Application of mung bean ethanol dehydrogenase coding gene VrADH in resisting mung bean yellow mosaic disease

Abstract

The invention relates to the technical field of molecular genetic breeding, in particular to application of a mung bean ethanol dehydrogenase coding gene VrADH in resisting mung bean yellow mosaic disease. The mung bean ethanol dehydrogenase encoding gene VrADH can enhance the resistance to MYMIV, the mung bean VrADH is subjected to up-regulation expression in leaves after being induced by the MYMIV, meanwhile, it is found through tobacco instantaneous conversion verification that the VrADH can positively regulate the resistance to the MYMIV, and a material, carrying the VrADH, in a mung bean material created through hybridization shows the resistance to the MYMIV; therefore, the VrADH can be used as a target spot for regulating the MYMIV by the mung beans and is used for modifying the MYMIV resistance of the mung beans.

Description

Technical Field

[0001] This invention relates to the field of molecular genetic breeding technology, specifically to a gene encoding mung bean alcohol dehydrogenase. VrADH Application in combating mung bean yellow mosaic disease. Background Technology

[0002] Mung bean [Vigna radiata (L.) Wilczek] is an important edible legume crop, rich in protein, dietary fiber, vitamins, and minerals. During its growth and development, mung bean is frequently subjected to various environmental stresses and infections by a variety of diseases. Among these diseases, yellow mosaic disease (YMD), caused by Mungbean yellowmosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV), can severely damage both the yield and quality of mung beans, with yield losses reaching as high as 85% in some years.

[0003] YMD is one of the main factors restricting mung bean production. At present, the main control measures for mung bean YMD include spraying insecticides and cultivating disease-resistant varieties. However, due to its wide distribution, serious damage and difficulty in eradication by chemical agents, the most economical and effective control method is to explore the resistance gene of mung bean YMD and apply it to resistance molecular breeding. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention aims to provide a mung bean alcohol dehydrogenase encoding gene. VrADH In its application against mung bean yellow mosaic virus, this gene was upregulated in leaves after being induced by MYMIV. VrADH It can be used as a target gene for transient conversion into Nicotiana benthamiana, and exhibit resistance to MYMIV.

[0005] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a gene encoding mung bean alcohol dehydrogenase. VrADH Application in combating mung bean yellow mosaic disease.

[0006] Furthermore, the mung bean alcohol dehydrogenase encoding gene VrADH The nucleotide sequence is shown in SEQ ID NO.1; the gene encoding the mung bean alcohol dehydrogenase is... VrADH The encoded amino acid sequence is shown in SEQ ID NO.2.

[0007] Furthermore, the mung bean alcohol dehydrogenase encoding gene VrADH The specific primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0008] Furthermore, it includes the gene encoding the mung bean alcohol dehydrogenase. VrADH The primers for the ORF are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0009] Furthermore, the mung bean alcohol dehydrogenase encoding gene VrADH The primers for determining the expression level are shown in SEQ ID NO. 9 and SEQ ID NO. 10; the mung bean alcohol dehydrogenase encoding gene... VrADH The characteristic primers for the CDS are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0010] Secondly, this invention provides an overexpression vector pFGC5941- VrADH Including the mung bean alcohol dehydrogenase encoding gene. VrADH .

[0011] Thirdly, the present invention provides an overexpression engineered bacterium, comprising the overexpression vector pFGC5941- VrADH .

[0012] Fourthly, the present invention provides a method for cultivating plants resistant to mung bean yellow mosaic virus, characterized in that the overexpression vector pFGC5941- is used. VrADH Alternatively, the overexpressed engineered bacteria may be transferred into mung bean plants.

[0013] Fifthly, this invention provides a method for screening mung bean yellow mosaic disease-resistant plants, using the mung bean alcohol dehydrogenase encoding gene. VrADH After PCR amplification of mung bean plant samples using specific primers, sequencing is performed. If the sequencing result is as shown in SEQ ID NO.1, then the plant is a resistant mung bean yellow mosaic virus.

[0014] Sixthly, this invention provides a gene encoding mung bean alcohol dehydrogenase. VrADH Or the overexpression vector pFGC5941- VrADH Or the application of the overexpressed engineered bacteria in the preparation of a reagent for cultivating plants resistant to mung bean yellow mosaic disease.

