RNAi提高家蚕对BmNPV的抗性及家蚕精细胞发育相关基因Achi功能的研究
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摘要
本研究构建了基于piggyBac转座子、表达有BmNPV复制必需基因ie-1和lef-1短片段dsRNA和双筛选基因（gfp和neo基因）的转基因载体piggyantiIE-lef1-neo。转染有该载体的稳定转化细胞在接种病毒稀释度为10-9时，感染后2d天细胞中ie-1基因的转录表达量比对照组下降了97倍。采用精子介导并结合G418的筛选，我们获得了可稳定遗传的转基因家蚕。转基因家蚕在接种106/mL的BmNPV多角体时，感染11d后转基因家蚕的死亡率仍比对照组下降了40%。这些表明稳定转化细胞和转基因家蚕对BmNPV的抗性显著提高。
本研究同时展开了对家蚕Bmachi基因功能的研究。研究发现：Bmachi主要在精巢和卵巢中表达，且精巢中表达有四种剪接体（FJ643616，FJ913885，FJ913886和GQ120180），卵巢中有两种剪接体表达存在(FJ502823和GQ120180)。对精巢中存在的四种剪接体进行qPCR，结果显示FJ913885剪接体的表达量最高。
qPCR和RNA原位杂交显示了Bmachi基因在三龄时期的精巢大量表达，该时期是初级精母细胞大量形成时期。凝胶阻滞实验显示复性后的BmAchi蛋白和带有该蛋白结合位点的DNA序列有结合作用。BmAchi含有两个相邻的酸性结构域，荧光素酶的光子数检测提示这两个酸性结构域对报告基因的表达起转录激活作用。培养细胞免疫荧光检测结果显示BmAchi蛋白主要位于BmN细胞的细胞核内；家蚕精巢组织的免疫荧光检测显示BmAchi在初级精母细胞和游动的精子中均有表达。家蚕注射Bmachi-siRNA的实验显示出该基因为一个减数分裂阻滞基因，注射组家蚕的未受精率比对照组高出35%左右，且抑制Bmachi的表达可导致精巢中精细胞的发育减缓。免疫共沉淀的结果显示BmAchi与BmAly有相互作用，提示了他们可能和其他减数分裂阻滞因子一起形成了一个复合物，调节与减数分裂和精子形成有关基因的转录。
Our study successfullly constructed the transgenic vector congtaing short fragmentdsRNAs of two essential genes (ie-1and lef-1) of BmNPV and two screening genes(gfpand neo).The stale transformed cells exhibited obvious resistance to BmNPV wheninoculated10-9diluted wild type BmNPV; in addition, the identified transgenicsilkworms also showed high efficacy of RNAi while infected BmNPV hydropolyvirusat a concentration of106/mL as a result of the mortality of transgenic silkwormsdecreased40%compared to the control groups.
In this study, we also investigated the characteristics and functions of BmAchi, aBombyx mori homolog of DmAchi. Like DmAchi, BmAchi is predominantly expressedin the testes and ovaries. Four alternatively spliced isoforms could be isolated fromtestes, and two isoforms from ovaries. Quantitative polymerase chain reaction indicatedthat Bmachi was abundantly expressed in the testis of3rd instar larvae, when the testisis almost full of primary spermatocytes. The results of luciferase assays indicated thatBmAchi contains two adjacent acidic domains that activate the transcription of reportergenes. Immunofluorescence assay in BmN cells showed that the BmAchi protein waslocated mainly in the nucleus, and paraffin sections of testis showed that BmAchi wasgrossly expressed in primary spermatocytes and motile sperms. Consistent with the roleof DmAchi in Drosophila development, BmAchi significantly affected spermatiddifferentiation, as indicated by both hematoxylin-eosin staining of paraffin sections oftestis from Bmachi-small interfering RNA (siRNA)-injected male silkworms, and by theincidence of unfertilized eggs produced by crossing Bmachi-siRNA-injected males withnormal females. Co-immunoprecipitation experiments suggested that BmAchiinteracted with BmAly, and that they may recruit other factors to form a complex toregulate the genes required for meiotic divisions and spermatid differentiation.
本研究同时展开了对家蚕Bmachi基因功能的研究。研究发现：Bmachi主要在精巢和卵巢中表达，且精巢中表达有四种剪接体（FJ643616，FJ913885，FJ913886和GQ120180），卵巢中有两种剪接体表达存在(FJ502823和GQ120180)。对精巢中存在的四种剪接体进行qPCR，结果显示FJ913885剪接体的表达量最高。
qPCR和RNA原位杂交显示了Bmachi基因在三龄时期的精巢大量表达，该时期是初级精母细胞大量形成时期。凝胶阻滞实验显示复性后的BmAchi蛋白和带有该蛋白结合位点的DNA序列有结合作用。BmAchi含有两个相邻的酸性结构域，荧光素酶的光子数检测提示这两个酸性结构域对报告基因的表达起转录激活作用。培养细胞免疫荧光检测结果显示BmAchi蛋白主要位于BmN细胞的细胞核内；家蚕精巢组织的免疫荧光检测显示BmAchi在初级精母细胞和游动的精子中均有表达。家蚕注射Bmachi-siRNA的实验显示出该基因为一个减数分裂阻滞基因，注射组家蚕的未受精率比对照组高出35%左右，且抑制Bmachi的表达可导致精巢中精细胞的发育减缓。免疫共沉淀的结果显示BmAchi与BmAly有相互作用，提示了他们可能和其他减数分裂阻滞因子一起形成了一个复合物，调节与减数分裂和精子形成有关基因的转录。
Our study successfullly constructed the transgenic vector congtaing short fragmentdsRNAs of two essential genes (ie-1and lef-1) of BmNPV and two screening genes(gfpand neo).The stale transformed cells exhibited obvious resistance to BmNPV wheninoculated10-9diluted wild type BmNPV; in addition, the identified transgenicsilkworms also showed high efficacy of RNAi while infected BmNPV hydropolyvirusat a concentration of106/mL as a result of the mortality of transgenic silkwormsdecreased40%compared to the control groups.
In this study, we also investigated the characteristics and functions of BmAchi, aBombyx mori homolog of DmAchi. Like DmAchi, BmAchi is predominantly expressedin the testes and ovaries. Four alternatively spliced isoforms could be isolated fromtestes, and two isoforms from ovaries. Quantitative polymerase chain reaction indicatedthat Bmachi was abundantly expressed in the testis of3rd instar larvae, when the testisis almost full of primary spermatocytes. The results of luciferase assays indicated thatBmAchi contains two adjacent acidic domains that activate the transcription of reportergenes. Immunofluorescence assay in BmN cells showed that the BmAchi protein waslocated mainly in the nucleus, and paraffin sections of testis showed that BmAchi wasgrossly expressed in primary spermatocytes and motile sperms. Consistent with the roleof DmAchi in Drosophila development, BmAchi significantly affected spermatiddifferentiation, as indicated by both hematoxylin-eosin staining of paraffin sections oftestis from Bmachi-small interfering RNA (siRNA)-injected male silkworms, and by theincidence of unfertilized eggs produced by crossing Bmachi-siRNA-injected males withnormal females. Co-immunoprecipitation experiments suggested that BmAchiinteracted with BmAly, and that they may recruit other factors to form a complex toregulate the genes required for meiotic divisions and spermatid differentiation.
引文
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