgenic wheat with the GLO gene 14 days after inoculation with Fusarium head blight. Left: control wheat plants; right: wheat plants with silenced GLO gene.

[0016] Figure 4 Represents wheat TagLO Detection of gene expression. WT: indicates non-transgenic wheat plants; RNAi-1, RNAi-2, RNAi-3: indicates transgenic silenced wheat plants.

[0017] Figure 5 Overexpression TagLOThe phenotype of Fusarium head blight resistance in transgenic wheat expressing the gene 14 days after Fusarium head blight inoculation. Left: control wheat plants; Right: overexpression TagLO Genetically modified wheat plants.

[0018] Figure 6 Represents wheat TagLO Detection of gene expression. WT: non-transgenic wheat plants; OE-1, OE-2, OE-3: transgenic over-expressing wheat plants. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1 1. Construction of pGBKT7-TaHis bait vector and screening of wheat library: According to Li et al. (Li G, Zhou J, Jia H, et al. Mutation of a histidine-rich calcium-binding-protein gene in wheatconfers resistance to Fusarium headblight. Nature genetics, 2019, 51(7): 1106-1112) TaHis Gene sequence primers were used to amplify the disease-resistant wheat variety Bainong 4299 using PCR technology. TaHis Gene, PCR was a 25 μL reaction system, including 1 μL template cDNA, 1 μL upstream and downstream primers, 12.5 μL 2× EsTaqMasterMix enzyme, ddH 2 O 9.5 μL. PCR amplification reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 55 s, extension at 72 ℃ for 50 s, 30-35 cycles; final extension at 72 ℃ for 5 min. TaHis Recombined into plasmid pGBKT7, constructed bait vector pGBKT7-TaHis, double enzyme digestion detection positive clones, and sent the positive clones to Jinkairui Company for sequencing verification. Fhb1The wheat yeast two-hybrid library was constructed using the Fusarium graminearum-resistant variety Bainong 4299 as the material and wheat ears infected with Fusarium graminearum for 3 days. The library plasmids were transformed into yeast cells Y2HGold containing the pGBKT7-TaHis bait vector by PEG / LiAC-mediated transformation, and then spread on two-deficiency (SD / -Leu / -Trp) and four-deficiency (SD / -Leu / -Trp / -His / -Ade) solid culture media, respectively. The cells were inverted and cultured in a 30 ℃ incubator for 3-5 days. The well-growing single clones were picked and placed in four-deficiency culture medium for expansion culture. The plasmids were extracted after 3-5 days and sequenced using the universal primers of the pGADT7 vector (the vector used to construct the library). The sequencing results were analyzed and compared in the NCBI database to exclude plasmids containing repetitive sequences. The final plasmids were further transferred back into yeast cells Y2HGold with the pGBKT7-TaHis vector, and spread on four-deficiency (SD / -Leu / -Trp / -His / -Ade) solid culture medium for verification. If the yeast grew normally, it was a real interacting protein.

[0021] After screening the wheat yeast two-hybrid library using the above-mentioned yeast two-hybrid technology, it was identified that wheat TaGLO interacted with His.

[0022] 2. Wheat TagLO Gene cloning Based on the His-based screening of the wheat yeast two-hybrid library, the wheat TagLO The gene sequence was XM_044476205.1, and amplification primers were designed (forward: 5'-ATGGATGGTGATTTGCACAG-3'; reverse: 5'-TTACAAGCGGGACGGCATGA-3'). The "Bainong 4299" cDNA was used as a template for amplification. The PCR reaction conditions were: 94℃5min, 94℃ 30s, 55℃ 30s, 72℃ 55s, a total of 30 cycles; 72℃ 10min. After PCR was recovered by 1.2% agarose gel electrophoresis, it was connected to the pMD18-T vector and transformed into Escherichia coli DH5α. The extracted plasmid was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing after identification. TagLO The gene sequence is shown in SEQ ID NO: 1, and the protein sequence encoded by the gene is shown in SEQ ID NO: 2.

[0023] 3. TagLO Gene silencing vector construction according to TagLO Primers GLO-F (VIGS) and GLO-R (VIGS) were designed for the gene forward sequence. The 5' end of the primer pair was introduced SmaI digested the vector end sequence so that the amplified fragment can be seamlessly connected to the BSMV-γ vector. The sequences of GLO-F (VIGS) and GLO-R (VIGS) are as follows: GLO-F(VIGS): 5'-TAGCTGATTAATTAACCCGGGTGGAAGAGGTCGTGAAGGGT-3'; GLO-R(VIGS): 5'-TAGCTGAGCGGCCGCCCCGGGAATTTGTCGGCCTCGGTGAC-3'.

[0024] Then, GLO-F (VIGS) and GLO-R (VIGS) were used as primers to clone the above TagLO PCR was performed using the gene as a template to amplify TagLO 277 bp fragment. PCR program: 1 μL TagLO Gene template (100ng / μL), 2μL upstream primer, 1μL downstream primer, 25μL Max Premix, add water to 50μL. PCR reaction conditions: 98℃ pre-denaturation for 3min; 98℃ denaturation for 10s, 55℃ annealing for 15s, 72℃ extension for 20s, 35 cycles, 72℃ extension for another 5min.

