Cloning And Stress Response Analysis Of The Promoter Of9-cis-epoxycarotenoid Dioxygenase Gene(CrNCED1)in Citrus Reshni

In the growth process of plants, it will suffer from all kinds of adversity inevitably, when the plants subject to drought, cold, salt and all kinds of stresses, it will rapidly accumulate ABA to protect themselves from damage, so ABA was considered to be the very important adversity hormone. NCED is the key rate-limiting enzyme in ABA biosynthesis. In our lab, we have cloned the CrNCED1gene from Citrus reshni, and it was a stress-induced gene.But the regulation mechanism about the gene in transcription level is still not clear. So analysing the specific components and studying in the promoter of CrNCEDl gene, studying its expression in transcription level are of crucial importance to elucidate the regulation mechanism of CrNCEDl and separate stress inducible promoters.1.According to Orange databases, we cloned the1989bp promoter of CrNCEDl gene by multiple alignments. PLACE database analysis showed that this segment sequence regions not only containing basic components, like TATA box and CAAT box, but also contains ABRE, MYBCORE, BIHD10S, ERELEE4, GT1GMCAM4S that are closely related to stress. They are concentrated in about1000bp upstream of the ATG.2. A1058bp length promoter were amplified by designed primer, and connect it to pCAMBIA1391binary expression vector which was cut off the CaMV35S promoter, Then this1058bp fragments were transfered into wild-type tobacco with the help of Ti-plasmid of Agrobacterium tumefaciens. At last we selected the transgenic tobacco by hygromycin. In our experiments, we found that we can not get the transgenic plants with GUS activity by PCR amplify as DNA is the template, but as we use cDNA that obtained by RNA, we finally get transgenic lines#7and#8.3.For tissue-specific analysis, we selected transgenic lines (#7,#8) for GUS staining. The results showed that there was no GUS expression in seeds and pods, but there was a high GUS expression in the leaves and stem end. Then from the roots staining we found that there was a high GUS expression in the column parts of root, but in the apical and other regions there was no GUS expression.4.We treated transgenic lines (#7,#8) with dehydrated, ABA and mannitol, then we found that all of these three treatments can enhance GUS activity in transgenic lines, especially in young leaves GUS activity was significantly enhanced. GUS fluorescence activity also proved that this promoter is able to respond to dehydration, ABA and mannitol. These results above all showed that this promoter is a stress induced promoter.