普通野生稻抗褐飞虱基因Bph33(t)的克隆及育种利用

The brown planthopper is the most serious migratory pest of rice.Guangxi is located in Southernmost Point of our mainland.Which makes it become to the fist station of migration, and be called “bridgehead” and the source of breeding ground.Guangxi is the route of the brown planthopper migration and is also the main pest source place of the rice paddies in the middle and lower reaches of the Yangtze River. It is a great threaten to food security for a large outbreak of brown planthopper every year in China. Therefore, it is of great significance to explore the resistance genes of brown planthopper in Guangxi wild rice germplasm and breeding resistant varieties to the control of brown planthopper. In our previous study, two resistance loci Bph33(t) and Bph34(t) have been identified in BPH2170 by using the software MapQTL 5.0, which were mapped on short arm and long arm in chromosome 4, and explained the phenotypic variance of 58% and 63.7% , respectively. Both of them are novel resistant genes. A total of 2 candidate genes were identified in the region of 116Kb by using whole-genome sequencing BSA and fine mapping. In this project, we will confirm the target candidate gene by RT-PCR. Functional complementation test were used to confirm the isolated target gene Bph33 (t) and functional analysis will be carried out, including morphological and physiological investigation, subcellular locatization analysis, quantitative expression analysis. Marker assisted selection of brown.planthopper resistance gene for improving Taifeng B and R4684. Through observation and analysis of BPH on host selection, honeydew, group productivity, survival rates, etc., which will be determined the resistance mechanism of Bph33 (t) gene in rice (i.e. antibiosis, avoid insect repellent and insect resistance). These results will be lay the foundation for further elucidating the molecular mechanism of rice - brown planthopper interaction.

褐飞虱是为害水稻的重大迁飞性害虫，广西是褐飞虱境外虫源迁入中国的“桥头堡”和北往南返的必经之地，也是长江中下游稻区的主要虫源地，连年大规模的暴发给我国粮食安全构成严重威胁。因此，挖掘广西野生稻资源中的抗褐飞虱基因、培育抗性品种对褐飞虱的防控具有重要的意义。前期研究在高抗褐飞虱普通野生稻BPH2170的第4染色体短臂和长臂上，各鉴定了1个抗性位点Bph33(t)和Bph34(t)，均为新的抗性基因。全基因组重测序BSA和精细定位相结合将Bph33(t)限定在116Kb的范围内，共2个候选基因。本研究利用RT-PCR确定候选基因，进行转基因功能验证，克隆Bph33(t)基因和功能分析。标记辅助选择培育泰丰B和R4684的抗褐飞虱改良新品系，并分析褐飞虱对寄主的选择、蜜露量、群体生产率、存活率等确定Bph33(t)的抗虫机制，为进一步揭示水稻-褐飞虱互作的分子机制奠定基础。

褐飞虱是为害水稻的重大迁飞性害虫，广西是褐飞虱境外虫源迁入中国的“桥头堡”和北往南返的必经之地，也是长江中下游稻区的主要虫源地，连年大规模的暴发给我国粮食安全构成严重威胁。因此，挖掘广西野生稻资源中的抗褐飞虱基因、培育抗性品种对褐飞虱的防控具有重要的意义。前期研究在高抗褐飞虱普通野生稻BPH2170的第4染色体短臂和长臂上，各鉴定了1个抗性位点Bph33(t)和Bph34(t)，均为新的抗性基因，并将Bph33(t)精细定位在116Kb的范围内。本研究利用qRT-PCR分析对Bph33(t)候选基因1-ORF38基因在抗感虫亲本BPHR2170和R4684中取食前后表达水平变化。结果表明1- ORF38基因在感虫亲本R4684上取食前后的表达水平都非常低或基本不表达；而1-ORF38基因在抗虫亲本BPHR2170中接虫前后的表达水平都很高，接虫24 h后的达到最高表达水平，比其他时间点表达水平高了1倍。接虫前后的1-ORF38基因在抗虫亲本BPHR2170上的表达水平要显著或极显著高于R4684的表达水平。因此，最终确定1-ORF38基因为目标候选基因。通过对抗褐飞虱基因Bph33(t)的候选基因结构分析和克隆，抗虫亲本普通野生稻BPHR2170 Bph33(t)基因CDS全长996bp，编码332个氨基酸,感虫亲本 R4684对应Bph33(t)等位基因CDS的长度也是996bp，但在启动区域有大片段插入（10976bp），因而使R4684丧失抗性。利用基因敲除载体pYLCRISPR-cas9-MH-Bph200/400对抗性亲本BPHR2170中的目标候选基因1-ORF38进行敲除，获得37个转基因株系，抗性鉴定表明，所有转基因株系均高感褐飞虱，表明1-ORF38基因为目标抗性基因。亚细胞定位表明Bph33是内质网定位蛋白。通过观察和分析褐飞虱泰丰B和R4684的抗性改良新品系的选择、蜜露量、群体生产率、存活率等，确定Bph33(t)基因的抗虫机制为抗生性。在该项目的资助下，发表SCI论文2篇，获授权国家发明专利2项，培养博士研究生1名，晋升正高级职称人员1名。

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