Identification Of Drought Resistance Function Of Aspartate Transaminase Gene VfAAT From Vicia Faba L.

Fava beans(Vicia faba L.)is the third most important winter edible bean crop in the world.It is one of the crops widely cultivated in the world.It has important nutritional value and commercial value because of its rich nutrition.Fava beans was susceptible to drought stress at germination,growth and maturity stages,which made fava beans show obvious abnormal morphological characteristics and greatly reduced its yield.Identification of drought tolerance genes in fava beans is beneficial to breeding droughtresistant varieties through marker-assisted breeding and genetic engineering.In previous studies,we identified a class of aspartate aminotransferase genes(ASPAT)responding to drought stress in fava beans by transcriptome sequencing.This study identified the members of ASPAT gene family from the whole genome level,explored its expression mode,further verified its drought-tolerance function and explored the drought-tolerance mechanism.The main research results are as follows:1.Analysis of ASPAT gene families and their expression patterns: Eight ASPAT gene were identified from the transcriptomes,which includes three eukaryotype ASPAT(AAT)genes and five prokarytoype ASPAT(PAT)genes with mitochondria and chloroplast location,respectively.Phylogenetic analysis showed that ASPAT could be divided into two subfamilies Iα and Iβ.Iα is AAT protein including Vf ASPAT1-Vf ASPAT3,and Iβ is PAT protein consisting of Vf ASPAT4-Vf ASPAT8.The ASPAT family members of Vicia faba contain motif 1,motif 3 and motif 9,and had similar gene structures and common protein domain aminotran-1-2.Vf ASPATs protein interaction network suggested that the members may be involved in stress metabolism.The analysis of transcriptome and fluorescence quantitative PCR showed that Vf ASPAT gene might have a positive regulatory effect on drought stress,and Vf ASPAT3(DN8046＿c0＿g1)participated in the16 h and 64 h drought regulatory response.2.Based on the known Vf ASPAT3 CDS gene sequence,corresponding primers were designed to amplify the sequence,the PVX-LIC-Vf ASPAT3 overexpression vector was constructed by LIC method and injected into tobacco leaves,and Vf ASPAT3 was overexpressed in tobacco by viral vector replication.The physiological indexes and amino acid contents of control plants,empty vector plants and overexpressed Vf ASPAT3 plants under drought stress showed that overexpression of Vf ASPAT3 in Nicotiana benthamiana can reduce MDA content and water loss,improve enzyme activities of AAT and POD,and increase cysteine and isolucine contents,so as to enhance drought resistance of tobacco.Based on the analysis of physiological and amino acid changes of over-expressed Vf ASPAT3 Nicotiana benthamiana,a physiological mechanism model for enhancing drought tolerance through osmotic stress,antioxidant stress and amino acid synthesis regulation was proposed.3.Vf ASPAT3 gene was cloned,overexpression vector was constructed,Vf ASPAT3 gene was transferred into Nicotiana benthamiana by Agrobacterium transformation method to obtain transgenic strains.T1 transgenic Nicotiana benthamiana seeds were screened,and the stress resistance of T1 transgenic Nicotiana benthamiana lines with Vf ASPAT3 gene was identified under drought conditions.The results showed that the drought resistance of transgenic Nicotiana benthamiana lines was significantly stronger than that of wild-type Nicotiana benthamiana plants.Specifically,under the treatment of200 m M and 300 m M mannitol,the germination rate of transgenic Nicotiana benthamiana seeds was higher than that of WT,indicating that the overexpression of Vf ASPAT3 promoted the germination of transgenic tobacco Nicotiana benthamiana seeds.The phenotype of Nicotiana benthamiana plants grown for 40 days was observed after15 days of drought treatment and 3 days of rehydration.It was found that transgenic tobacco lines grew normally,with leaves filled with water,while WT leaves wilted.The physiological index analysis at 0 and 15 days after drought treatment showed that PRO content,POD and SOD activity were higher and MDA content was lower in transgenic Nicotiana benthamiana plants.These results indicated that overexpression of Vf ASPAT3 could eliminate reactive oxygen species and accumulate osmotic regulatory substances to maintain normal life activities by enhancing antioxidant enzyme activity,and improve drought resistance of transgenic Nicotiana benthamiana.The results of this study preliminarily revealed the function and mechanism of Vf ASPAT3 in drought resistance of fava beans,laying a foundation for further utilizing this gene to improve drought resistance of fava beans.