Cloning And Functional Analysis Of AhWRI1 Transcrition Factors From Peanut

With the increasing demand for plant oils in the world,improving the fatty acid composition and oil content of peanut seeds has become the top priority of peanut breeding.Transcription factors can promote the over-expression of a series of genes in related metabolic pathways,which can greatly improve the content of lipids.In this study,two transcription factors,AhWRI1-1 and AhWRI1-2,were cloned from the leaves of peanut variety Huayu 33,and the genes and their encoded proteins were analyzed by bioinformatics.The qRT-PCR method were used to analyze the expression of two genes in different tissues,different stages of seed development,under stress and hormone treatment.The Arabidopsis protoplast transient expression technology was used to identify the subcellular localization of encoded protein.The target proteins were obtained through prokaryotic expression system.Transgenic Arabidopsis and tobacco were obtained by Agrobacterium-mediated genetic transformation.The main research is as follows:1.AhWRI1-1 and AhWRI1-2 genes were cloned from leaves of peanut variety Huayu 33.The ORF of AhWRI1-1 gene is 1101bp,encoding 366 amino acids.The ORF of AhWRI1-2 gene is 1128bp,which encodes 375 amino acids.Bioinformatics analysis showed that the encoded proteins by the two WRI1 genes were similar to those of Arabidopsis,Brassica napus and soybean,and the sequence similarity was all more than 60%.Both of these two proteins contain two AP2/EREBP domains.Among them,the two AP2 domains of AhWRI1-1 protein are located at 65¹37 and 167²31 sites,and AP2 domains of AhWRI1-2 protein are located at 65¹37 and 167²31 sites.Therefore,we speculate that these two genes may belong to the AP2 transcription factor family,and have the same function as them.2.The qRT-PCR method were used to analyze the expression of AhWRI1-1 and AhWRI1-2 in different tissues,different stages of seed development,under abiotic stress?high salt,cold and drought?and hormone treatment.We found that AhWRI1-1had the highest expression level in seeds,and the highest expression of AhWRI1-2 was in hypocotyls.AhWRI1-1 gene responded to three kinds of abiotic stresses.Its expression level was significantly up-regulated under high salt and cold stress,and was significantly down-regulated under drought stress.The expression of AhWRI1-2 gene was up-regulated under high salt,cold and drought stress.These results indicate that the two genes may be involved in the resistance regulation of peanut to high salt,cold and drought stress.Moreover,AhWRI1-2 is a positive regulatory factor in ABA signal regulation pathway,and is induced by ABA signal.3.The recombinant expression vector of pGBKT7-AhWRI1-1 and pGBKT7-AhWRI1-2 were transferred into AH109 yeast strain by Yeast One-hybrid technique.The results showed that it could grow in the nutrient-deficient culture medium?SD/-Trp/-His?and was changed to blue in the?-galactosidase assay.These indicated that AhWRI1-1 and AhWRI1-2 had transcriptional activator activity,which belonged to the transcriptional activator.4.The fused expression vector?pAN580-GFP-AhWRI1-1,pAN580-GFP-AhWRI1-2,pAN580-GFP?were transformed into the Arabidopsis protoplasts.The expression of GFP in nucleus was observed by laser scanning confocal microscope.The results showed that both AhWRI1-1 and AhWRI1-2 proteins were located in nucleus.TheprokaryoticexpressionvectorsofpET-28b-AhWRI1-1and pET-28b-AhWRI1-2 were constructed,and the optimal expression condition of fusion proteins were detected.It was as follows:Rosetta strain,0.5 mM IPTG,220rpm,37?,8 h.The expression of fusion proteins have been successfully induced under this condition.5.We constructed plant expression vectors pCAMBIA1300-AhWRI1-1 and pCAMBIA1300-AhWRI1-2,and transferred them into Agrobacterium EHA105 strain.Six transgenic AhWRI1-2 Arabidopsis lines were obtained by inflorescence transformation,and six transgenic AhWRI1-2 tobacco lines were obtained by plant tissue culture.The oil content of T₄ seed of Arabidopsis was analyzed.The results showed that the seed oil content of Arabidopsis was increased by 94.90%compared with the wild control,indicating that AhWRI1-2 could promote the improvement of seed oil content in transgenic Arabidopsis.