家蚕氨基酸转运蛋白与其抗BmNPV功能的研究

BmNPV (Bombyx mori L. nucleopolyhedrovirus) is a key pathogeny to silkworm with highly infective. And the reciprocity between virus and host is an important issue which was paid many attentions by researchers all the time. Mechanism associated with BmNPV resistance has not yet entirely clear, but it could be attributed to both host and viral. To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we investigated variations in the gene expression in the midguts of BmNPV-infected BC8 and 306 at 12 h pi using the microarray. Here, we defined the role of the amino acid transporter in insect antiviral. The complete cDNA sequence was 2520 bp long, potentially encoding a protein of 445 amino acids, and consisted of nine exons and eight introns, prediction of transmembrane helices showed that the candidate gene had 10 putative transmembrane domains. The amino acid transporter was similar to SLC32、SLC36 and SLC38, which belong to the ß group of SLC families. The intracellular compartmentalization events involving endomembrane transporters may be important to maintain intracellular absorption of essential amino acids and related metabolic processes in the group. We speculated that the identified amino acid transporter may play an important role in the anti-NPV. However, the function of the amino acid transporter and the molecular mechanism of the anti-NPV were not known, so far. The DNA sequences、cDNA sequences and the differential expression of resistant and susceptible genes were analyzed in this project. The resistant genes and molecular mechanism in Bombyx mori against BmNPV in Silkworm will be got.

昆虫抗NPV虽然研究多年，但一直未鉴定出抗性基因，申请人利用基因芯片筛选出一种新颖的氨基酸转运蛋白，该蛋白跟抗性相关，并初步进行了验证。该基因位于Z染色体，该蛋白相似于SLC32、SLC36和SLC38，属于SLC家族ß亚族。在多细胞动物中，该亚族有关的内膜转运蛋白的细胞内的区室化对维持必需氨基酸的细胞内的吸收和代谢过程起非常重要作用。查阅相关文献，我们猜测筛选出的氨基酸转运蛋白在抗NPV中起着重要作用。然而，该氨基酸转运蛋白的基因功能及抗NPV的分子作用机制迄今尚不清楚。本项目拟分析该基因的组织表达分布和表达时相；鉴定在抗性、感性家蚕的基因组序列、cDNA序列及基因调控的差异，最后通过构建转基因家蚕进一步进行验证。最终希望鉴定出家蚕抗NPV的基因及阐明抗核型多角体病毒家蚕的抗性分子机理。

氨基酸转运蛋白是一类调控氨基酸转运的膜蛋白，参与细胞的各种生理活动。然而氨基酸转运蛋白在病毒感染尤其是在家蚕BmNPV感染中的作用还未知。本项目前期通过基因芯片研究发现家蚕氨基酸转运蛋白在抗性家蚕品系NB中高表达，推测该基因在抗BmNPV中起到关键作用。本项目为了研究该基因在抗BmNPV中作用，分别设计了过表达和干扰实验进行功能验证。定量PCR和western结果表明，家蚕氨基酸转运蛋白在BmNPV感染的抗性品系NB中明显比感性品系表达水平高。细胞过表达氨基酸转运蛋白实验结果表明在过表达细胞系中能抑制BmNPV增殖。而RNA干扰实验结果表明抑制氨基酸转运蛋白表达的细胞中BmNPV增殖显著增加。这些结果说明了家蚕氨基酸转运蛋白在抑制BmNPV增殖中起到关键作用。后续的研究将更深入的研究家蚕氨基酸转运蛋白在抗BmNPV增殖的具体机制。