Study On The Role Of A Cotton (Gossypium Hirsutum) Blue Copper Protein (BCP4) In Fiber Cell Elongation

Cotton fiber as a natural textile fiber, which can be processed by modern textile technology into apparel and household cotton products, are deeply loved by consumers. But cotton fiber quality from our country original varieties is uneven, unable to satisfy the market demand of high yield and quality. From the perspective of sustainable development, improving the quality of cotton fiber is imminent. With the rapid development of modern biological technology, through cloning identification control key genes in cotton fiber development, studying the molecular regulation mechanism of cotton fiber development, and then quickly get high quality cotton by using transgenic technology is a commonly used research and breeding method at present.Blue copper proteins (BCP) are a kind of type I copper protein, originally found in bacteria. It is widely distributed and plays an important function in living organisms, such as electron transfer, oxygen activation, etc. Recent studies have shown that it is also play an important regulatory role in plant normal development. GhBCP4(accession no. in Genebank:GU451702.1)protein is a blue copper protein, which isolated from cotton (Gossypium hirsutum). Tissue expression spectrum analysis showed that GhBCP4 highly expressed in 10 days post anthesis fiber, suggesting that this gene may have an important function in cotton fiber elongation. Arabidopsis trichome, and cotton fiber belong to category of trichome in botany, several studies show that heterologous expression of genes associated with the cotton fiber development in Arabidopsis trichome also helps to understand the biological function of the gene in cotton fiber development.In this study, from subcellular localization of GhBCP4 protein, isolation and characterization of GhBCP4 promoter, identification and phenotype analysis of GhBCP4 transgenic Arabidopsis and cotton, We conducted a preliminary study on the role of GhBCP4 in cotton fiber elongation process, the main results were described as follows:1. Subcellular localization of GhBCP4 proteinThe constructed GhBCP4::GFP fusion expression vector was transformed into competent Agrobacterium LBA4404. By the Agrobacterium mediated DNA transfer into cotton, we get cotton callus through genetic transformation. Picking up cotton callus with fluorescent signal and making temporary slide, we observed under a confocal microscope and found that GhBCP4 protein located not only in the cytoplasm, but also on the cell membrane.2. Isolation and characterization of GhBCP4 promoterThe acquaintance of GhBCP4 upstream regulatory sequences contribute to the study of GhBCP4. Therefore, according to the cotton genome sequence previously published, primers were designed. GhBCP4 promoter sequence was isolated by using PCR technology, which has a size of 1985bp. The element analysis was conducted by PLACE and PlantCARE two online promoter prediction software, we found that the promoter not only had the basic features of the promoter elements, but also had the important regulatory elements involved in the growth and development, resistance response.The constructed GhBCP 4Pro::GUS vector was transformed into Arabidopsis, then we get transgenic lines. GUS staining was carried out on the screened green seedlings, but we did not get the expected blue signal. Related experiments need to be further verified.3. Phenotype analysis of GhBCP’4 overexpression transgenic ArabidopsisThe constructed GhBCP4 overexpression vector was transformed into Arabidopsis, then we get transgenic lines. GhBCP4 expression detection and phenotype analysis were conducted in purified transgenic T2 generation lines. RNA of T2 generation seedlings were extracted from the different lines, we selected 3 lines (GhBCP4 expression level from high to low), counted the length and number of trichomes on 5/6/7 rosette leaves, and found that transgenic Arabidopsis trichomes had an increasing trend in length and density, compared to the wild type.4. Phenotype analysis of GhBCP4 transgenic cotton1) Phenotype analysis of CaMV 35S promoter driving GhBCP4 RNAi (RNA interference) transgenic cotton plantsFirstly, we identified three generations of GhBCP4 RNAi (T0, T1, T2) transgenic cotton in gDNA levels, used Quantitative RT PCR technique to detect the GhBCP4 gene expression in transgenic cotton, results suggesting that its expression in RNAi transgenic lines was inhibited by different degree and significantly lower than wild type. Moreover, with transgenic cotton fiber development blocked, mature fiber length was obviously shorter than wild type. This showed that GhBCP4 play an important regulating role in cotton fiber development.2) The construction of RDL1 promoter driving GhBCP4 RNAi (RNA interference) vector and identification of transgenic cotton plantsIn order to better illustrate GhBCP4 function during cotton fiber development, we used a cotton fiber specific promoter RDLlp to drive GhBCP4 specific expression in cotton fiber. By the genetic transformation of cotton, we get 7 transgenic embryonic callus lines, most of these lines had already got massive TO generation transgenic seedlings. We transplanted them into the field, waiting for their development into maturity. The preliminary identification and analysis were proceeded in these transgenic lines.3) Identification of CaMV 35S promoter driving GhBCP4 overexpression transgenic cotton plantsIn addition to overexpression GhBCP4 in Arabidopsis trichome, we also constructed the GhBCP4 overexpression vector and constructed a genetic transformation in cotton. We had already got massive TO generation transgenic lines, most of them grew and developed normally. Through extracting gDNA from these lines for positive identification, we found that almost lines were real transgenic seedling. Phenotype analysis of GhBCP4 overexpression transgenic lines are in progress.