Clone And Its Prokaryotic Expression Analysis Of Melatonin Synthesis Key Enzyme VvSNAT Gene In Vitis

Grape is one of the most widely grown tree species in the world.According to the statistics of the International Organisation of Vine and Wine(OIV),the global grape growing area in 2016 has reached 7.5 million hectares.In China’s agricultural planting structure,the grape cultivation and processing industry have an important role.However,in the Northwest of China,where water resources are scarce,drought is an important factor limiting the grapes growth and increasing yields.Previous studies have confirmed that exogenous melatonin can significant-ly increase the drought resistance of grape seedlings.Therefore,melatonin may play an important role to resist grape drought.However,whether endogenous melatonin participates in grapes drought-responsive process,the key enzymes regulating the synthesis of melatonin and its biological functions have not yet been reported.Melatonin is a tryptophan derivative widely found in organisms.It plays an important role not only in plant growth,but also in response to stress.Based on this,in this experiment,we study the effects of drought stress on melatonin synthesis and related enzyme gene expression levels,Using wine grape Cabernet Sauvignon as material.Then,screening out the key enzyme genes that regulate melatonin synthesis during grape drought response,and analysis its biological functions.the following is research results:1.Using LC-MS analyze the synthetic changes of melatonin and its precursors in grapes treated with 15% PEG simulated drought.The results showed that L-tryptophan,tryptamine,serotonin,N-acetyl-5-hydroxytryptamine,and 5-methoxyhydroxytryptamine tend to from rise to decrease,while melatonin rises significantly with increasing stress time.of.This indicates that drought stress induces the synthesis of melatonin in grape seedlings and thus resists the stress environment of plants.2.Using Real-time q PCR analyze the expression of melatonin synthesis-related genes in grapes under simulated drought stress.The results showed that the genes(VvASMT1,VvT5H1,VvM3 H,VvSNAT,VvT5H2,VvTDC1)were all expressed in roots,stems and leaves.The expression levels of all genes in all stages were stem>root>leaf.and the expression of four genes VvM3 H,VvSNAT,VvT5H2,VvTDC1 showed the same trend.Therefore,drought stress by PEG-treated has a significant effect on the expression of genes in the process of synthetic melatonin,and the appropriate concentration and time will increase the amount of gene expression.3.By analyzing the correlation analysis between melatonin and its precursors and synthesis-related genes in different tissues of grape under drought stress.The results showed that VvSNAT,VvASMT,VvT5H1 significantly correlated with melatonin content.4.We cloned the key gene VvSNAT of melatonin synthesis,from Cabernet Sauvignon grapes and analysed bioinformatics.The closest relatives to the grape VvSNAT protein are jujube,followed by lotus and walnut,and the most distantly related is the Hevea brasiliensis.The grape SNAT protein has a conserved domain of the NAT＿SF family,the VvSNAT protein is located in the chloroplast,It is water-soluble non-secretory protein and no transmembrane structure.5.According to the obtained VvSNAT CDS region and the prokaryotic expre-ssion vector p ET28 a,primers were designed to carry out double digestion of the Bam HI and Sal I sites on the vector.The homologous recombination method was used to connect the target fragment with the digested vector and transform it into E.coli.Double enzyme digestion was performed in DH5α and sequenced.The correctly identified plasmids were transformed into the E.coli Rossete(DE3)for induction and expression,and detected by SDS-PAGE.By enzymatic activity experiments confirmed that the VvSNAT protein can catalyze the production of N-acetyl-5-hydroxy tryptamine by serotonin.