Chinese

With the development of plant disease-resistant genetic engineering, more and more disease-resistant genes have been found. In 2004, Taler et al. cloned two melon downy mildew resistance genes At 1 and At2 with serine glyoxylate aminotransferase (SGT) activity from Indian wild melon varieties. Taler et al. found that At 1 and At2 have serine glyoxylate transaminase activity related to plant photorespiration pathway, and do not belong to any known R gene, and their resistance to pathogens is not species-specific, and their resistance is related to H_2O_2. Based on this phenomenon, they put forward a new disease resistance mechanism "enzyme disease resistance", which is the first report to give plants disease resistance by changing the expression of enzymes. It is speculated that At 1 and At2 may also be resistant to many other plant leaf diseases except downy mildew, and they are disease-resistant genes with great research value. Moreover, the mechanism of "enzyme disease resistance" needs more genetic evidence and experimental support. Based on this, At 1 and At2 homologous genes GmSGT were cloned from soybean with strong photorespiration, and their sequence analysis, enzyme activity center prediction and prokaryotic expression analysis were carried out, and they were transferred to recipient plants for disease resistance identification. The main research results and innovations of this paper are summarized as follows: 1. The gene sequence of GmGGT 1 encoding serine glyoxylate transaminase was cloned from soybean varieties resistant to downy mildew for the first time by using soybean EST sequence and 5'-Race technology (obtained 1 national invention patent, patent number: ZL2005 100887 83.4). At the same time, two homologous genes GMS GT 2 and GMS GT 3 of GmSGT 1 were cloned from the susceptible variety Heinong 10 induced by SA. Sequence analysis showed that GmSGT 1 had 88.03% homology with At 1 and At2, respectively, and 83.33%-85.79% homology with serine glyoxylate transaminase of Arabidopsis thaliana, rice, Fritillaria and Lemna Multiflora. The sequence analysis of protein deduced from GmSGT 1 showed that it had a pyridoxal -5- phosphate binding site GSQKAL and a strong peroxisome localization signal SRI, which indicated that the gene might play a role in plant peroxisome through photorespiration. The nucleotide sequence homology of GmSGT2 and GmSGT3 with GmSGT 1 is as high as 96.38% and 99. 17%, and the deduced amino acid sequence homology is 97.03% and 99.25%. The enzyme activity centers of GMS GT 1, GMS GT 2 and GMS GT 3 were analyzed by bioinformatics method. The results showed that all three protein sequences had serine glyoxylate transaminase activity. 2. By simulating the plant photorespiration pathway in E.coli, GmSGT 1 was first found to have serine glyoxylate transaminase activity. Reaction in photorespiration pathway: glycolic acid (? H2O 2+ glyoxylic acid; Glyoxylic acid (? ) glyoxylic acid. Because Escherichia coli contains glycolate oxidase (GOX), the above reaction can be completed by adding glycolate to the prokaryotic expression strain. The serine glyoxylate transaminase function of GmSGT 1 gene can be determined by detecting the change of H2O 2 content. The results showed that only the fraction expressing GmSGT 1 protein could induce a large amount of H2O 2, but the control group could not. Therefore, it can be confirmed that GmSGT 1 has serine glyoxylate transaminase activity. 3. The correlation between GmSGT protein and resistance of different soybean varieties was analyzed. The results of Western hybridization showed that the expression of target protein could be detected in downy mildew resistant varieties Zaofeng 5 and Jiunong 9, but not in susceptible varieties Heinong 10. Semi-quantitative RT-PCR results before and after SA induction showed that the expression of susceptible variety Heinong 10 was not detected before SA induction, but it was slightly expressed after SA induction, and the resistance of soybean to freezing injury was greatly improved. Therefore, we infer that the expression of GmSGH is indeed related to SA-induced pathway, and with the increase of GmSGT 1 expression, the resistance of soybean to downy mildew is also obviously improved. 4. Plant expression vector GmSGT 1 was constructed, transgenic plants were obtained by tobacco transformation, and their resistance to brown spot, black shank and bacterial wilt were identified. The results showed that transgenic tobacco significantly improved the resistance of tobacco to brown spot, black shank and bacterial wilt. This study provides new experimental evidence for the plant "enzyme resistance gene" proposed by Taler et al. The temporal and spatial expression characteristics of transgenic soybean GmSGT 1 gene were studied with disease-resistant variety Zaofeng 5 as the material. The gene was expressed in soybean leaves, but not in roots and stems. The expression level increased with the increase of growth period, and it was the strongest in reproductive growth period, and then weakened until it disappeared with the aging of cells. This shows that the change trend of GmSGT 1 is consistent with the change trend of plant photorespiration. At the same time, the expression characteristics of glycolate oxidase (GOX) upstream of serine glyoxylate transaminase in photorespiration pathway were detected. The results showed that the expression level of GOX was consistent with that of GmSGT 1, indicating that the upstream reaction of GmSGT 1 and H2O 2 expression were enhanced at the same time. This is consistent with our conclusion that GmSGT protein plays a role in plant photorespiration pathway. 6. The genetic transformation system of soybean was determined. Tissue culture systems of soybean Zaofeng No.5 and Heinong 10 were established, and the constructed plant expression vectors PIM1.1-GMSGT-plus were transformed into Zaofeng No.5 and RNAi plant expression vectors PIM1.1-GMSGT-plusf by Agrobacterium-mediated method. At the same time, the effects of regeneration frequency, regeneration time, K screening concentration and different infection time of Agrobacterium on the regeneration frequency of regenerated buds were studied, which provided experimental basis for further optimizing the tissue culture and transformation system of Zaofeng 5.

