Cloning,Functional Analysis And Isolation Of Transcription Factors Of N-Methyltransferase Gene Promoter In Tea Plants(Camellia Sinensis)

Tea is one of the three major non-alcoholic drinks in the world,and rich in tea polyphenols,alkaloids,tea polysaccharides,theanine and other active substances.As an important alkaloid in tea,caffeine has been paid more and more attention because of its exciting nerve,diuretic and other medical effects.N-methyltransferase is a key enzyme involved in caffeine biosynthesis.Cloning the promoter of NMT gene and studying its function,and analyzing the transcriptional regulatory factors interacting with it,have positive significance for further revealing the regulation of tea caffeine metabolism.In this study,important functional gene yhNMT1 cloned from Camellia sinensis Yinghong 9 was selected,and the promoter of yhNMT1 gene was cloned by hiTAIL-PCR.The cis-acting elements were analyzed by bioinformatics.The functional characteristics of the promoter and the effect of environmental stress factors on its function were studied by transient expression and stable expression in transgenic tobacco.The transcription factors interacting with the promoter of yhNMT1 gene were cloned by screening yeast one-hybrid library,and the transcription factor genes were subjected to subcell localization analysis.The main results obtained were as follows:?1?Cloning and bioinformatics analysis of yhNMT1 gene promoterThe cloned yhNMT1 gene promoter was 767bp,which was named P_NMT1,The sequence was analyzed by online software such as Plant CARE and PLACE,The predicted transcription initiation site?TSS?was located at the 63 bp base upstream of the translation initiation site.P_NMT1MT1 contained basic cis-acting elements of eukaryote promoter such as TATA-box,CAAT-box and a number of response elements related to abiotic stress in plants,for example:ABRE element for abscisic acid response,ARE element for the anaerobic induction,CGTCA-motif element involved in the MeJA-responsiveness,HSE element involved in heat stress response,ERE element for ethylene response,TCA element involved in salicylic acid response,AE-box element for light response.?2?Transient expression analysis of yhNMT1 gene promoter in tobacco leavesAccording to cis-elements composition,the CaMV35S promoter in pBI121 was replaced by the 5'shorten yhNMT1 promoters,resulted in 4 new vectors fusion with GUS and marked as pA,pB,pC and pD.Transient expression of GUS gene in tobacco leaves was analyzed.The results of GUS histochemical staining on leaves showed that the staining of the leaves transformed by four vectors deepened with the increase of the length of the inserted promoter in vectors,which revealed that yhNMT1promoters with different lengths all could regulate the expression of GUS,and the activity of GUS enhanced along with promoter length increased.?3?Stable expression analysis of yhNMT1 gene promoter in transgenic tobaccoThe pA vector which owned full cloned NMT1 promoter was selected for transgenosis and successfully gained transgenic tobacco.The results of GUS histochemical staining showed that the expression of GUS could be detected in different tissues of transgenic tobacco.The expression levels of GUS were detected by quantitative PCR in transgenic tobacco and its expression in leaf>stem>root,and the expression level was the highest in leaves and reached 3 times than that in roots,When transgenic tobacco treated with light,temperature,simulated drought and abscisic acid,there was a significant change in the expression of GUS in leaves,except at the temperature of 40?.The results showed that the function of yhNMT1promoter is influenced by the stress of environmental factors.?4?Cloning of transcription factors interacting with yhNMT1 gene promoter screened in yeast one-hybrid libraryAccording to yeast one-hybrid technique,yhNMT1 gene promoter was constructed into bait plasmid and transformed into yeast Y₁H,then resulted in Y₁H?PNMT1-AbAi?resistant to AbA.Next the tea cDNA and pGADT7-Rec vector were co-transformed intoY₁H?PNMT1-AbAi?,the yeast one-hybrid library was successfully constructed,the capacity of the library was 2.7×10⁶ cfu.Four transcription factors that could interact with promoter P_NMT1MT1 were obtained by further screening,and they were named yhTF1,yhTF2,yhTF3,yhTF4.Analysis and prediction of its protein function,yhTF1 belongs to the NAC family transcription factor,yhTF2 belongs to the HD-Zip family transcription factor,yhTF3 has about 45%homology with NAC domain protein,which may be involved in abiotic stress,yhTF4contains DUF1685 protein domain whose function is unknown.It is predicted that yhTF4 may be involved in plant photoreaction system.?5?Transcription factor subcellular localizationSubcellular Localization of proteins were by predicted by online tool PSORT,The probability of the four transcription factor proteins being located in the nucleus is73.9%,91.3%,65.2%,26.1%respectively.Four transcriptional factor genes were constructed into transient expression vectors pGreen-C18-GFP and transformed into tobacco mediated by agrobacterium for subcellular localization in subepidermal cells of tobacco leaves.The results showed that the yhTF1 and yhTF3 protein located in the nucleus,the yhTF2 protein located in the nucleus and cytoplasm,the yhTF4 protein located in cytoplasm.