[0025] The PCR products were recovered after detecting the amplified bands by 1.5% agarose gel electrophoresis. TagLO Fragment. TagLO After the fragments are amplified and recovered, the seamless connection technology is used to TagLO The fragment was connected to the BSMV-γ vector, transformed into E. coli DH5α competent cells, and single clones containing the target fragment were selected for sequencing. TagLO The gene was inserted into BSMV-γ Sma I restriction site, the γ-GLO recombinant vector was successfully constructed and can be used for the next step of in vitro transcription and induced gene silencing.

[0026] 4. In vitro transcription According to the instructions for plasmid extraction (Tiangen Biochemical Technology Co., Ltd.), BSMV viral vectors α, β, γ (Song P, Zhang L, Wu L, et al. A ricin B-like lectin protein physically interacts with TaPFT and is involved in resistance to fusariumhead blight in wheat. Phytopathology, 2021, 111(12): 2309-2316.) and γ-GLO recombinant vectors carrying exogenous insertion fragments of target genes were extracted respectively, and then the linearized vectors were transcribed in vitro using the mMESSAGEmMACHINE T7in vitro transcription kit (AM1340, Ambion). After in vitro transcription, α, β, γ and γ-GLO transcription products were obtained respectively. After mixing equal volumes of α, β, and γ transcription products, they were diluted with three times the volume of DEPC water, and then 2×GKPBuffer (50mM glycine, 30mM K 2 HPO4, pH 9.2, 1% bentonite, 1% celite) as the empty control group (BSMV:00). The α, β, and γ-GLO transcription products were mixed in equal volumes, diluted with three times the volume of DEPC water, and then 2×GKP Buffer (50mM glycine, 30mM K 2 HPO4, pH 9.2, 1% bentonite, 1% celite) as the gene silencing experimental group (BSMV: TaGLO). The above mixed solutions were stored in a -80℃ refrigerator for future use.

[0027] 5. Application test Referring to the method of Song et al. (Song P, Zhang L, Wu L, et al. A ricin B-like lectinprotein physically interacts with TaPFT and is involved in resistance tofusariumhead blight in wheat. Phytopathology, 2021, 111(12): 2309-2316.), BSMV:00 and BSMV:TaGLO were used to friction inoculate Bainong 607 wheat at the booting stage. When the wheat to be inoculated entered the poplar flowering stage, the single flower drip method was used to identify the resistance to fusarium head blight. Ten days after the virus inoculation, the fusarium head blight inoculated spikelets of plants inoculated with the empty vector control BSMV:00 and the gene fragment virus BSMV:TaGLO were sampled and RNA was extracted, and fluorescence quantitative PCR was used to detect TagLO Silent efficiency.

[0028] The results showed that BSMV: TaGLO was compared with the control BSMV: 00. TagLO The relative expression level decreased significantly ( Figure 2 ). The number of diseased spikelets was investigated 21 days after Fusarium graminearum inoculation. The results showed that the number of diseased spikelets in BSMV:TaGLO silenced plants 21 days after Fusarium graminearum inoculation was significantly lower than that in the control BSMV:00 ( Figure 1 ).

[0029] The above results show that silence TagLO It can significantly increase wheat's resistance to ergot.

[0030] 6. TagLO Transgenic wheat positive plants were obtained (1) Construction of vector: Using wheat spike cDNA of Bainong 4299 as template, TagLO Primers GLO-F (OE) and GLO-R (OE) were designed for the gene forward sequence for amplification. The amplified product was connected to the T vector using a DNA ligation kit (DNA Ligation KitVer.2.1, Takara) (specifically, 1μL T4 ligase, 4μL gel recovery product, 5μL Solution), and transformed into Escherichia coli DH5α after overnight connection at 16°C. The bacteria were picked and sent for testing the next day. After the correct sequencing bacteria were expanded, plasmids were extracted, and the extracted plasmids and the stored PTCK303 vector were digested with enzymes. The restriction sites were BamH I and SacI, then gel recovery, the digestion product of the recombinant T vector and the digestion product of the PTCK303 vector were connected at a ratio of 2:1, and then transformed into E. coli DH5α, a single clone was picked for bacterial liquid PCR verification, and the bacterial liquid was sent to the company for sequencing, and the bacterial liquid with successful sequence alignment was saved, and then the overexpression vector (OE-GLO) was obtained. The sequences of primers GLO-F (OE) and GLO-R (OE) are as follows: GLO-F(OE): 5'-CGACTCTAGAGGATCCATGGATGGTGATTTGCACAG-3'; GLO-R(OE): 5'-GATCGGGGAAATTCGAGCTCTTACAAGCGGGACGGCATGA-3'.

