| Brassica napus, as one of the most important oil crops, has become the largest domestic source of edible vegetable oil in China. In 2009, the rapeseed oil had accounted for more than 57%, but the self-sufficiency rate is less than 40% in the supply of domestic edible oil in our country. Rapeseed which is grown in the southern winter fallow fields has a lower yield and oil content, weaker resistance and lower-level mechanization. Separating the tissue-specific promoters by using the genetic engineering techniques could modify specific traits of crops, such as improving the resistance and photosynthetic efficiency, and then to expand the planting area and increase yield, which will be an effective measure to solve the shortage of vegetable oil. Tissue-specific promoters can specially regulate the gene expression in specific tissue site or organ, not only overcoming the waste of the non-specific sustained expression of the exogenous gene driving by constitutive promoters and its insufficient expression at 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 B. napus, and making clear of the molecular mechanisms of transcriptional regulation will be helpful to lay the foundation to study of genetic engineering in rapeseed and its relatives.In this study, to obtain vegetative organ-specific promoters(root, stem, leaf and flower bud, silique pericarp, seed) in B. napus, the organ-specific candidate genes were 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 had been determined by using 5’RACE method. Then, with the homology-based cloning strategy, the sequences of SP3-1, SP6-1, ST-8-1, SC-2 and FL-2 promoters were isolated and its cis-regulatory elements were predicted by PlantCARE databases. Finally, the promoter expression vector of candidate genes were constructed by double digestion technology and transformed into A. thaliana with Agrobacterium tumefaciens strain GV3101. The transgenic plants were identified by PCR, and those organ-specific expression pattern and expression levels were analyzed. Meanwhile, in order to study the specific gene expression in flowers and seeds of B. napus, the Illumina sequencing technology was used(based on RNA-seq). The transcriptomes of different organs and the seeds of different developmental stages in B. napus cultivar ZS11 were obtained. Then we compared the flowers and seeds transcriptome with other organs transcriptomes, and analyzed the specific genes which were chosen. Finally, 10 genes were selected from the selected seed and flower specific expression genes. After that, designing the primers to carry out quantitative PCR experiments to verify and analyze the specific candidate genes expression pattern were done. The main conclusions are as follows:1. Ten organ-specific candidate genes were identified by bioinformatic analysis from A. thaliana microarrays and transcript profiles of B. rapa and B. oleracea, and real-time quantitative PCR detection showed 5 of those genes were organ-specific. SP3-1 and SP6-1 showed specific expression in the silique pericarp, ST-8-1 was detected with a specific expression in stem, SC-2 was specifically expressed in seed, respectively. Amplification fragments of SP3-1, SP6-1 and ST-8-1, SC-2, and FL-2 showed nucleotide sequence identities with orthologous genes, indicating that the qPCR amplification fragments were derived from target candidate genes. 5’ RACE results showed that the length of 5’ cDNA ends of the 5 tissue-specific promoter genes were range from 1173-2400 bp(including 5’-UTR sequence), and accurate transcription start sites were found range from 53 to 269 bp of the upstream of the translation initiation site. Sequencing results of these promoters showed that the length were from 1173 to 2400 bp. Prediction of cis-regulatory element in promoters showed that core promoter element TATA-box and several cis-acting element of CAAT-box in enhancer regions 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 promoters were identified and named SP3-1(2151 bp), then engineering strains were obtained.3. Five plant expression vectors were transformed into Arabidopsis wildtype Col through Agrobacterium-mediated floral dip transformation method. GUS staining and PCR amplification showed that 28 and 35 transgenic plants were obtained for p1305.1-FL-1P and p1305.1-SC-2P vectors, respectively. The T3 generation of transgenic plants were used for GUS staining, and assessments of GUS protein activity and GUS gene expression. The results showed that FL-1 mainly expressed in flower, was considered as the flower specific promoter, but SC-2 is not expressed in seeds, and don′t have seed specificity.4. The genes expression level of the root, hypocotyls and cotyledons of the 48 h of seedlings, and roots, stems, leaves, flowers and flower buds, and the 5d, 9d, 30 d, 46 d seeds and silique pericarp in mature plants were studied by using high-throughput RNA-seq technology. The transcriptional level of the genome-wide gene expression was calculated and represented as FPKM(fragments per kilobase of exon model per million mapped reads value) by bioinformatic analysis to the high quality sequencing data of different organs and different periods of seeds and pericarp samples. The specific genes expressed in flowers and seeds, which expression level was 10 times higher than other tissues, were selected. And 1380 specific genes of flower and flower bud, and 2842 seed-specific genes can be obtained. The GO enrichment analysis was performed for those organ-specific expression genes by using Cytoscape. And The GO classification and enrichment analysis of the specific expression genes were also performed according to the corrected P-Value from the aspects of the cellular component ontology, the molecular function ontology and biological process ontology. To confirm the transcriptome data generated by RNA-seq, qRT-PCR was carried out on ten flower or seed-specific expression genes. The expression patterns of these detected genes showed similar expression patterns with RNA-seq results, confirming the reliability and accuracy of the RNA-seq data in this study. |
PDF Full Text Request