Clonging And Functional Preliminary Study Of CsWRKY57 And CsWRKY40 Transcription Factor Intea Plant(camellia Sinensis)

Tea plant [Camellia sinensis(L.)O.Kuntze] is an important perennial,woody crop,which is widely planted in the south of China,and is one of the important economic crops.Tea is a kind of natural beverage,rich in tea polyphenols,caffeine,lipopolysaccharide,etc.,with anti-cancer,anti-cancer effect,therefore,by the people enjoy of the world.However,the tea plant is subject to various organisms(such as bacteria,fungi)and abiotic(such as drought,high temperature and other factors)stress,seriously affect the output and quality of tea in the growth process,which causing serious economic losses.Therefore,it is of great significance to study the regulation mechanism of tea plant under stress and the functional analysis of the stress resistance gene.The climate is dry and soil salinization is more serious in Shanxi province,the tea cultivars ‘Shachayihao' as the experimental materials,with leaf cDNA as template,cloned from tea plant WRKY57 and WRKY40 two genes,and the sequence of the two genes by bioinformatic analysis,subcellular localization of encoding protein,polyethylene glycol 6000(PEG 6000),sodium chloride(NaCl)and abscisic acid(ABA)treatment,expression pattern analysis and validation of gene transcription activity.The main results are as follows:1.The WRKY transcription factor CsWRKY57 and CsWRKY40 were cloned from tea plant(Camellia sinensis)cultivars ‘Shanchayihao' by homologous cloning technology using cDNA template.Bioinformatics analysis showed that CsWRKY57 was contained a 912 bp Open Reading Frame(ORF),encoding 303 amino acids,the molecular weight of Cs WRKY57 was 33.5 kD and theoretical isoelectric point was 5.49.The BLAST results showed that CsWRKY57 contained one typical WRKY domains and one zinc finger motifs Cx4Cx23 HxH,suggesting that it was member of the WRKY group IIc family.CsWRKY40 was contained a 963 bp Open Reading Frame(ORF),encoding 320 amino acids,the molecular weight of CsWRKY40 was 35.06 kD and theoretical isoelectric point was 8.80.The BLAST results showed that CsWRKY40 was contained one typical WRKY domains and one zinc finger motifs Cx5Cx23 HxH,suggesting that it was member of the WRKY group IIa family.2.Quantitative real-time PCR(q RT-PCR)analysis results showed that the expression of Cs WRKY57 and CsWRKY40 were induced by salt,drought and ABA stresses,and showed a trend of increasing first and then decreasing,implying that Cs WRKY57 and CsWRKY40 involved in the process of tea plant respondes to salt,drought and ABA stresses.3.To investigate the transcriptional activation activity,yeast expression vectors of constructed by ligating the cloned cDNA of CsWRKY57 and CsWRKY40 to vector pGBKT7.pGBKT7 was negative control and pCL1 was positive control.Transcriptional activation activity assays indicated that Cs WRKY57 and Cs WRKY40didn't have transcriptional activation activity.