丝蛋白基因的分子设计及过表达RaslCA基因提高实用品种产丝量研究
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摘要
家蚕（Bombyx mori）属鳞翅目蚕蛾科，可大规模饲养，遗传背景比较清楚，其丝腺具有合成和分泌丝蛋白的能力。蚕丝有纤维皇后之美誉，但也存在易霉变，黄化、皱缩、褪色和脆化等缺陷。为了探讨通过家蚕丝素基因（fib）的分子设计与遗传改造，从基因水平改善丝纤维的性能，本研究利用家蚕丝素轻链基因（fib-L）上游约1.2kb的第5~6外显子片段和约0.5kb的第7外显子及其下游序列作为基因打靶的左右同源臂，将gfp基因和人工合成的家蚕抗菌肽基因（cec）融合克隆插入到两同源臂之间，并使其与fib-L基因处于同一读码框；将由ie-1驱动的DsRed基因克隆至同源右臂右侧，作为负选择标记，构建基因打靶载体pSK-Fib.L-L-GFP-cec-Fib.L-R-IE-DsRed-PolyA。将该载体转染BmN细胞后，发现部分转染细胞呈现绿色荧光而无红色荧光，表明载体构建成功。将该载体通过精子介导转入家蚕卵，在G0代筛选茧呈绿色荧光的家蚕，PCR检测证实筛选出的蚕为转基因家蚕；同蛾圈转基因家蚕杂交传代，用GFP抗体对G6代家蚕后部丝腺组织进行western blot检测，可观察到Fib-L-GFP融合蛋白的特异性条带；抗菌性实验显示，转基因家蚕丝表面的菌落数明显少于对照组；转基因家蚕丝在紫外光的激发下发出绿色荧光，表明通过基因打靶gfp-cec已与fib-L融合并表达。
为了探讨提高实用品种的产丝水平和叶丝转化率的可能性，将后部丝腺特异性表达Gal4的Fil-Gal4/3XP3-DsRed2转基因家蚕系与实用品种菁松回交6代，获得Fil-Gal4转基因家蚕菁松系统；将带有UAS-Ras1~(CA)元件的UAS-Ras1~(CA)/3XP3-EGFP转基因家蚕系与实用品种皓月回交6代，获得UAS-Ras1~(CA)转基因家蚕皓月系统。将Gal4菁松系与UAS皓月系杂交，得到在后部丝腺特异性表达Ras1~(CA)的转基因实用品种Fil-Gal4-菁松×UAS-Ras1~(CA)-皓月，调查结果显示，与菁松×皓月相比，转基因家蚕的的后部丝腺重和蚕丝产量分别提高了11%、1.3%，叶丝转化率提高0.57%。实时定量PCR检测结果显示Fil-Gal4-菁松×UAS-Ras1~(CA)-皓月后部丝腺Ras1mRNA水平提高了4.08倍。
The silkworm Bombyx mori belonging to Lepidoptera Saturniidae, can be raised ona large-scale, and its genetic background is clear. Its silk glands can synthesize andsecrete silk protein. To improve the performance of silk fibre by molecular designingand genetic modification of the fibrion gene of silkworm in molecular level, a1.2kb(between exon5and exon7)and a0.5kb (between exon7and its following partial) of thesequence located at fibroin light chain (fib-L) gene as homologous arms, a greenfluorescent protein gene (gfp) and artificialaynthesis silkworm antibiotic peptide genewas flanked by fib-LL and fib-LR, the DsRed gene with ie-1promoter was clonedfollowed by the right homologous arm as the negative chosing factor, to generate agene-targeted vector: pSK-Fib.L-L-GFP-cec-Fib.L-R-IE-DsRed-PolyA, in which the gfpgene and fib-L gene were in same open reading frame. BmN cells with greenfluorescence but without red fluorescence could be found after being transfected withthe vector, indicating the vector was successfully constructed. The targeted vector wasintroduced into the eggs of silkworm using sperm-mediated gene transfer, somesilkworms with green fluorescent cocoons were screened out, PCR results confirmedthat the silkworm was transformant. In the posterior silk glands of the G6generationtransformation silkworms, a specific band representing the fusion protein of GFP andFib-L could be detected by Western blotting with an antibody against GFP; antibacterialexperiment was done with the silk fibre spanned by transformation silkworms showedless clonies than the control;
the silk fibre spanned by transformation silkworms emit fluorescent under theexcitation of ultraviolet light. These results indicated that the gfp gene could beintegrated into the fib-L gene locus of silkworm genome by gene targeting and fusion protein of Fib-L and GFP was expressed successfully.
