Study On The Activity Of Cotton D - 7 Promoter

Cotton is an important economic crop, which has significant application value in daily life. D-7 protein is the member of the third group of the Late-embryogenesis-abundant proteins (LEA) family. Researchers have shown that the third class of LEA is an inducible gene, which has high expression in the late-mature seeds, and can be induced by drought, ABA, high temperature, low temperature and other stress conditions in vegetative tissues. We guessed that the gene would possess more induced elements located in the upstream sequence. In earlier work, we had acquired 1876bp D-7 gene promoter sequence in LEA gene upstream by bioinformatics and genetic transformation. We found 1876bp D-7 promoter contains CAAT box, TATA box, LTR, ABRE, HSE cis-elements, and responses to some different abiotic stress, such as drought, low temperature, salt and ABA. On the basis of previous research, we established a series of D-7 gene promoter with 5’ terminal deletion fragment (392bp, 355bp,312bp,247bp,173bp,92bp) and eGFP fused expression vectors. We detected these promotoers activity by using transient expression system and transgenic Arabidopsisto analysis core zone of D-7 promoter, which could lay the foundation for transformation and the utilization of the promoter. The main results are as follows1. Six pair of primers of 5’ terminal deletion fragment were designed based on D-7 gene promoter sequence, by the method of PCR, we constructed six 5’ terminal deletion fragments (392bp,355bp,312bp,247bp,173bp,92bp) with eGFP fused expression vectors. Then we successfully transfered the fused expression vectors into agrobacterium GV3101 and LBA4404.2. Detection report gene eGFP expression level by transient expression system in cotton cotyledons, determine 4 days as the best co-culture days. Analysis of series D-7 promoter activity, preliminary determine truncated 247bp and 173bp fragment have the strongest eGFP fluorescence.3. Arabidopsis genetic transformation method mediated by agrobacterium GV3101, we acquire six transgenic Arabidopsis thaliana with D-7 gene promote of 5’terminal deletion fragments fused with eGFP. After the resistance selection and molecular identification, T2 generation pure line is obtained.4. The green fluorescence is observed in the different organization parts in T2 generation of transgenic Arabidopsis thaliana by fluorescence microscope, results show the eGFP only expresses in transgenic Arabidopsis leaf and seed. The fluorescence expression of the T2 generation transgenic Arabidopsis seeds enhanced increasingly, green fluorescent began to appear in the 15 days transformed 392bp,312bp promoter Arabidopsis seeds, then the fluorescence intensity; In transformed 247bp,173bp,92bp promoter of Arabidopsis seeds, green fluorescence began to appear at day 12, in which the line transformed 247bp promoter, the fluorescence intensity of seed expression is stronger than the other five truncated, in 12 to 18 days.5. Series D-7 promoter of transgenic Arabidopsis drought resistance analysis results show the drought treatment can increase the expression of eGFP strength, and eGFP in turn to the 247bp promoter strongest expression. Through Real-time PCR method to detect the T2 generation of transgenic Arabidopsis leaf relative expression of eGFP, results showed that the drought induced series D-7 promoter drive eGFP expression, under drought stress, the 247bp and 312bp D-7 promoter drive eGFP the highest amount of expression. But the 247-247bp sequence whether is D-7 promoter core region, has yet to be further in-depth study.