[0015] The beneficial effects of this invention are: mung beans VrADH It is an alcohol dehydrogenase encoding gene that enhances resistance to MYMIV; mung beans VrADHVrADH was upregulated in leaves after MYMIV induction. Subcellular localization analysis revealed that VrADH is a nuclear membrane co-localized protein. Functional validation through transient tobacco transformation further confirmed this. VrADH It can positively regulate resistance to MYMIV, and the mung bean materials created through hybridization carry this... VrADH The material exhibits resistance to MYMIV; therefore VrADH It can be used as a target for regulating the MYMIV response of mung beans, and can be used to modify mung beans to resist MYMIV. Attached Figure Description

[0016] Figure 1 for VrADH PCR amplification diagram of the gene; Marker: 5000 bp. VrADH The target gene band is 1143 bp.

[0017] Figure 2 for VrADH Subcellular localization map; GFP: green fluorescent protein, BF: bright field, Merge: fusion protein, 35S:GFP: empty vector control, 35S: VrADH-GFP, VrADH protein with GFP tag.

[0018] Figure 3 7, 14, and 21 days after inoculating MYMIV onto resistant mung bean material NM10 and susceptible mung bean material JL7 VrADH Expression level; error bars represent standard deviation, ns: not significant. Significance level P <0.01.

[0019] Figure 4 for VrADH Gene expression levels at 3, 7, and 14 days after transient overexpression of Nicotiana benthamiana; CK was the control, and the error bars represent the standard deviation. Significance level P <0.05, Significance level P <0.01.

[0020] Figure 5 for VrADH The transient overexpression of the resistance phenotype of Tobacco Benedict's tobacco to MYMIV was observed; CK was the control, and photos were taken 14 days after inoculation.

[0021] Figure 6 for VrADH The expression level of the gene encoding the viral coat protein (CP) in *Nycium benzoate* was determined by transient overexpression; error bars represent standard deviation. Significance level P <0.05, Significance levelP <0.01.

[0022] Figure 7 To construct a MAGIC genetic population map of mung beans, including traits such as MYMIV resistance.

[0023] Figure 8 To identify the disease resistance phenotype of the mung bean MAGIC genetic population L202 against MYMIV; photographs were taken 21 days after inoculation. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0026] Example 1 green beans VrADH Analysis of gene cloning and expression characteristics 1) Gene encoding mung bean alcohol dehydrogenase VrADH Cloning according to VrADH Gene number EVM0001128 The corresponding base sequence of the gene was found in the database (SEQ ID NO.1), and the encoded amino acid sequence is shown in SEQ ID NO.2. Specific primers were designed based on this sequence for gene cloning; the primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. Using the disease-resistant mung bean material NM10 as the sample, its leaves were crushed in a mortar and pestle, added to a 1.5 mL EP tube containing lysis buffer, and after thorough shaking, transferred to another 1.5 mL EP tube to extract total RNA. Using the obtained total RNA as a template, reverse transcription was performed according to the instructions of the reverse transcription kit provided by Takara. After obtaining the first strand of cDNA, PCR amplification was performed. The PCR program was as follows: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 1 minute 30 seconds, for a total of 35 cycles, followed by incubation at 72℃ for 5 minutes, and then isothermal treatment at 12℃. Subsequently, the PCR product was purified by gel extraction, ligated, and transformed. Positive single clones were selected for sequencing. Sequencing yielded a 1143 bp mung bean gene with a complete CDS (Coding Sequence). EVM0001128 Named VrADH ( Figure 1 ).

[0027] SEQ ID NO.1:

[0028] SEQ ID NO.2: MSSTAGQVIKCKAAVAWEAGKPLVIEEVEVAPPEAGEVRLKILYTSLCHTDVYFWEAKGQTPLFPRIFGHEAGGIVESVGEGVTHLKPGDHALPVFTGECGECAHCKSEESNMCDLLRINTDRGVMIHDSQTRFSIKGQPIYHFVGTSTFSEYTVVHAGCVAKVNPAAPLDKICVLSCGICTGLGATVNVA KPKPGSSVAIFGLGAVGLAAAEGARISGASRIIGVDLVSSRFEEAKKFGVNEFVNPKDHDKPVQQVIAEMTNGGVDRAVECTGSIQAMVSAFECVHDGWGVAVLVGVPNKDDAFKTHPVNFLNERTLKGTFYGNYKPRTDLPSVVEKYMNGELELEKFITHTVPFSEINKAFDYMLKGESIRCIIRMGE*.

[0029] SEQ ID NO.3: TTTCATCTTTTCTTCTCACC.

[0030] SEQ ID NO.4: AAGCCTCCTATCGTTCTCTA.