English

With the development of plant disease-resistant genes, more and more disease-resistant genes have been found in human body. In 2004, Taler and other wild melon varieties from India had cloned two genes, At 1 and At2, which were active in serine glyoxylate aminotransferase (SGT). Taler At 1 and At2 are genes with serine glyoxylate aminotransferase activity and plant photorespiration pathway, which do not belong to any known R gene and have no special resistance to pathogens. Their disease resistance is related to H2O 2. Based on this phenomenon, they put forward a new disease resistance mechanism "enzyme resistance", which is the first report to give plants disease resistance by changing the expression of enzymes. It is speculated that At 1 and At2 may have stronger resistance to als o downy mildew than many other plant leaf diseases, indicating that the resistance gene has great research value, and the mechanism of "resistance enzyme" needs more genes to support and test. Accordingly, the homologous gene GmSGT was cloned from soybean photorespiration clones At 1 and At2, and its sequence analysis, enzyme activity site prediction and prokaryotic expression analysis were carried out, and it was transferred into recipient plants for disease resistance identification. The main findings and innovations of this paper are summarized as follows: 1. Sequence gmg gt 1 (national invention patent: ZL2005 100887 83.4) encoding serine glyoxylate transaminase was cloned for the first time from soybean varieties resistant to downy mildew by using 5'-Race technology. From two soybean varieties susceptible to downy mildew induced by SA, 10 was black. Sequence analysis showed that GmSGT 1 had 88.03% and 87.78% amino acid sequence homology with At 1 and At2 reported by other melon downy mildew resistance genes. At the same time, GmSGT 1 has 83.33%-85.79% homology with serine glyoxylate aminotransferase from Arabidopsis thaliana L., Oryzae sativa L., Fritillaria, and duckweed. Protein sequence analysis derived from GMS gt 1 showed that it had a pyridoxal-5- phosphate binding site GSQKAL and a strong peroxisome targeting signal SRI, indicating that the homology of GmSGT2 and GmSGT3 with GmSGT 1 nucleotide sequence was as high as 96.38% and 99. 17% in plant peroxisome photosynthesis. The enzyme active sites of GmSGT 1, GmSGT2 and GmSGT3 were analyzed by bioinformatics method. The results showed that all three protein sequences had serine glyoxylate transaminase activity. Photorespiratory pathway reaction occurs: glycolic acid (? ) H2O 2+ glyoxylic acid; Glyoxylic acid (? ) Gan helium acid. Because Escherichia coli contains glycolate oxidase (GOX), the original strain is expressed by adding acid, which is completed by the above reaction. The function of GmSGT 1 serine glyoxylate transaminase gene can be determined by detecting the change of H2O 2 amount. The results showed that only the expression of GmSGT 1 protein component could induce the production of H_2O_2 in large quantities, but the control group could not. Therefore, serine can be used to determine the activity of GmSGT 1 glyoxylate transaminase. The results of Western blotting showed that the downy mildew resistant varieties Zao 5 and Zao 9 Nong 9 could be detected with targeted expression, while Heinong 10 was infected and had no expression. Semi-quantitative RT-PCR results before and after SA induction showed that the expression of Heinong 10 was not detected before SA induction, but the resistance of soybean expressed by SA induction to butterzyme was greatly improved. Therefore, we concluded that the induced expression of GmSGT 1 was true and relevant, and with the increase of GmSGT 1 expression, the resistance of soybean to downy mildew was also significantly improved. 4. The GMS gt 1 plant expression vector was constructed for tobacco transformation, and transgenic plants were obtained, and the resistance of tobacco to brown spot disease, black shank disease and bacterial wilt was identified. The results showed that transgenic tobacco significantly improved the resistance of tobacco to brown spot disease, black shank disease and bacterial wilt. Taller, etc. This study puts forward the "enzyme resistance gene" in plants, which provides new experimental evidence. 5. The spatio-temporal characteristics of GmSGT 1 abundant gene expression in soybean were explored by using the first five disease-resistant varieties. The gene was expressed in soybean leaves, but not in roots and stems, and the expression increased with the growth period, most of them were expressed in reproductive stage, and then decreased with cell aging until it disappeared. This shows the trend line of plant GmSGT 1 photorespiration. At the same time, the expression characteristics of glycolate oxidase (GOX) under the action of serine glyoxylate transaminase in the upstream photorespiration pathway were detected. The results showed that the expression level of GOX GmSGT 1 changed consistently, indicating that the expression of GmSGT 1 was enhanced, and the upstream reaction was also enhanced, and the expression of H_2O_2 was also increased. We speculate that GmSGT, a protein of plant photorespiration pathway, plays a role in the conclusion. 6. The genetic transformation system was studied and identified. Hsbc established an embryo tip tissue culture system on 5th, soybean and Heinong 10th, and transformed the excellent member PIM1.1-GMS gt-plus into the early 5th, RNAi plant expression vector PIM1./kloc-with the plant expression vector mediated by Agrobacterium tumefaciens LBA4404. At the same time, the regeneration frequency, regeneration time, K screening concentration and Agrobacterium infection time of three explants of HSBC 5 were studied, which provided experimental basis for further optimization of tissue culture and transformation system of HSBC 5.