稻飞虱性别决定分子机理研究

Sex determination is one of the fundamental problems in biology. Revealing the sex determination molecular mechanisms for important insect pests is not only very valuable for basic biology, but also very important for insect pest control both in theory and practice. Sex determination cascade in several known holometabolous insects but silkworm is: sexual instruction signal→ female switch gene Transformer(tra)/ Fem→doublesex (dsx) →executive genes. In the cascade, tra and dsx are two conserved genes. However, up to date, no gene homologous to tra/fem are found in any hemimetabolous insect genomes, and no sexual instruction signal is revealed in hemimetabolous insects. In our pilot studies, we confirmed a dsx in the brown planthopper, and more importantly, we luckily found a gene we tentatively named Pfm which could regulate the sex-specific alternative transcription of dsx, maternal RNAi of Pfm resulted all offspring becoming male or pseudomale. Based on these finding, we will further conduct studies on cascade relationship among Pfm and other sex determination genes, so as to reveal if the planthoppers also possess a tra→dsx -like sex determination cascade as that in holometabolous insects, and to understand the specificity and conservation in the sex determination of hemimetabolous insects. We will also compare the differences and similarities in the sex determination mechanisms in three rice planthoppers, the brown planthopper, white backed planthopper and small brown planthopper, which differ in chromosome numbers. Finally, we will edit the genome of the brown planthopper using CRISPR/Cas9+Gene-drive technology to explore a way for using sex determination genes in rice planthopper control.

性别决定是生物学基本问题，研究重要害虫性别决定机理，不仅具重要理论意义，对害虫防治也具重要实际价值。已知全变态昆虫性别决定基本级联为：初始因子→性别开关基因Tra→双性基因dsx→执行基因，其中tra和dsx是保守的。但至今不完全变态昆虫基因组中还未发现tra的同源基因，初始因子更是不清。我们在前期研究中，确定了褐飞虱dsx，特别是幸运地筛到了能调控dsx转录本性别特异性剪切的1个上游因子（暂名为Pfm），对该基因进行母体RNAi可使后代全为雄/假雄性。本研究拟在此基础上，深入研究Pfm和其它性别基因的级联关系，以明确稻飞虱性别决定是否存在类似于全变态昆虫的tra→dsx保守级联，以揭示不全变态昆虫性别决定级联特性，并比较染色体数量存在差异的褐飞虱、白背飞虱和灰飞虱性别决定异同点。最后通过CRISPR/Cas9+Gene drive探索将性别决定基因应用于稻飞虱遗传防控的途径。

不完全变态昆虫的性别决定至今所知甚少。我们在半翅目昆虫褐飞虱Nilaparvata lugens中鉴定到竟有多达4个的双性基因doublesex（dsx），但只有其中一个较为接近于线虫mab-3的Nldsx具有雌(dsx-F)雄(dsx-M)特异性的可变剪切本和雌雄共有可变剪接本（dsx-c）， 并且RNAi干扰表明其是性别发育所必须，而与完全变态昆虫的dsx接近的Nldsx-like1及与哺乳动物Dmrt1接近的Nldsx-like2和Nldsx-like3则与性别发育无关。与完全变态的dsx不同，褐飞虱的Nldsx蛋白含有一个不保守的OD1结构域，而OD2则鉴定不到。对褐飞虱雌虫的Nldsx进行RNAi会导致个体变大，但生殖正常，而若虫期对雄虫Nldsx-M进行RNAi会导致雄成虫雌性化和不育。我们深入研究进一步鉴定到了1个雌性化分子开关female determiner (Nlfmd) ，其编码一个富含丝氨酸和精氨酸的蛋白。在若虫期用RNAi敲低雌虫的Nlfmd会导致雄dsx-M特异性转录本上升和雌虫雄性化表型。 卵中的雌性特异性的Nlfmd转录本Nlfmd-F最先从母体带到子代卵中，在子代合子阶段自己再开始转录。 RNAi和CRISPR-Cas9基因编辑敲除Nlfmd会导致雌性胚胎特异性死亡。敲低 hnRNP40 家族的一个基因（我们命名为female determiner 2，Nlfmd2) 也导致雌虫雄性化。体外实验表明Nlfmd2 的一个转录本NlFMD2-340编码蛋白能与Nldsx pre-mRNA 的重复基序RAAGAA 结合并与 NlFMD-F 互作而形成一个复合体，从而调控Nldsx 可变剪接进而控制性别分化，这表明NlFMD2 可能是作为雌性分子开关NlFMD的配体蛋白与RNA结合。 我们的研究结果揭示了一种新的昆虫性别决定调控机制。

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