[0031] 2) Subcellular localization study of VrADH The design includes VrADH Primers for the complete ORF (Open Reading Frame) of the gene (excluding the stop codon) are shown in SEQ ID NO.5 and SEQ ID NO.6. The specific PCR procedure is the same as in step 1). Then, the primers excluding the stop codon are obtained by double digestion with Xhol and BamHI. VrADH The complete ORF of the gene was homologously recombinated into the expression vector 1305, thereby... VrADH The complete ORF gene was fused to the 3' end of the reporter gene GFP on expression vector 1305, thus constructing the subcellular localization vector 35S:VrADH-GFP. This vector and the empty vector were transformed into *Tobacco Bengal* leaf cells using *Agrobacterium* transformation, respectively. The results showed that VrADH protein was localized in the nucleus and cell membrane. Figure 2 ).

[0032] SEQ ID NO.5: CGGAGCTAGCTCTAGAATGTCGAGCACAGCTGGT.

[0033] SEQ ID NO.6: TGCTCACCATGGATCCCTCTCCCATACGGATAAT.

[0034] 3) VrADH Expression analysis after MYMIV induction The resistant mung bean material NM10 and the susceptible mung bean material JL7 were inoculated with MYMIV, and the differences between the resistant and susceptible materials were tested. VrADH Differences in expression levels. Leaves were collected at 7, 14, and 21 days after treatment, flash-frozen in liquid nitrogen, and stored at -80°C. Total RNA extraction was performed as in step 1). Actin The primer sequences for the internal reference gene are shown in SEQ ID NO.7 and SEQ ID NO.8. Total RNA from leaves of two mung bean materials, NM10 (resistant) and JL7 (susceptible), under different treatment conditions was used as a template. After being reverse-engineered into cDNA, real-time quantitative PCR was performed to detect the virus. VrADH Changes in expression levels after MYMIV induction VrADH The primer sequences for expression level determination are shown in SEQ ID NO. 9 and SEQ ID NO. 10. The study found... VrADH Expression in the disease-resistant material NM10 was strongly induced by MYMIV, with expression levels at 14 and 21 days post-infection being approximately 5-fold and 18-fold higher than at 7 days, respectively. Figure 3 );and VrADH The induced expression in the susceptible material JL7 was significantly weaker than that in the resistant material NM10. The expression levels in the susceptible material JL7 at 14d and 21d after infection were only 1.2 times and 5 times that at 7d, respectively. Figure 3 This result indicates VrADH It can actively respond to MYMIV induction in disease-resistant materials, thereby significantly increasing expression levels.

[0035] SEQ ID NO.7: CAGTGTCTGGATTGGAGGCT.

[0036] SEQ ID NO.8: GTCCTCGACCACTTGATG.

[0037] SEQ ID NO.9: GGAGTGCCAAACAAAGATG.

[0038] SEQ ID NO.10: GAACGGTGTGAGTGATGAA.

[0039] Example 2 VrADH Applications of genetic engineering 1) Construction of plant overexpression vectors Using SEQ ID NO.11 and SEQ ID NO.12 as primers, mung beans containing the sample shown in SEQ ID NO.1 were extracted. VrADH PCR amplification was performed on the CDS of the gene, and recombination was used to... VrADH Linked into the pFGC5941 vector, pFGC5941- is obtained. VrADH Plant overexpression vector. The plant transformation vector pFGC5941 contains a strong 35S promoter, which can strongly induce the target gene. VrADH The vector was expressed in the receptor. Then, the vector was transformed into Agrobacterium tumefaciens strain EHA105 using a freeze-thaw method, and Nicotiana benthamiana was infected using a transient transformation method.

[0040] SEQ ID NO.11: TTACATTTACAATTACCATGGATGTCGAGCACAGCTGGT.

[0041] SEQ ID NO.12: CTCTAGACTCACCTAGGATCCCTACTCTCCCATACGGAT.

[0042] 2) VrADH Identification of disease resistance phenotypes for MYMIV The constructed overexpression vector pFGC5941- VrADH The empty control and its precursor were used to infect *Nicotiana benthamiana* leaves for 6-8 weeks via transient transformation. Leaves were harvested at 3, 7, and 14 days post-infection, flash-frozen in liquid nitrogen, and stored at -80°C. Total RNA extraction was performed as described in step 1 of Example 1. NbEF1a As an internal reference gene, the primer sequences are shown in SEQ ID NO.13 and SEQ ID NO.14. Using total RNA as a template, the DNA was reverse-engineered into cDNA and then subjected to real-time quantitative PCR. VrADH Primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, and primer sequences for the viral coat protein (CP) encoding gene are shown in SEQ ID NO.15 and SEQ ID NO.16.

[0043] SEQ ID NO.13: CCCAAGAGGCCCTCAGACA.

[0044] SEQ ID NO.14: CACACGACCAACAGGGACAGT.

[0045] SEQ ID NO.15: ACGTGACTAGAGGTAATGG.

[0046] SEQ ID NO.16: TGGACTTAATGTTTTCGTC.

