基于DGEP解析家蚕高温耐受力差异的关键基因及表达特征

South China region is located in tropical and subtropical climate zones, where high temperature and humidity environment makes a high claim for the varieties used in sericulture production. Significantly increasing the resistance level of varieties under the premise of maintaining economic character had become the bottleneck of upgrading and development of sericulture industry. Based on the problem in the current sericulture production, the project was carried out. In this project, a pair of varieties, high-temperature tolerant '932G' and sensitive 'HY', was selected. High-throughput sequencing was used for building the digital gene expression profiling of the midgut of 932G and HY after high temperature treatment, and the differential expression genes and discrepant metabolism or function pathways were screened by the bioinformatics analysis, which would be further verified and identified by bioinformatics analysis, molecular biology techniques including real time PCR, Western blot, immune histochemistry and RNA interference (RNAi) etc.. It is hoped to obtain the key genes related to high temperature stress tolerance and its experession profiles between silkworm varieties, and lay a good theoretical for the further use of the silkworm transgenic breeding high resistance varieties, and has important theoretical and practical significance to further promote the stable development of sericulture industry.

华南蚕区地处热带亚热带，高温多湿的环境对实用化蚕品种提出了很高的要求。在保持经济性状的前提下显著提高品种的抗逆性水平已经成为当前蚕桑产业升级发展的瓶颈。本项目正是基于当前蚕业生产中的这一突出问题开展相关基础研究。本项目以一对高温耐受力强的"932G"和高温敏感的"HY"作为试验材料，采用转录基因组高通量测序数字基因表达谱技术（DGEP）对高温处理后的试验蚕品种中肠组织的数字基因表达谱进行生物信息学分析，筛选品种内及品种间中肠组织的差异表达基因和代谢途径特征，经Real Time -PCR、Western blot、RNAi等分子生物学验证与鉴定，以期获得影响家蚕品种间高温耐受力差异的基因表达特征和关键基因，为进一步利用家蚕转基因技术选育高抗逆性强健品种提供试验素材和理论参考，对推动蚕桑产业的稳定发展具有重要的理论和现实意义。

针对华南蚕区家蚕实用品种抗逆性水平难以通过常规育种技术提高的问题开展了本项目的研究。项目通过对不同品种高温胁迫后中肠组织基因表达谱的分析，初步解析了高温对不同家蚕品种中肠的分子代谢的影响，发现氧化磷酸化、糖酵解、三羧酸循环等途径受高温影响显著，且这些代谢途径在高温耐受性家蚕品种中肠中的mRNA活性水平较高；探讨了高温胁迫后热激蛋白及化学感应蛋白家族成员等的基因表达模式，克隆鉴定了CSP7、CSP8和CSP9三个基因的上游启动子部分序列并对其进行了一致性分析；利用western blot及RNAi技术对分析了LP-C6等三个基因在高温胁迫后的蛋白质表达变化分析及其基因部分沉默后蚕体高温耐受性的变化分析，发现三个基因RNAi部分沉默后，影响了家蚕个体的高温耐受力，高温刺激后早期死亡率显著增加，因此我们推测其可能在高温耐受中起一定的作用。项目研究结果对进一步解析家蚕品种间高温耐受差异，及生物技术辅助家蚕高抗品种选育奠定了理论基础。