棉花超矮杆基因(du)的精细定位与候选基因的克隆

A special dwarf mutant cultivar of GA-sensitivity was found in isolated population of upland cotton C119-2. Cotyledon and leaf of the mutant was curly and the height was extremely dwarf. Without manual spraying exogenous gibberellin, the mutant’s development process of flowering, boll, etc. can not be started. Conversely, exogenous gibberellin can make it finish the normal flowering and bolling. The mutant gene – du was primary mapped on Chr.06 of the cotton used SSR markers. Based on the basis of preliminary studies, the fine mapping of mutant gene – du will be launched on the use of "Hai7124" and "ultra-dwarf 101" hybrid produced F2 population. Combined with the results of GA treatment between ultra-dwarf mutant "ultra-dwarf 101" and the wild-type material by the expression profiling of sequencing, the candidate genes controlling the trait will be screened. We hope to get the muntant gene contronling the ultra-dwarf mutant followed by virus induced gene silencing(VIGS) and functional complementation experiment in Arabidopsis.

本课题组在陆地棉C119-2分离群体中发现一种新的对赤霉素敏感的矮杆突变体材料。该突变体的子叶与真叶卷缩并极度矮化。若不进行人工喷施外源赤霉素，该突变体不能启动开花、结铃等发育过程；但通过外源赤霉素的施加，可以正常开花结铃。通过SSR分子标记，将控制该突变体的基因du初步定位在棉花第6染色体上。本研究在前期研究的基础上，利用“海7124”与“超矮101”杂交产生的F2作图群体，对该突变基因进行精细定位。同时结合超矮突变体 “超矮101”和野生型材料经赤霉素处理前后的表达谱测序分析来筛选控制该性状的候选基因。将得到的候选基因利用病毒诱导的基因沉默技术和拟南芥突变体植株的功能互补实验来对候选基因进行功能验证，以期获得控制该突变性状的基因。

本项目利用超矮杆突变体与野生型构建的分离群体将控制该性状基因-du定位于SSR标记JESPR-194与dPL0811之间，这两个标记在陆地棉遗传标准系TM-1基因组之间的距离约1Mb，其间含有49个预测基因。通过转录组测序，这49个基因中的Gh_A06G1386基因在突变体与野生型之间具有显著性表达差异。并且该基因编码一个细胞色素P450家族蛋白-贝壳杉烯酸氧化酶(ent-kaurenoic acid oxidase, KAO)。KEGG分析表明，该基因为赤霉素合成代谢途径中的关键酶之一。同时，本研究对GhCYP701A1基因通过病毒介导的转基因沉默(VIGS)技术进行了沉默。结果表明，该基因沉默后植株高度显著低于对照植株，并且赤霉素含量也显著低于对照。通过本项目的研究，为棉花的矮化育种提供更多的矮源候选基因。未来通过CRISPR技术对这些基因进行定点突变，将会进一步加快培育适合机械化种植的棉花新品种。