Expression Analysis Of The Seed-specific Promoter Of α-Globμlin A Gene From Cotton(Gossypium Hirsutum L.)

Cotton is one of the indispensable crops in the world, not only provide the main raw material for the textile industry but also the cooking oil for human needs. Cottonseed oil can be extracted as feed, but also cotton is important genetic material for engineering. Cotton seed-specific promoter cloning and expression analysis will not only help elucidate the regulatory mechanism of cotton seed development and seed-specific gene, but also facilitate the use of plant genetic engineering to improve cotton seed. In this paper, the promoter deletion mutation analysis methods, deletion series promoter by converted into Arabidopsis, full-length study of the relevant 1640 bp pαGloA promoter regulatory elements, obtained the following results:1. Sequence analysis of pαGloA. Bioinformatics website implement bioinformatics analysis pαGloA full-length, the results show that include a TATA-box and CAAT-box, which is an essential component element of the promoter; there are six E-box(CANNTG), one G- box(CACGTG), one Skn-1 motif(GTCAT), two RY motif(CATGCA) and two DPBFCOREDCDC3 motif(ACACNNG), the expression of these functions essential element driving the seed; there are a SEF1 motif, three SEF4 motif, one AT-rich region, and fourteen(CA) n, these elements are mainly associated with the promoter activity. Marking element in accordance with the layout of the full-length pαGloA, using a 5 ’terminal deletion method, which was truncated to-1640 bp,-1402 bp,-684 bp,-208 bp four segment sequences and connected GUS gene construct into a plant expression vector, clearly named pαGloA-1640, pαGloA-1402, pαGloA-684, pαGloA-208.2. The Arabidopsis transgenic plants. Infected buds using methods to carry a target gene is introduced into agrobacterium growing well in WT Arabidopsis. Screened by Basta resistant seedlings, continued to train with the new nutritional soil resistant seedlings. In order to avoid the appearance of false-positive plants, Arabidopsis plants resistance obtained using leaf extract DNA PCR or method of re-identification, access to the GUS gene fragment 477 bp, the electrophoretic bands show has received positive plants.3. Transgenic Arabidopsis staining at different time in different organs. Transgenic Arabidopsis chemical testing different organs and tissues, GUS gene expression only in the embryo, grin seeds and seedlings of two cotyledons in different periods, but not in leaf, bud and other express nutrition organs, to prove that the promoter is a seed expression have specificity.4. Transgenic Arabidopsis plant positive seeds of GUS enzyme activity results analysis. Arabidopsis seed GUS activity measurement results on GM showed that with continuous truncated promoter pαGloA their expression activity showing a high-low-high mode. According to the results of inference in activity between-684 and-208 presence of inhibitory negative regulatory element of the promoter activity, specific element needs further proved.-1402 And-684 in the promoter activity with increased functional elements, these elements include SEF1 motif, SEF4 motif, AT-rich region, and(CA) n elements, SEF1 motif, SEF4 motif and AT-rich region as a positive regulator element,(CA) n can be used as a positive regulatory element as a negative regulatory element. In addition, expression of the promoter activity pαGloA-1640 was significantly higher than pαGloA-1402, five(CA) n elements present between-1640 and-1402, description(CA) n elements in the promoter may promote the promoter from active role.