Molecular Mechanism On Alternative Promoter Of Terpene Synthase Gene CsLIS/NES In Tea Plant

There are more than 700 aroma compounds in tea,in which terpenoids is dramatically abundant.Among them,linalool as the abundant monoterpene,contributes to floral and fruity character of Oolong tea,and actively responds to adversity.Previous studies on a linalool synthase gene CsLIS/NES in our lab showed that it can produce two isoforms:CsLIS/NES-1 and CsLIS/NES-2,and these two genes have different subcellular localization and biological functions,hence producing linalool and nerolidol,respectively.In present study,the genome walking kit was used to clone the promoter of CsLIS/NES,then the upstream sequence of 1492bp was obtained,proved that this part is the promoter of CsLIS/NES.The noncoding region of CsLIS/NES-1 is from the first ATG,while that of CsLIS/NES-2 is from the second ATG.In our study,these promoters from different ATG and TATA-box sites were respectively cloned,and combined with a reporter gene GUS or LUC for the sake of validating the function of these promoters.Meanwhile,The deletion cloning was also used in our study.The results show that these two isoforms possess respective promoter,which indicate that the promoter of CsLIS/NES-2 were comprised by the first exon and intron of CsLIS/NES-1,located in the region between the two ATG.Furthermore,the transcription of CsLIS/NES-2 not depend on the promoter of CsLIS/NES-1.These results confirmed that the CsLIS/NES-2 isoform not depend on the post-transcriptional processing,but occured at the beginning of transcription.In addition to,CsLIS/NES-1 was induced by MeJA and significantly up-regulated,while CsLIS/NES-2 did not respond to JA.We supposed that the differential response of these two genes to MeJA is related to the structure and cis-elements of CSLIS/NES promoter.The interaction experiment between CsMYC2 and G-box confirmed that two G-box,-1458bp and-1379bp,which is a key for CsLIS/NES-1 responding to JA,but the interaction could not activate the promoter activity of CsLIS/NES-2.This result well explained that CsLIS/NES-1 was induced by JA but not CsLIS/NES-2.The TATA-box mutation results proved that the TATA-box located in-108bp(TATAAATA)involved the transcription of CsLIS/NES-1.However,there is no TATA-box in the promoter of CsLIS/NES-2.In addition,these two genes expressed differently in different developmental stages of tea flowers.The transcription level of the two genes increased gradually with the blooming of tea flower.But the transcription level of CsLIS/NES-2 was significantly higher than that of CsLIS/NES-1 at each stage.In our study,according to the promoter identification,we found that the promoter of CsLIS/NES gene is an alternative promoter,which could regulate the products of CsLIS/NES gene,such as linalool or nerolidol.This study provide insight into the formation mechanism of terpenoid compounds in tea plant.