棉花抗黄萎病相关基因的精细定位及克隆

Verticillium dahliae is one of the most crushing diseases to cotton production. Due to the lack immune varieties to Verticillium wilt, the progress of breeding was slow. We acquired a chromosome segment introgression line (CSILs), SuVR043 with the higher resistance to Verticillium dahliae, which were characterized by carrying one homozygous chromosome 22 (D4) segment from G. barbadense cv Hai7124 in the background of the upland cotton, Sumian8. Using the recently released cotton D genome sequence information and EST database of G. barbadense and G. hirsutum, we extracted the sequence of segment introgression and designed 92 SSR primer pairs, and mapped QTL of the resistance Verticillium dahliae on the segment introgression using the F2 population containing 176 generations which was constructed by using CSIL SuVR043 corssing with Sumian8, and explained 68.4% of the phenotypic variation. Moreover, we predicted sixty-four genes to be as candidate genes. The result of GO analysis indicated that these genes involved in different biological processes, and 7 of them participated in the reflection of resistance stresses and one of them participated the process of biotic stress. In this study, we will be fine mapping the resistance genes using the F2 and BC1 populations; meanwhile we will pick up the candidate genes from predicted sixty-four genes by using Virus-Induced Gene Silencing (VIGS) in G. barbadense cv Hai7124 and screening different expression and sequence divergence in the genome DNA of the genes between SuVR043 and Sumian8. Combined with the gene fine mapping results, the target genes will be defined. Then we will analyze the functions of the target genes by methods of transgene. The completion of the study will establish foundation for the study of cotton Verticillium wilt resistance mechanisms, and provide candidate genes for transgenic breeding to improve the resistance of cotton to Verticillium dahliae.

黄萎病是棉花生产中的主要病害，由于缺少对黄萎病免疫的常规棉花品种，研究进展缓慢。本研究室经过多年选育获得一个以苏棉8号为背景携带海岛棉海7124 D4染色体片段的具有较高黄萎病抗性的染色体单片段代换系苏VR043。利用棉花已经公布的D组序列以及海岛棉和陆地棉的EST数据库，提取并分析置换区段的基因组序列，开发92对SSR引物，以苏VR043×苏棉8号的F2群体初步定位抗病QTL于该片段上，解释64.8%的表型变异。我们预测该区段包含64个基因，GO分析表明其中7个基因参与棉花抗逆反应，1个参与棉花抗病反应。在本研究中，拟利用F2和BC1群体对抗病基因进行精细定位；同时利用病毒诱导的基因沉默方法及序列和表达差异分析来挑选基因。结合定位结果确定与抗病相关的候选基因。随后我们将通过转基因方法来验证基因功能。该研究的完成将为今后棉花抗黄萎病育种提供候选基因。

棉花是重要的经济作物，近年来，随着棉花黄萎病的爆发，显著影响棉花的生产。而棉花抗黄萎病病机制研究进展缓慢，没有找到对黄萎病抗性起关键作用的基因。本研究针对这一关键问题，开展以下研究：.1..利用最近释放的雷蒙德氏棉、陆地棉和亚洲棉基因组序列全基因组序列信息，对Hsp70基因家族进行鉴定、分类和系统进化分析。共获得115个Hsp70基因，非均匀性地分布在棉花基因组染色体上，部分HSP70基因在染色体上成簇分布。聚类分析结果显示，115个HSP70基因可以分为29个亚组。参照拟南芥对Hsp70基因的分类方法，将棉花115个Hsp70基因分为两个家族，91个属于DnaK基因家族，24个属于Hsp110/SSE基因家族。根据亚细胞定位结果，将棉花115 Hsp70基因分为6个亚组。分别定位于细胞质、内质网、线粒体和质体。以海岛棉黄萎病抗源品系海7124、陆地棉背景的雷蒙德氏棉染色体片段代换系苏远045和陆地棉遗传标准系TM-1为供试材料分析其中29个热激蛋白70受黄萎病诱导后表达情况。结果显示，除Hsp70-07，Hsp70-16，Hsp70-25和Hsp70-26基因的表达不受黄萎病菌诱导外，其它25个Hsp70基因均受黄萎病菌的诱导而上调表达，说明Hsp70基因在棉花应对黄萎病为害的过程中发挥重要作用。该研究为今后分析棉花热激蛋白基因功能提供基础，为进一步开展棉花抗黄萎病基因的表达调控以及棉花抗黄萎病机制的研究提供候选基因。.2..利用携带海岛棉海7124 D4染色体单片段导入系，构建群体，定位了位于该片段上的2个主效的QTL，平均解释表型变异分别为16.38%和22.36%，LOD值在3.2-8.8和4.9-9.6之间。根据已经释放的棉花四倍体陆地棉和海岛棉基因组序列，提取定位区段基因组序列，预测和分析包含的基因信息，选择11个基因进行表达分析。根据表达结果，选择候选基因进行功能分析，确定2个基因在棉花抗病过程中扮演关键角色，分别是GbTMEM214和GbCYP450。同时，获得4份优质高产的陆地棉抗黄萎病种质材料。本研究结果可以为今后抗病研究提供候选材料，同时为今后通过基因工程的方法创制抗病材料和研究棉花抗病机制提供候选基因。