Functional Analysis Of The CsS40 Transcription Factor In Tea Plan

In this study,the yeast single hybrid point-to-point experiment was used to verify the binding of CsS40 to the promoter of caffeine synthase;an overexpression vector of the gene was constructed,and the physiological indicators of the transgenic tobacco were determined;Caffeine content and expression of different aging degrees of tea leaves had been analysised.The main results are as follows:1.The biological information of the CsS40 gene was clarified: CsS40 gene is1211 bp in length and consists of 212 amino acids.CsS40 gene constitutes a Senescnece＿reg domain from amino acids 289 to 825,which belongs to the S40 family of cellular senescence regulators;the relative molecule of CsS40 gene The mass and isoelectric point are 23369.73 DA and 5.98;the CsS40 gene is located in the nucleus;the promoter sequence of the CsS40 gene contains an ARE element(anaerobic induction regulation)and a circadian element(circadian rhythm regulation)related to the environment;2 ABRE elements related to hormones(abscisic acid response),1 GARE-motif element(involved in gibberellin response),1 TGA-element element(involved in auxin response)1 AE-box related to light regulation element,1G-box element,1 Gap-box element,1 I-box element and 1 TCT-motif element.2.CsS40 binding to TEA022575(TCS4)promoter was identified: Through the yeast single hybrid point-to-point experiment,the successfully constructed vector p GADT7-CsS40 and the promoter linked to the vector p HIS2 were co-transformed into yeast strain Y187,and then plated on the three-deficiency medium SD--Trp/-Leu/-His+3-AT.The results showed that yeast could continue to grow on the three-deficient medium with different gradients of 3-AT,indicating that the transcription factor CsS40 has a binding effect on the TEA022575(TCS4)gene.3.The subcellular localization of CsS40 was clarified: construct subcellular localization PCAMBIA1300-CsS40-35S-GFP fusion expression vector,and transfer the constructed plant fusion expression vector PCAMBIA1300-CsS40-35S-GFP and empty vector PCAMBIA1300-35S-GFP into agricultural Bacillus GV3101 for transient expression in tobacco.The results showed that CsS40-GFP was expressed in the nucleus of tobacco leaf cells,indicating that CsS40 was localized in the nucleus.4.Clarified the function of CsS40 in transgenic tobacco: The tea plant CsS40 overexpression vector p SH-35S-CsS40 was constructed,and tobacco leaves were genetically transformed by Agrobacterium-mediated leaf disc transformation,and 20 transgenic tobacco strains were obtained by PCR identification Tie.The xanthine content of transgenic plants was between 0.32 and 1.85 mg/g,and that of wild-type plants was 1.10 mg/g;the hypoxanthine content of transgenic plants was between 0.05 and 0.23 mg/g,and that of wild-type plants was 0.12 mg/g.5.The correlation between CsS40 expression and caffeine content was clarified:Through the correlation analysis between CsS40 expression and caffeine content in tea leaves,the results showed that the caffeine content of young leaves,mature leaves and old leaves were 1.55%,1.13% and 0.30%,respectively.The relative expression levels of leaves,mature leaves,and old leaves were 23.82,49.35,and 124.13,indicating that the higher the expression level of CsS40 gene,the more senescent tea leaves and the lower the caffeine content,showing a negative correlation.Through the correlation analysis between the expression of CsS40 in tea leaves and the content of caffeine,the results showed that the content of caffeine in young,mature land old leaves were 1.55%,1.13% and 0.30%,respectively.The expression levels were 23.82,49.35,and 124.13,indicating that the higher the expression level of CsS40 gene,the more senescent tea leaves and the lower the caffeine content,showing a negatively correlation.6.Clarified the function of CsS40 in caffeine synthesis in tea plant callus: The CsS40 overexpression vector was genetically transformed into tea plant stem segments by Agrobacterium-mediated leaf disc transformation to form callus,which were identified by GUS histochemical staining and PCR.Obtained an overexpressed transgenic callus.The results of caffeine content determination showed that the caffeine content of wild-type callus was 2.17%,respectively,and the caffeine content of transgenic callus was 4.09%.It shows that CsS40 gene can increase the caffeine content of tea plant.