褐飞虱分子细胞遗传图构建

Brown planthopper (Nilaparvata lugens) is one of most serious insect pests of rice (Oryza sativa) in the world, whose occurrence area reaches to hundreds of millions of acres of times every year, and often causes serious yield loss. The chemical insecticide and resistant rice varieties are usually used to control this important pest and give rise to drug resistance and virulence which make the damage much worse. It is the reason that the genetic variation and virulence mechanism of N. lugens are currently unknown. Thus, it is greatly necessary to study on the basic genetics of brown planthopper. .In this study, we have constructed a high density genetic linkage map in the previous work. We will use the probes of repetitive sequence based on the information of high-throughput genomic sequence of N. lugens for fluorescence in situ hybridization (FISH) analysis, and construct a standard molecular karyotype map of N. lugens. Then we will construct the BAC (bacterial artificial chromosome) library for the genome of N. lugens, and screen the BAC clones for probes of FISH by using the molecular markers from the linkage groups of our genetic map. At the same time, the sequences of full-length cDNA from single copy gene are cloned for probes of FISH. We construct a complete cytogenetic map of brown plalnthopper by using FISH analysis which makes a linkage group correspond to a specific chromosome. The results of this study not only supply important technique for the research of integrated pest management of N. lugens, but also provide essential information for the improvement of sequence assembly and offer molecular tools for brown planthopper genomics research, comparative genomics and evolutionary study.

褐飞虱是我国水稻生产中发生和为害面积最大的害虫，每年发生面积达几亿亩次，造成水稻严重减产。利用化学杀虫剂和抗性水稻品种进行控制会使其产生抗药性和致害性，导致防治效果明显下降，究其原因是褐飞虱的遗传变异规律和致害机理不明了。因此，对褐飞虱进行基础性遗传学研究显得十分必要。.本项目在我们已成功构建褐飞虱高密度遗传连锁图的基础上，拟根据褐飞虱基因组的高通量序列信息制备重复序列探针并进行荧光原位杂交（FISH），建立褐飞虱标准分子核型图；用连锁群上的分子标记筛选基因组BAC文库获取特异BAC克隆并作为探针，同时以克隆单拷贝基因cDNA全长序列为探针，利用FISH技术将连锁群定位于染色体，从而构建褐飞虱的完整分子细胞遗传图。本研究结果不仅可以为褐飞虱的综合防治研究提供重要技术支持，而且能够为褐飞虱全基因组测序工作提供必要信息，为褐飞虱的基因组学、比较基因组学和进化等基础性研究提供强有力的分子工具。

褐飞虱是水稻生产中的首要害虫，而褐飞虱分子遗传方面的基础研究较为薄弱。前期研究构建了褐飞虱的包含886个分子标记的高密度遗传连锁图谱。本项目对褐飞虱基因组进行高通量测序，生物信息学分析后寻找到褐飞虱的24条高丰度重复序列，然后以重复序列为探针的FISH技术初步建立褐飞虱标准分子核型图。在褐飞虱性染色体的基因（YC基因）功能研究中，已获得YC基因的cDNA序列全长，序列分析表明此基因包含与精子形成相关的保守结构域，推测与褐飞虱生殖相关，然后根据cDNA序列信息合成用于RNAi干涉技术的dsRNA，并进行褐飞虱的显微注射，观察此基因表达降低后表型，完成功能验证工作。在褐飞虱致害性基因定位研究中，以生物型1与生物型Y为研究对象，这两种生物型褐飞虱在致害性表型（包括寄主选择性，蜜露量，生长率和产卵量）上存在显著性差异。构建了两种褐飞虱的近交系，采用遗传定位方法已将致害性基因定位于褐飞虱的染色体某一区段内。本项目研究为褐飞虱的分子遗传学研究奠定坚实基础，为防控褐飞虱提供理论依据。在项目实施过程中，目前已在Plant Physiology，Current Opinion in Insect Science 和Applied Entomology and Zoology等国际期刊发表SCI源论文5篇。