高温介导葡糖脱氢酶Glucose dehydrogenase (GLD)在班氏跳小蜂性别分配中的作用机制

The invasive mealybug Phenacoccus solenopsis (Hemiptera: Pseudococcidae) has been recognized as an important pest in China, and Aenasius bambawalei (Hymenoptera: Encyrtidae) is the predominant accompanying parasitic wasp of this mealybug. Moreover, as an excellent biocontrol agent, the parasitization rate of this parasitic wasp could up to more than 60% in field. However, in the mass rearing, there are more male offspring in the population. Our previous findings have shown that 36 ℃could significantly enhance the proportion (45.5%) of female offspring of A. bambawalei . In addition, the expression of Glucose dehydrogenase (GLD) gene in this temperature is the most. On the other hand, GLD could facilitates both sperm uptake and release , and plays a critical role in the sex allocation. Thus, make clear the mechanism of high temperature mediate GLD for sex allocation of A. bambawalei is very important for solving the problem of more male offspring. In this study, 1)CRISPR/Cas 9 gene edit technology will be used to tested the comprehensive function of GLD; 2) the function of signal pathway which GLD involved in will be verified ; 3) mechanism of sex allocation resulted from the role of high temperature on GLD expression will be demonstrated. Our expected findings will uncover the mechanism involved in sex allocation in A. bambawalei, and on the other hand, will helpful for solving the problem of more male offspring in future.

班氏跳小蜂Aenasius bambawalei是重要入侵害虫扶桑绵粉蚧Phenacoccus solenopsis的优势伴迁天敌，野外寄生率达60%以上，应用前景广阔。室内饲养条件下，该蜂存在子代偏雄化现象。本课题组前期研究发现，36℃高温条件下该蜂的子代雌性比例可提高45.5%，且其体内葡糖脱氢酶Glucose dehydrogenase(GLD)表达量显著上调。GLD可参与精子的吸收和释放，与寄生蜂的性别分配密切相关。因此，研究高温介导GLD的作用机制，对于解决该蜂偏雄化问题具有重要指导意义。本项目将1）应用CRISPR9基因编辑方法，分析GLD功能；2）明确GLD参与的信号通路在性别分配中的作用；3）论证高温介导与GLD表达的关系。以上研究结果既有助于揭示班氏跳小蜂性别分配的内在机制，又可为解决该蜂偏雄化问题提供重要科学依据，以利于其规模化饲养及利用。

班氏跳小蜂Aenasius bambawalei 是重要入侵害虫扶桑绵粉蚧Phenacoccus solenopsis 的优势种伴迁天敌，野外寄生率达60%以上。室内饲养条件下，该蜂存在子代偏雄化现象。本课题组前期研究发现，36℃高温条件下该蜂的子代雌性比例可提高45.5%，且其体内葡糖脱氢酶Glucose dehydrogenase (GLD) 表达量显著上调。前人研究表明，GLD基因可参与精子的吸收和释放，与寄生蜂的性别分配密切相关。因此，研究高温介导GLD基因的作用机制，对于解决该蜂偏雄化问题具有重要指导意义。本项目分别利用CRISPR/Cas9和基因工程技术构建班氏跳小蜂GLD基因缺失和过表达突变体并验证其功能，结果均表明，高温通过影响GLD基因的表达量升高，从而使得其子代雌性比例升高；使用String数据库构建与GLD蛋白质相互的蛋白网络，并搜寻与GLD互作的上下游靶基因及其参与的信号通路，结果表明，Gaph2和Pgi参与的脂肪酸生物合成（map00061）与代谢（map01212）通路中包含若干与生殖相关的基因，如 elongation of very long chain fatty acids protein， fatty acyl-CoA reductase 1-like 和serine/ threonine-protein kinase等；利用CRISPR/Cas9技术构建班氏跳小蜂以上3个基因缺失突变体，荧光定量PCR结果表明，fatty acyl-CoA reductase 1-like 和serine/ threonine-protein kinase基因缺失个体的GLD基因表达量及子代雌性比例均显著下降，而elongation of very long chain fatty acids protein基因敲除之后，GLD基因表达量和子代雌性比例均无明显变化；通过比较高温和常温下GLD基因缺失个体的生殖腺发育速率、卵子和精子数量，结果表明，高温虽然能促进班氏跳小蜂GLD基因缺失个体雌雄生殖腺的发育，但因为GLD基因的缺失，使得精子的成熟与转运均受到影响，从而最终影响到班氏跳小蜂的子代雌雄性比。

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