棉花矮化突变体AS98差异基因表达cDNA-AFLP分析

摘要

[目的]为了探究矮化突变体AS98发生变异的分子生物学机制,[方法]本研究利用正常陆地棉品种LHF10W99(简称LHF)与AS98杂交后代分离出的正常表现型植株和极端矮化表现型植株,在现蕾期提取2种株型的茎尖、茎和根的RNA,利用cD-NA-AFLP技术分析2种植株茎尖的表达差异.[结果]筛选到差异表达的片段32条,对这32条片段进行克隆测序,利用生物信息学进行分析发现其中一条(HD61)与拟南芥的阿拉伯半乳聚糖蛋白的同源性较高.用棉花的阿拉伯半乳聚糖蛋白(GhAGP)的序列设计引物,用RT-PCR方法检验阿拉伯半乳聚糖蛋白在2类植株的茎尖、茎和根等组织上的表达量,结果表明,GhAGP基因在突变型和正常型材料的茎、根上的表达量基本相同,但是在茎尖上却表现出明显的差异,突变型茎尖上的表达量显著低于正常型茎尖的表达量,说明GhAGP可能参与棉花茎尖细胞的伸长生长过程.[结论]AS98的半乳糖代谢的改变可能与赤霉素(GA)合成中基因发生突变有关,不能合成正常具有生物活性的GA,使得阿拉伯半乳聚糖蛋白等基因在AS98茎尖的表达减少甚至缺乏.%[Objective] The present paper aims to get the molecular biology mutation mechanism of dwarf mutant AS98.[Method] The normal upland cotton LHF10W99 was crossed with AS98,and the normal plant and extreme dwarf plant from the hybrid offspring plants were isolated.The RNA of stem tip,stem and root from these two kind plants in the budding period were extracted,and the expressing difference in the stem tip of two type plants was analyzed by cDNA-AFLP.[Result] 32 fragments were amplified and sequenced,and one of fragment HD61 exhibited high homology with Arabidopsis Arabinogalactan protein using bioinformatics analysis.Then the special primers were designed according to the cotton Arabinogalactan Protein (GhAGP) sequences,the RT-PCR was done in stem tip,stem and root to verify the expressing difference between the two type plants.The GhAGP expression were the same in stem and root of normal and dwarf mutant plants,while there were obvious difference in the stem tip:the expression of mutant stem tip was significantly lower than normal stem tip,indicating that the GhAGP might participate in the process of cell elongation growth of cotton stem tip.[Conclusion] The change of AS98 galactose metabolism maybe has relationship with the gibberellin (GA) synthesis gene mutation;The AS98 cannot synthesize bioactive GA normally and reduce or lack the gene expression of GhAGP in AS98 stem tip.