[0047] Through testing VrADH The expression level of the gene in Nicotiana benthamiana can be seen VrADH The expression level in the overexpression material of Nicotiana benthamiana was significantly higher than that in the wild type. Figure 4 To verify VrADH Resistance to MYMIV was subsequently assessed using *Nicotiana benthamiana* for phenotypic identification. Compared to wild-type *Nicotiana benthamiana* uninfected with MYMIV (CK), wild-type *Nicotiana benthamiana* infected with MYMIV exhibited obvious mosaic symptoms (MYMIV). VrADH Overexpression of Nicotiana benthamiana did not result in obvious mosaic symptoms (MYMIV+). VrADH () Figure 5 Meanwhile, the study found that at 3, 7, and 14 days after MYMIV infection, VrADH Overexpression of Benedictine tobacco CP Gene expression levels were significantly lower than those of wild-type Nicotiana benthamiana (Nicotiana benthamiana). Figure 6 ).

[0048] The above results indicate that overexpression VrADH The gene can reduce the accumulation of the virus and alleviate the corresponding symptoms, thereby increasing the plant's resistance to MYMIV.

[0049] Example 3 VrADH Breeding applications This study constructed a mung bean MAGIC (Multiparent Advanced Generation Inter-Cross) population with eight parents, including the MYMIV-resistant parent NM10 and the susceptible parent JL7. This population comprised seven subpopulations, and through multiple generations of hybridization and continuous self-pollination over several years, a total of 1500 F7 generations of families were formed. Figure 7Mung bean leaves were crushed in a mortar and pestle, added to a 1.5 mL EP tube containing lysis buffer, and thoroughly shaken. The mixture was then transferred to another 1.5 mL EP tube to extract total RNA. Using the obtained total RNA as a template, reverse transcription was performed according to the instructions of the reverse transcription kit provided by Takara. After obtaining the first strand of cDNA, specific primers were designed for PCR amplification. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The PCR program was as follows: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 1 minute and 30 seconds, for a total of 35 cycles, followed by incubation at 72℃ for 5 minutes. The PCR products were then sequenced. The sequencing results showed that the mung bean with SEQ ID NO.1 was resistant to MYMIV, while the mung bean with SEQ ID NO.17 was not resistant to MYMIV. Through gene cloning and disease resistance phenotype identification, it was found that the mung bean carried the same gene as NM10. VrADH The population material L202 with genotype (SEQ ID NO.1) showed strong resistance to MYMIV. Figure 8 ).

[0050] SEQ ID NO.17:

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Gene encoding mung bean alcohol dehydrogenase VrADH Application in combating mung bean yellow mosaic disease.

2. The application according to claim 1, characterized in that, The mung bean alcohol dehydrogenase encoding gene VrADH The nucleotide sequence is shown in SEQ ID NO.1; the gene encoding the mung bean alcohol dehydrogenase is... VrADH The encoded amino acid sequence is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, The mung bean alcohol dehydrogenase encoding gene VrADH The specific primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. The application according to claim 2, characterized in that, Contains the gene encoding the mung bean alcohol dehydrogenase VrADH The primers for the ORF are shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. The application according to claim 2, characterized in that, The mung bean alcohol dehydrogenase encoding gene VrADH The primers for determining the expression level are shown in SEQ ID NO. 9 and SEQ ID NO. 10; the mung bean alcohol dehydrogenase encoding gene... VrADH The characteristic primers for the CDS are shown in SEQ ID NO.11 and SEQ ID NO.

12.

6. An overexpression vector pFGC5941- VrADH Its characteristics are, Including the mung bean alcohol dehydrogenase encoding gene as described in claim 1 VrADH .

7. An overexpression engineered bacterium, characterized in that, Including the overexpression vector pFGC5941 as described in claim 6- VrADH .

8. A method for cultivating plants resistant to mung bean yellow mosaic virus, characterized in that, The overexpression vector pFGC5941- as described in claim 7 VrADH Alternatively, the overexpression engineered bacteria described in claim 8 may be transferred into mung bean plants.

9. A method for screening mung bean yellow mosaic virus resistant plants, characterized in that, The mung bean alcohol dehydrogenase encoding gene described in claim 3 is used. VrADH After PCR amplification of mung bean plant samples using specific primers, sequencing is performed. If the sequencing result is as shown in SEQ ID NO.1, then the plant is a resistant mung bean yellow mosaic virus.

10. Gene encoding mung bean alcohol dehydrogenase VrADH Or the overexpression vector pFGC5941 as described in claim 6. VrADH Or the application of the overexpressed engineered bacteria as described in claim 7 in the preparation of a reagent for cultivating plants resistant to mung bean yellow mosaic disease.

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