荒漠甲虫准噶尔小胸鳖甲低温响应转录组的研究及重要基因的功能鉴定

Tenebrionid insects in Junggar Basin account for more than 70% species of the desert insects in Xinjiang. Microdera punctipennis dzungarica is an endemic species in this region, its mechanism in cold hardiness is representative in the desert insects in northern Xinjiang. In this project，we will carry out transcriptomic study on M. punctipennis adults treated at 4℃ for different time lengths by extracting the total RNA and then RNA-seq sequencing so that to systematicaly study the transcritomic changes among the M.punctipennis beetles treated with different conditions. The differentially expressed genes will be screened by deep bioinformatic analyses through comparing the transcriptomic data of the different samples, and the regulatary net work will, then, be constructed. The up-regulated genes will be analyzed by qRT-PCR for the transcriptional expression profile under different low temperatures and under certain low temperature for different time period. In addition, cold related functions of the genes which are significantly up-regulated by low temperature in the transcriptional profile will be identified by analyzing the biological features and the physiological and biochemical indexes under low temperature of the transgenic flies which will carry the candidate genes. Meanwhile, we will also do functional identification of the candidate genes in cold sensitive yeast which will be transformed with these genes and with RNA interference. By combining the results from the above experimental plans, it is possible to discover the major genes in response to low temperature, and to shed light into the molecular mechanism that insect adapts to desert environment. In the application, the discovered new genes can be used to breed cold tolerant new species through biotechnology method, and to develop new types of cryoprotectant.

准噶尔盆地的拟步甲科昆虫占新疆荒漠昆虫种类的70%以上。准噶尔小胸鳖甲是该地区的特有种，其耐寒机制在新疆荒漠昆虫中具有代表性。本项目采用转录组学的研究策略，从低温处理不同时间小胸鳖甲基因表达的差异入手，提取4℃处理不同时间（0h、3h、9h）样品的总RNA，进行RNA-seq测序和深入的生物信息学分析，系统研究低温处理不同时间小胸鳖甲的转录组变化，通过比较不同样品的转录组数据，找出与小胸鳖甲低温响应有关的基因，构建相关基因的调控网络。对转录表达谱中明显受低温上调表达的基因进行功能鉴定，分析转基因果蝇在低温下的生物学特性和生理生化指标。以酵母转基因和RNA干涉法同步鉴定基因功能，分析低温处理过程中相关基因在耐寒性形成中的作用，揭示小胸鳖甲耐寒的分子机理。通过本研究可发现重要的低温响应基因，揭示荒漠昆虫的低温响应机制。在应用上，可利用相关基因培育耐低温生物新品种和开发新的生物抗冻保护剂。

利用高通量测序技术建立了小胸鳖甲的4℃和25℃转录组，获得51712个平均长度为984 bp的非冗余基因，约57.87％有同源序列（E-value<10-5）。低温响应上调基因有514个，富集到35个GO类群，18个KEGG途径。其中胁迫响应、生物调节、免疫系统过程等类群都占有重要比例，嘌呤代谢、硫胺素代谢、糖酵解/糖异生等代谢通路显著富集。在胁迫响应类群中，热激蛋白Hsp90、超氧化物歧化酶(SOD)，钙联接蛋白Calnexin等8个非生物胁迫相关基因显著上调表达，提示小胸鳖甲可能从分子伴侣、细胞周期阻滞、线粒体稳定性、抗氧化胁迫等多方面应对低温胁迫。嘌呤代谢和硫胺素代谢在其他昆虫低温转录组没有发现，表明小胸鳖甲在低温下的转录组代谢通路与其他物种有较大差异。研究了27个基因在4℃和-4℃的表达谱，主要为典型的胁迫响应的双峰型，MAPK通路上游和中游基因的低温响应十分迅速。SOD、几丁质酶等基因响应的程度很强烈。昆虫几丁质酶基因响应低温胁迫为本研究首次发现。利用大肠杆菌和酵母表达系统对几丁质酶、甘油合成酶MpGPDH和MpGK、p38MAPK、SOD、水通道蛋白AQP等基因进行了耐寒性功能验证，均能显著提高转化菌的耐寒性。综合研究结果，从代谢转换、生物大分子保护等方面绘制了小胸鳖甲低温响应的调控网络图，初步阐明了荒漠甲虫小胸鳖甲低温响应的分子机制，研究结果为深入挖掘耐寒性相关基因、与模式昆虫进行基因组学比较研究、深入研究昆虫耐寒性的分子机制都具有重要价值。