陆地棉ghsh1a基因克隆及功能的初步分析-生物化学与分子生物学专业论文.docxVIP

末端序列杂交的带较多，表明3’一末端序列较保守。表明棉花基因组中 末端序列杂交的带较多，表明3’一末端序列较保守。表明棉花基因组中 至少含有两个GhSHla同源基因。 为了分析该基因产物在棉花组织发育过程中的功能，构建了可被雌 二醇p-estradiol诱导的植物RNA干扰表达质粒pER8．GhSHla—RNAi。 利用农杆菌介导法将GhSHla．RNAi片段转化到棉花细胞中，以敲除基 因组中内源GhSHla的表达，观察和分析转基因棉株的表型变化。 获得了7个GhsHla—RNAi转基因株系，PCR和Southern blot杂交 检测都证明GhSHIa．RNAi稳定整合到棉花基因组中。7个转基因系中 有3个育有种子。13-estradiol诱导GhSHla．RNAi表达的Tl代6天无 菌苗的侧根比野生型的较长，根系较发达。侧根被剪去之后，转基因无 菌苗的侧根再生能力也比野生型的强。根据转基因棉株的这一表型变化 初步分析，GhSHla—RNAi的表达促进侧根的发育，即GhSHla抑制棉 花侧根的发育。这说明GhSHla的功能与拟南芥中的SINAT5抑制侧根 发育的功能是一致的。 将对T1代植株的棉纤维表型包括纤维强度和长度等做进一步的研 究，以分析GhSHla—RNAi的表达是否在纤维的发育过程起作用。 该研究的意义在于确定GhSHIa是否与棉花纤维的发育或者与根系 发育有关，以便通过基因工程改善棉花纤维品质或提高纤维产量，或者 得到根系发达的耐旱品种。 关键词：陆地棉；GhSHla；发育；序列分析；RNAi；遗传转化；侧根 表型分析；功能 ABSTRACTCotton ABSTRACT Cotton iS all important commercial crop as a maln source of natural fiber，Studies on the functions of cotton gene are to provide theoretic foundition for cotton genetic modi fication、A SINA homologous clone was screened from cotton(Gossypium hirsutum L．)cDNA library in this study and was termed GhSHid．Sequencing analysis showed that GhSHla cDNA was 1394 bp in length，encoding a 339一AA peptide．Cotton GhSHla protein shares about 30％amino acid identity with Drosophila S1NA protein、and 78％amino acid identity with Arabidopsis SINAT5 protein．GhSHl a protein is a member of SINA(Seven in absentia)protein family which containing a highly conserved N-terminal C3HC4 Ring finger motif．N—terminal C3HC4 Ring finger motif of SINA protein is required to promote ubiquitin— proteasome pathway of target protein degradation，and C—terminal sequence plays a role in interaction with target protein． SINA gene was firstly identified in Drosophila．SINA protein is critically involved in regulating the cell fate decision of R7 photoreceptor cell in Drosophila eyes．Human SIAH l is involved in cell apoptosis． Arabidopsis SINAT5 protein has ubiquitin—ligase E3 activity for attenuating auxin signals to regulate lateral root development．Howeve r，to our best knowledge，there is still no publication on the f