[0031] Using wheat spike cDNA from Bainong 4299 as template, TagLO Primers GLO-F (RNAi) and GLO-R (RNAi) were designed for the gene forward sequence to amplify. After gel recovery, the amplified product was connected to the T vector to obtain RNAi-GLO-T. The recombinant plasmid was transformed into Escherichia coli DH5α. The bacteria were selected for sequencing the next day. After the correct sequencing bacteria were expanded, the plasmid was extracted. The extracted plasmid and the stored PTCK303 vector plasmid were digested with enzymes. The restriction sites were Spe I and Sac I, then gel recovery, the cut PTCK303 vector and the cut RNAi-GLO-T were connected at a ratio of 1:2, and then transformed into E. coli DH5α, and then the bacteria were picked and shaken for sequencing. After the sequence alignment was successful, the plasmid was expanded by shaking, and after the sequencing was successful, it was preserved to obtain the RNAi silencing vector (RNAi-GLO). The sequences of primers GLO-F (RNAi) and GLO-R (RNAi) are as follows: GLO-F(RNAi):5'-GGGGTACCACTAGTTGGAAGAGGTCGTGAAGGGT-3'; GLO-R(RNAi): 5'-CGGGATCCGAGCTCAATTTGTCGGCCTCGGTGAC-3'.

[0032] (2) Genetic modification: The two recombinant vectors obtained in (1) were transformed into Bainong 607 by Agrobacterium-mediated genetic transformation.

[0033] (3) Identification of transgenic positive plants: Transgenic lines were planted to the T2 generation, and positive plants were identified by PCR in the T0, T1, and T2 generations. DNA was extracted by CTAB method, and PCR was performed on the plants using transgenic detection primers Test-F and Test-R. The sequences of Test-F and Test-R are as follows: Test-F: 5'-TGGATCGCGAAAACTGTGGA-3'; Test-R: 5'-CGGTGATATCGTCCACCCAG-3'.

[0034] In T2 generation positive plants, wheat TagLO The gene was analyzed by real-time fluorescence quantitative analysis, and qPCR was used to verify the expression of the overexpression vector OE-GLO and the RNAi silencing vector RNAi-GLO in the T2 generation transgenic positive strains during the flowering period of wheat and poplar. The results showed that TagLO The expression level of the gene in OE1-OE3 positive lines was significantly higher than that in wild-type wheat, and the expression level in RNAi1-RNAi3 positive lines was significantly lower than that in wild-type wheat, indicating that the expression of the gene in the OE-GLO transgenic wheat lines was significantly higher than that in wild-type wheat. TagLO Genes can be overexpressed and RNAi-GLO can be used in wheat TagLO The gene expression was suppressed, further clarifying The Reliability of GLO genetically modified wheat.

[0035] (4) Identification of resistance to fusarium scaber in transgenic plants The T2 transgenic plants were inoculated with Fusarium graminearum by single flower dripping method during the flowering period of wheat for resistance identification. The results showed that 14 days after inoculation, the silent TagLO The resistance of transgenic plants expressing the gene to Fusarium head blight was significantly higher than that of the control ( Figure 3 , Figure 4 ), while overexpression TagLO The resistance of transgenic plants expressing the gene to Fusarium head blight was significantly lower than that of the control ( Figure 5 , Figure 6 ), further proving that TagLO Genes positively regulate wheat ergot resistance.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. TaGLO Gene, TaGLO Genes encode proteins that contain TaGLO The use of a recombinant vector of a gene, a cell line containing the recombinant vector or a recombinant bacterium in regulating wheat fusarium resistance is characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: Said TaGLO The cDNA sequence of the gene is shown in SEQ ID NO:

1.

3. The use according to claim 1 or 2, characterized in that: By inhibiting TaGLO Gene expression or reduction of TaGLO protein content to improve wheat ergot resistance.

4. The use according to claim 3, characterized in that: The inhibition TaGLO Gene expression silencing using RNAi TaGLO Gene.

5. A method for cultivating wheat resistant to scab, characterized in that: Including wheat TaGLO The step of genetically expressing or reducing the content of TaGLO protein; the amino acid sequence of the protein is shown in SEQ ID NO:

2.

6. The method according to claim 5, characterized in that Silencing of wheat mitochondria by RNAi TaGLO Gene.

7. The method according to claim 6, characterized in that include: (1) Construction of RNAi silencing vector: Using wheat cDNA as a template, amplification was performed using GLO-F (RNAi) and GLO-R (RNAi) primers, and the amplified product was cloned into a plant expression vector to obtain an RNAi silencing vector; the sequences of GLO-F (RNAi) and GLO-R (RNAi) are as follows: GLO-F(RNAi):5'-GGGGTACCACTAGTTGGAAGAGGTCGTGAAGGGT-3'; GLO-R(RNAi): 5'-CGGGATCCGAGCTCAATTTGTCGGCCTCGGTGAC-3'; (2) The RNAi silencing vector was transformed into wheat through Agrobacterium-mediated genetic transformation, and positive transgenic plants were obtained by screening and identification.

8. The method according to claim 7, characterized in that The plant expression vector in step (1) is a PTCK303 vector.

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