In addition, the transgenic silkworm had a high silk yield and converting food intosilk based on a binary Gal4/UAS system from the Chinese Academy of Sciences inShanghai. But it is difficult to apply this method in sericulture because the Ras1~(CA)wasexpressed in non-diapause silkworm. So, we designed the Gal4transgenic silkwormhybridized with stock JingSong and screened out some silkworms withred fluorescent eyes, then the next generation transgenic silkworm was constantlybackcross with JingSong to generate G6-Gal4diapause JingSong silkworm.
we also designed the UAS transgenic silkworm hybridized with stock HaoYue andscreened out some silkworms with green fluorescent eyes, then the next generationtransgenic silkworm was constantly backcross with HaoYue to generate G6-UASdiapause HaoYue silkworm.Then the transgenic Gal4JingSong silkworm is
hybridized with the transgenic UAS HaoYue silkworm to get the Ras1~(CA)specifically expressed. The results showed Ras1~(CA)overexpression improved posteriorsilk gland weight and silk yield11%、1.3%, confirming substantial increment of silkproduction capacity in transgenic silkworm; But food consumption only increased0.57%, which has potential in sericulture. As measured by quantitative real-timePCR,Ras1~(CA)mRNA level in the posterior silk gland of Fil-Gal4/UAS-Ras1~(CA)silkwormwas4.08fold higher than in controls. These results ensured Ras1~(CA)is successfullyoverexpressed in the posterior silk gland.
为了探讨提高实用品种的产丝水平和叶丝转化率的可能性，将后部丝腺特异性表达Gal4的Fil-Gal4/3XP3-DsRed2转基因家蚕系与实用品种菁松回交6代，获得Fil-Gal4转基因家蚕菁松系统；将带有UAS-Ras1~(CA)元件的UAS-Ras1~(CA)/3XP3-EGFP转基因家蚕系与实用品种皓月回交6代，获得UAS-Ras1~(CA)转基因家蚕皓月系统。将Gal4菁松系与UAS皓月系杂交，得到在后部丝腺特异性表达Ras1~(CA)的转基因实用品种Fil-Gal4-菁松×UAS-Ras1~(CA)-皓月，调查结果显示，与菁松×皓月相比，转基因家蚕的的后部丝腺重和蚕丝产量分别提高了11%、1.3%，叶丝转化率提高0.57%。实时定量PCR检测结果显示Fil-Gal4-菁松×UAS-Ras1~(CA)-皓月后部丝腺Ras1mRNA水平提高了4.08倍。
The silkworm Bombyx mori belonging to Lepidoptera Saturniidae, can be raised ona large-scale, and its genetic background is clear. Its silk glands can synthesize andsecrete silk protein. To improve the performance of silk fibre by molecular designingand genetic modification of the fibrion gene of silkworm in molecular level, a1.2kb(between exon5and exon7)and a0.5kb (between exon7and its following partial) of thesequence located at fibroin light chain (fib-L) gene as homologous arms, a greenfluorescent protein gene (gfp) and artificialaynthesis silkworm antibiotic peptide genewas flanked by fib-LL and fib-LR, the DsRed gene with ie-1promoter was clonedfollowed by the right homologous arm as the negative chosing factor, to generate agene-targeted vector: pSK-Fib.L-L-GFP-cec-Fib.L-R-IE-DsRed-PolyA, in which the gfpgene and fib-L gene were in same open reading frame. BmN cells with greenfluorescence but without red fluorescence could be found after being transfected withthe vector, indicating the vector was successfully constructed. The targeted vector wasintroduced into the eggs of silkworm using sperm-mediated gene transfer, somesilkworms with green fluorescent cocoons were screened out, PCR results confirmedthat the silkworm was transformant. In the posterior silk glands of the G6generationtransformation silkworms, a specific band representing the fusion protein of GFP andFib-L could be detected by Western blotting with an antibody against GFP; antibacterialexperiment was done with the silk fibre spanned by transformation silkworms showedless clonies than the control;
the silk fibre spanned by transformation silkworms emit fluorescent under theexcitation of ultraviolet light. These results indicated that the gfp gene could beintegrated into the fib-L gene locus of silkworm genome by gene targeting and fusion protein of Fib-L and GFP was expressed successfully.
In addition, the transgenic silkworm had a high silk yield and converting food intosilk based on a binary Gal4/UAS system from the Chinese Academy of Sciences inShanghai. But it is difficult to apply this method in sericulture because the Ras1~(CA)wasexpressed in non-diapause silkworm. So, we designed the Gal4transgenic silkwormhybridized with stock JingSong and screened out some silkworms withred fluorescent eyes, then the next generation transgenic silkworm was constantlybackcross with JingSong to generate G6-Gal4diapause JingSong silkworm.
we also designed the UAS transgenic silkworm hybridized with stock HaoYue andscreened out some silkworms with green fluorescent eyes, then the next generationtransgenic silkworm was constantly backcross with HaoYue to generate G6-UASdiapause HaoYue silkworm.Then the transgenic Gal4JingSong silkworm is
hybridized with the transgenic UAS HaoYue silkworm to get the Ras1~(CA)specifically expressed. The results showed Ras1~(CA)overexpression improved posteriorsilk gland weight and silk yield11%、1.3%, confirming substantial increment of silkproduction capacity in transgenic silkworm; But food consumption only increased0.57%, which has potential in sericulture. As measured by quantitative real-timePCR,Ras1~(CA)mRNA level in the posterior silk gland of Fil-Gal4/UAS-Ras1~(CA)silkwormwas4.08fold higher than in controls. These results ensured Ras1~(CA)is successfullyoverexpressed in the posterior silk gland.
引文
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