西葫芦ZYMV病毒病显性抗病基因ZYMV1的精细定位与克隆

Zucchini yellow mosaic virus (ZYMV) is one of the most destructive viruses that badly reduce the production of squash (Cucurbita pepo), which are economically important crops grown worldwide. Resistance is the best approach to control the disease. Thus, the dominant gene conferring resistance to ZYMV appeared to be very valuable in breeding new virus resistant squash. Squash inbred line ‘BS12’ showed a high-level of ZYMV resistance in our germplasm evaluations over several years. The genetic basis of the resistance in ‘BS12’ was elucidated through an inheritance study and molecular mapping. A total of 1172 F2:3 lines from the crosses of BS3, a susceptible parent, with BS12 were tested with ZYMV-CH, a highly predominant ZYMV strain in China, under the controlled greenhouse conditions, and Bulked segregant analysis was carried out to identify SSR markers linked to this resistance gene. Results indicated that resistance to this virus observed in ‘BS12’ was controlled by a single dominant gene, which was designated as ZYMV1, and closely flanked by SSR markers CpZ142 and CpZ23 at a genetic distance of 0.4 and 1.1 cM, respectively. Here, we propose quickly further fine mapping of ZYMV1 with RAD (restriction-site associated DNA) markers, which produced by sequencing of reduced-representation libraries. A BC7F2:3 populations with 1172 lines would be used for ZYMV1 gene mapping. And, ZYMV1 gene will be further cloning with the results of whole-genome sequencing of C. pepo, which is now available. Gene function will also be investigated by some strategies, such as overexpression or RNA interference etc.. This work will be very helpful to facilitate the use of this resistance gene, and also be valuable for the discovery of the molecular mechanism of resistance to ZYMV virus in plants.

小西葫芦黄化花叶病毒（ZYMV）是世界范围内危害西葫芦等葫芦科作物生产的主要病害之一，可造成60%以上的产量和经济损失。ZYMV显性抗病基因对抗病西葫芦新品种的培育具有重要价值，但西葫芦中抗性强的抗源较少。我们获得了1份高抗ZYMV的西葫芦材料，抗性性状由单显性基因控制，并已将该显性抗病基因—ZYMV1，遗传定位在1.5 cM的区域内。本项目拟通过极端性状分离群体的简化基因组高通量测序技术，结合大规模BC7F2：3家系（1172个）的性状鉴定及西葫芦基因组序列信息，快速精细定位西葫芦ZYMV显性抗病基因，利用全基因组基因注释结果筛查候选抗病基因，并进行基因克隆与初步的功能分析。该研究对拓展该抗病基因的应用，提高西葫芦抗病育种效率，以及阐明植物对ZYMV的抗病分子机理均具有重要价值。

课题按计划执行，完成了研究任务与目标，获得以下主要结果：（1）利用F2：3家系及回交群体，揭示了西葫芦ZYMV抗病自交系“08-1”的抗性受一对显性基因控制。（2）通过BAS-seq将ZYMV抗病基因定位在第16染色体1.46 Mb的区间内，并进一步定位在标记InDel 103与InDel 46之间457 Kb的区域内。（3）利用两侧标记筛查1677个F2单株，获得36个交换单株，自交获得F2：3家系鉴定抗、感表型，结合新标记开发，最终将ZYMV抗病基因精细定位在5.28 Kb内，包含两个候选基因，A20/N1锌指蛋白与叶泡分选蛋白4编码基因（VPS 4）。（4）抗病自交系“08-1”中，VPS 4基因启动子区（ATG上游-738）存在大片段插入，编码区抗、感材料间有22个SNP位点，氨基酸序列间有3个替换；抗、感材料间A20/N1锌指蛋白基因编码区有8个SNP位点，导致4个氨基酸替换；54个西葫芦品种中抗病品种均与“08-1”基因型一致。（5）两个候选基因在根、茎、叶、花、果实中均有表达，在抗、感材料间的表达量无显著差异，且表达丰度与ZYMV病毒的诱导无关；（6）对抗、感品种各10份分析，发现导致两个基因氨基酸替换的SNP位点是保守的。在不同植物来源蛋白序列中，A20/N1锌指蛋白的氨基酸替换位点不在功能区，且在其它植物中存在相似替换，而且抗、感材料间该基因诱导表达的下游抗病相关基因CpRdR1与CpNpR1的表达量并无差异。而不同植物来源的VPS4蛋白的氨基酸序列在203（W）位点非常保守；原核表达VPS4蛋白，抗病基因型的ATPase活性显著降低，表明VPS 4基因为西葫芦ZYMV抗病候选基因，VPS 4在包膜病毒复制侵染循环中的细胞跨膜转运外释放中具有重要作用；（7）构建了VPS 4的基因编辑及植物过表达载体，分别转化抗、感自交系。（8）开发了InDel及KASP分子标记，并利用标记辅助选择结合回交转育育成“翠葫336”、“京葫42”等西葫芦抗病新品种，两年累计推广1.5万亩，创造了显著的经济效益。（9）获北京市科技进步二等奖1项，发表文章5篇，授权及申请国家发明专利各1项，授权新品种权1个，新申请1个，毕业硕士生2名。