Cloning And Functional Verification Of RING Type Ubiquitin Ligase PnLOG2 Gene In Pharbitis Nil(L.)Choisy

In the process of growth and development,plants will be subjected to biological stress and abiotic stress which will adversely affect them drought,salt,alkali and other abiotic stresses.And these stresses can cause extremely complex molecular reactions in plants.The ubiquitin/26 S proteasome pathway(UPP)is an efficient and highly selective protein degradation pathway in cells.RING type E3 ubiquitin ligase is the most abundant enzyme in UPP,and it plays a key role in the process of specific selective degradation of substrates.Studies have shown that the RING type E3 ubiquitin ligase genes involved in the response to different abiotic stresses.In this study,the RING type E3 ubiquitin ligase gene PnLOG2 was cloned from the Ipomoea purpurea Lam.The expression pattern and function of this gene were preliminarily investigated in order to provide theoretical basis for elucidating the mechanism of RING type E3 ubiquitin ligase participating in plant abiotic stresses.The results of this study are as follows:(1)RING type E3 ubiquitin ligase gene PnLOG2 was homologous cloned from Ipomoea purpurea Lam.Its full length is 1022 bp.Through bioinformatics analysis: An open reading frame with a length of 941 bp(51-992 bp)was predicted.It encodes a total of 313 amino acid residues.It is a soluble non-transmembrane protein.There are no signal peptides and guide peptides.It is the closest relative to the tomato.The conserved zf-C3HC4 RING-Ubox superfamily domain was found in the sequences with high homology.The analysis of the components of cis-acting promoter showed that it might be involved in abiotic stress.(2)Tissue specificity and expression pattern of PnLOG2 gene were analysesed in roots and leaves under different stress treatments by qRT-PCR.The results showed that there displayed different expression in tissues.PnLOG2 gene expression was significantly upregulated after drought,salt,alkali and ABA stress.After drought,salt and alkali treatment,its expression was significantly upregulated in leaves.Gene expression in root was up-regulated earlier than that in leaf.(3)PYES2-PnLOG2 expression vector was constructed and introduced intoINVSC1 competent cells of s.cerevisiae.After drought,salt and alkali stresses,the phenotypes of control yeast and recombinant yeast were observed and their survival rates were determined.The results showed that the overexpression of PnLOG2 gene significantly improved the ability of recombinant yeast to withstand drought and salt stresses,but decreased the ability of recombinant yeast to withstand alkali stress(4)The expression vector of PCAMBIA-2300-PnLOG2 was constructed.When introduced into agrobacterium and infected with arabidopsis thaliana.When introduced into agrobacterium and infected with arabidopsis thaliana,the seeds and seedlings of two transgenic lines and WT were treatment under drought,salt and alkali stresses.The germination rate and survival rate of all lines in seedling stage were calculated and the fresh weight,chlorophyll content and MDA content were measured.The results showed that overexpression of PnLOG2 gene could improve the germination rate,survival rate,fresh weight and chlorophyll content of arabidopsis thaliana under drought,salt and alkali stresses,and reduce the content of MDA.ABA stress was used to treat the seeds of two transgenic lines and WT,and the germination rate was calculated after seedling formation,and the expressions of ABA related marker genes NCED,RD20,RD29 B and RAB18 were measured.The results showed that the germination rate of transgenic lines was significantly lower than that of WT,and the expression of transgenic lines was significantly different from that of WT.The above results indicated that the overexpression of PnLOG2 gene could improve the ability of arabidopsis thaliana to withstand drought and salinity,and the effect of overexpression was related to ABA signal transduction pathway.