Identification And Isolation Of Tissue-specific Promoters From Brassica Napus

Brassica napus L., as one of the most important oil crops, has become the largest domestic source of edible vegetable oil in China. In2009, the rapeseed oil acount for more than57%, but the self-sufficiency rate is less than40%in the supply of domestic edible oil in our country. Rapeseed is grown on the southern winter fallow fields with the lower yields and oil content, weaker resistance and lower-level mechanization. Nevertheless, using the genetic engineering techniques, tissue-specific promoters could be used to modify specific traits of crops, such as, improving the resistance and photosynthetic efficiency, gradually expanding planting acreage to increase yield, it will be an effective solution to solve the shortage of vegetable oil.Tissue-specific promoters can specialty regulate the gene expression in specific tissue site or organ, not only overcoming the waste caused by constitutive promoters driving the non-specific sustained expression of the exogenous gene and insufficient expression of the particular site, but also effectively avoiding the phenomenon of gene escape in transgenic crops and solving the transgenic environmental security issues. Therefore, isolation and identification of tissue-specific promoter from Brassica napus, and making clear the molecular mechanisms of transcriptional regulation, it will provide the foundation to study of gene function and genetic engineering in rapeseed and Its Allies.In this study, to obtain organ-specific promoters (root, stem, leaf and flower) in Brassica napus, firstly, the organ-specific candidate genes had been screened out based on Arabidopsis thaliana microarrays and transcript profiles of Brassica rapa and Brassica oleracea. Moreover, tissue-specific detection and expression pattern of candidate genes were identified by real-time quantitative PCR (qRT-PCR), and the precise transcription start site has been determined by using5’RACE. Secondly, using the homology-based cloning strategy, promoters sequences of RO-4, LE-7, FL-1, FL-4and ST-7promoters were isolated and the cis-regulatory element were predicted and by PlantCARE databases. Finally, the promoters expression vector of candicate genes were constructed by double digestion technology and transformed into hypocotyl of ZY821with Agrobacterium tumefaciens strain GV3101. PCR identification of expression specification and levels of promoters was used for selection of transgenic generation plants. Meanwhile, in order to further study the position and function of the core sequence of specific promoter, using gateway cloning technology, partial deletion of the core promoter vectors were successfully constructed and induced into Arobidopsis by floral dip method. The main conclusions are as follows:1.24organ-specific candidate genes were identified by bioinformatics analysis from Arabidopsis thaliana microarrays and transcript profiles of Brassica rapa and Brassica oleracea, and Real-time quantitative qRT-PCR detection showed14of those genes were vegetative organ-specific, mainly including RO-4, LE-7, FL-1and FL-4, showed specific expression in the root, leaf, and flowers, but ST-7was detected specific expression in root, stem and silique pericarp, respectively. Amplification fragments of RO-4, FL-1and FL-4, LE-7, and ST-7showed nucleotide sequence identities with orthologous genes, indicating qPCR amplification fragments were derived from target candidate genes.5’RACE results showed that the length of5’cDNA ends of5tissue-specific promoter genes were from53to269bp, and accurate transcription start sites were found from53to269bp upstream of translation initiation site. Sequencing results of promoters showed that the length were from1305to1566bp (including5’-UTR sequence). Prediction of cis-regulatory element in promoters showed that core promoter element TATA-box and several cis-acting element in enhancer regions CAAT-box were found in all cloned promoters. Besides, light-responsive elements, stress-responsive elements and elements involved in the hormone responsiveness were also widely existed in promoter, however only few cis-acting elements involved in organ-specific expression were detected.2. Five tissue-specific promoter fragments were cloned and the length of RO-4, LE-7, FL-1, FL-4and ST-7were1475,1566,1372,1517and1305bp, respectively. Furthermore, promoter-GUS expression vectors pl305.1-RO-4P, pl305.1-LE-7P, p1305.1-FL-1P and p1305.1-FL-4P were successfully constructed by inserting the promoter fragments into pCAMBIA1305.1, digesting with BamnHI and NcoI. Similarly, pl305.1-ST-7P was also constructed by the same means, digesting with SacI and Ncol. And then the expression vector were identified by multiple PCR and transformed into Agrobacterium tumefaciens GV3101strain to inform the engineering strains.3. Using the Agrobacterium-mediated transformation, the plant expression vectors p1305.1-RO-4P and p1305.1-LE-7P were transformed into B. napus cv. Zhongyou821and the transgenic positive generations were identified by multiple PCR. The results of GUS staining showed that LE-7promotor was mainly detected expression in the leaves, and nearly not detected in the flowers, buds, roots and silique pericarp; and the RO-4promotor showed GUS activity in edge of the leaf, but it still to be studied in the future.4. Six differential deletion fragments of RO-4promoter were cloned and named as R1(1301bp), R2(1030bp), R3(622bp), R4(481bp), R5(352bp) and R6(252bp) respectively; and five deletion fragments of LE-7promoter were named LI, L2, L3, L4and L5, and the lengths were925,755,526,350and231bp, respectively. Moreover, the expression deletion fragments vectors were also successfully constructed by LR recombination reaction with vector pMDC162, and then transformed into Agrobacterium tumefaciens GV3101strain to generate engineering strains.