Cloning, Sequence Analysis Of The Serine Glyoxylate Aminotransferase Gene Of Soybean, And Research On The Resistance Of Transgenic Plants

With the development of genetic engenier of plant resistance, more and more resistant genes were descovered. Taler et al. (2004) have cloned two genes At1 and At2 encoding peroxisomal serine glyoxylate aminotransferase (SGT) from the PI line of wild melon (Cucumis melo) resistant to downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis. The study has provided solid evidence that the expression of SGT gene confers protection against disease, by which they proposed a new type of defense mechanism called "enzymatic disease resistance (eR)", since the amino acid identity analysis revealed that these gene products differ from all the previously known R-proteins. eR is valuable and it is worth to be coloned from other plants to gain more evidence. So we coloned the eR gene from soybean, sequences analysis, expressed in E. coli, and the resistance identify of transgenec plant. The result and originality are shown as below:1. A GmSGT gene encoding serine glyoxylate aminotransferase has been cloned from a soybean variety resistant to downy mildew disease by using a short salicylic acid (SA)-inducted soybean EST and the 5'-RACE method. The full-length of GmSGT gene is 1206 bp encoding a serine glyoxylate aminotransferase. The ORF and deduced 402 amino acids and the 5'and 3'UTR sequences can be found in the GenBank (accession No. DQ167250). Notably, the ORF possesses two typical motifs for SGT that is a GSQKAL binding site (at position 198-203) for the pyridoxal-5-phosphate cofactor and a SRI peroxisomal targeting site at the 3'end, suggesting that the enzyme occurs in plant peroxisome and participates in the production of glycine during photorespiration. Sequence analysis has shown that the GmSGT does not belong to any known R genes. Comparison of nucleotide sequences between GmSGT and At1, At2 from melon (Taler et al., 2004) showed 80.76%and 78.77% homology, respectively, and the similarity of deduced amino acid sequence was as high as 88.30% and 88.28%. Comparison of GmSGT amino acid sequence with other known SGT or AGT proteins from Cucumis melo, Fritillaria agrestis, Spirodela polyrrhiza, Arabidopsis thaliana, Vitis vinifera and Oryza sativa showed 82.7-88.3% similarity. Interestingly, phylogenic analysis showed that the GmSGT and the melon CmAT1 were clustered in the same group, while the other SGTs or AGTs were formed in different groups.2. As SGT can catalyze L-serine and glyoxylate into glycine, the GmSGT expressed in E. coli showed enzymatic activity which produced 11.9 to 23 times of more glycine than that of the control, the empty vector without GmSGT gene. Meanwhile, the expression of GmSGT gene in E. coli dramatically increased H₂O₂ content and inhibited bacterial growth.3. Western blot analysis showed that the GmSGT protein was only produced in the downy mildew-resistant variety Zaofeng5 and Jiunong9, while not in the susceptible variety Heinong10. Transcriptional RT-PCR analysis showed that the GmSGT could be detected in the resistant variety (Zaofeng5) prior to SA treatment and it was increased six times more after SA induction. However, for the susceptible variety, the GmSGT could not be detected prior to or was only slightly increased after SA induction. Results indicated that the function of GmSGT gene was closely correlated with the soybean downy mildew resistance, which provided new evidence in supporting the enzymatic disease resistance (eR) proposed by Taler et al. in 2004.4. Set up the expression carrier of GmSGT and transfored it into tobacco. The resistance of the transgenic plants to Alternaria alternata, Phytophthora parsitica, Pseudomonas solanacearum Smithis notability increased.5. RT-PCR analysis of differrent parts during differrent periord showed that the GmSGT could be detected in the leaves but not in the root and stem. It also show that the expression is increased with the growth of soybean and the tendency is identical with photorespiration.6. Established the embryonic tip regeneration system of Zaofeng5 and Heinong10 and compaired the regeneration frequency of the embryonic tip, cotyledonary node and hypocotyl segment.Transformed the expression carrier of GmSGT and the RNAi carrier of GmSGT into Heinong10 and Zaofeng5 seperetly using Agrobacterium tumefaciens-medisted transformation and got the regeneration plant.