Ras1CA超表达提高蚕丝产量的分子机理解析

In the transgenic silkworm Fil-GAL4>UAS-Ras1CA, overexpression of the Ras1CA oncogene specifically in the Bombyx posterior silk gland increases cell size and fibroin protein synthesis, leading to silk yield improvement and promising application in sericulture. To identify more proper candidate genes to improve silk yield, the applicant tends to further illustrate the detailed molecular mechanism of Ras1CA overexpression in the posterior silk gland using transcriptomics and proteomics. Preliminary analysis of transcriptome results showed that Ras1CA overexpression in the posterior silk gland downregulates 20-hydroxyecdysone (20E) response genes during larval-pupal metamorphosis. The applicant will determine which Ras1 downstream signaling pathway (Raf-MAPK or PI3K-TORC1) suppressed the 20E-triggered transcriptional cascade as well as 20E-induced autophagy and apoptosis. Peliminary analysis of proteome results indicated that the protein level of cysteine protease inhibitor (BCPI) was increased in the posterior silk gland of Fil-GAL4>UAS-Ras1CA. The developmental profile of BCPI mRNA level suggested a regulation by the 20E signal. We will further identify which Ras1 downstream signal pathway collaborates with 20E to regulate BCPI mRNA or protein levels as well as BCPI-inhibited cell dissociation. As last, we will generate GAL4/UAS transgenic silkworm to overexpress EcRDN in posterior silk gland to understand whether decreasing the 20E signal might inhibit autophagy,apoptosis and cell dissocation as well as improve silk yield.

转基因家蚕FiL-GAL4>UAS-Ras1CA后部丝腺变大、丝蛋白合成增加、蚕丝产量提高，有较好的应用前景。为挖掘提高蚕丝产量的更佳候选基因，拟对Fil-GAL4>UAS-Ras1CA的后部丝腺进行转录组和蛋白组分析，进一步解析其提高蚕丝产量的分子机理。转录组学初步结果显示后部丝腺中超表达Ras1CA可抑制幼虫-蛹变态时期蜕皮激素（20E）反应基因的表达；将确定哪条Ras1下游信号(Raf-MAPK或PI3K-TORC1)抑制20E信号传导、以及20E诱导的细胞自噬和凋亡。蛋白组学初步结果显示半胱氨酸蛋白酶抑制剂BCPI的蛋白水平上调，BCPI mRNA发育变化结果暗示其受20E信号调控；将解析哪条Ras1下游信号与20E信号协同调控BCPI抑制的细胞解离。最终，在后部丝腺中超表达EcRDN，检测抑制20E信号及其促进的细胞自噬、凋亡和解离是否可以提高蚕丝产量。

利用GAL4/UAS双源转基因系统，在后部丝腺特异性超表达Ras1CA.转基因家蚕FiL-GAL4>UAS-Ras1CA 后部丝腺变大、丝蛋白合成增加、蚕丝产量提高，有较好的应用前景。为深入解析Ras1 超表达提高蚕丝产量分子机制，我们对野生型和Ras1CA超表达家蚕的后部丝腺进行了转录组和蛋白组比较分析。在转录组方面，利用Solexa高通量测序技术，比较上蔟早期转基因和野生型家蚕后部丝腺的转录本差异情况，对差异表达的基因进行了注释分析和实时定量PCR 验证，发现Ras1CA超表达促进Raf-MAPK和PI3K-TORC1信号途径大部分基因的表达，从而转录激活Ras1下游信号(Ma et al., 2014 BMC Genomics)。在蛋白质组学方面，利用2D-DIGE/MS-MS的方法，比较上蔟早期野生型和转基因家蚕后部丝腺的蛋白差异，发现在转基因家蚕后部丝腺中有一个可抑制组织蛋白酶活性的小分子蛋白BCPI 表达增多。进而阐明Ras1可通过其下游Raf-MAPK 和PI3K/TORC1信号通路上调BCPI的表达而抑制组织蛋白酶cathepsin促进的后部丝腺解离。这两部分研究工作的完成，不仅保证了申请人所主持自然基金青年项目的顺利结题，也为后续更为具体深入阐明Ras1CA超表达提高蚕丝产量的分子机理奠定良好的理论基础并指明了方向。