家蚕转基因载体元件的结构优化及在基因功能分析中的应用研究
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摘要
家蚕既是支撑蚕丝产业的生物基础,又是鳞翅目昆虫研究的典型模式种类,同时,也是开发新一代生物反应器和新型昆虫产业的材料。近几年来对其的科学研究所取得的成果具有较高的学术价值,代表了国际同领域的先进水平,特别是在遗传连锁图谱、家蚕基因组序列与结构等方面,使得结构基因组学开始过渡到了功能基因组学。在家蚕中基因功能的研究方法很多,转基因技术不乏为一个很优秀的手段。本文就有关基因功能研究中的家蚕转基因技术和方法开展了一些研究工作,主要的研究内容和得到的结果如下：
1、家蚕转基因效率与转座子内部序列的相关分析
自从基于piggyBac转座子方法的家蚕转基因成功问世以来,越来越多的研究学者利用该项技术来进行家蚕生物反应器和家蚕具有潜在经济性能的功能基因研究。然而该项技术的显微注射费时费力,而且成功率仅在3%～5%之间,不利于它的大规模推广与应用。为了提高家蚕转基因成功率,我们对piggyBac转座子载体进行了改造,敲除了载体中的冗余序列,构建得到了四种不同左右臂长度组合的转座子,分别进行了细胞水平和转基因家蚕水平的检测,其中左臂长333 bp、右臂长179 bp的转座子效率最高,经多次转基因实验证实,此种载体能将家蚕转基因效率提高2-7倍,更有利于开展家蚕转基因的研究。
2、家蚕hsp70启动子的克隆及功能研究
通过PCR的方法克隆得到家蚕热激蛋白70基因(Bombyx mori hsp70)的5'侧翼的两个长度分别为538 bp和305 bp的序列hsp70-538和hsp70-305。生物信息分析结果表明这两段序列在TATA序列的上游存在保守的热激元件(Heat Shock Element, HSE) CTnGAAnnTTCnAG。采用双荧光报告基因技术研究表明这两段序列在BmN细胞中都表现出热激活性,转基因家蚕实验证明hsp70-305在家蚕个体中也具有热激活性,可以认为这两个片段具有hsp70热激启动子特性,便于在家蚕中进行条件诱导的研究。
3. IRES在家蚕表达系统中的应用
通过化学合成的方法得到了来源于猪脑心肌炎病毒(Encephalomyocarditisvirus, EMCV)和禾谷缢管蚜虫病毒(Rhopalosiphum padi virus,RhPV)的四种不同长度的的内部核糖体进入序列(Internal Ribosome Entry Site, IRES) IRES,并将它们构建到pFastBac双荧光载体中,通过Bac-to-Bac的方法得到了相应的重组病毒后,在细胞及家蚕的3个组织中比较了它们的活性,结果显示IRES593的活性最高,家蚕组织中以中部丝腺的活性最高,其次是脂肪体和后部丝腺。
4、启动子陷阱在家蚕基因功能研究应用中的技术探讨
启动子陷阱是一种高通量、既能制造突变体同时又能得到被突变基因序列的研究方法,在陷阱系统的设计上我们综合了如上所述的3点成果,构建得到了两种转基因家蚕,一种是分别由hsp70、A3启动子控制表达转座酶的家蚕pBhsp70transposase3 xP3EGFP和pB A3 transpo sase3 xP3EGFP,另一种是带有检测基因EGFP的启动子陷阱家蚕。根据我们的初步研究结果表明：第一,由组成型启动子A3控制表达的转座酶能更有效地启动家蚕中转座子的再转座；第二,由于转座臂内可能残留有piggyBac转座酶启动子,因此陷阱中报告基因的方向应与原始转座酶的方向相反。
piggyBac转座子插入位点特性的分析
为更好的利用piggyBac转座子,本文还分析了piggyBac转座子在转基因家蚕基因组中的整合位点特征,得知有6%插入到内含子,12%插入到外显子,8%插入到基因的5'末端,3%插入到基因的3'末端,18%插入到了重复序列,53%插入到了基因间序列。
本论文所取得的成果为基因功能研究提供了参考,为家蚕转基因效率的提高提供了技术储备,同时也为基因陷阱研究奠定了一定的研究基础。
The silkworm, Bombyx mori, is not only the biological foundation of the sericulture, but also an important model for Lepidoptera. Moreover, it has been already explored as a new bioreactor and used as the alternative material for new type of insectical industry. Until recently, the research about the silkworm has achieved a higher scholarship value and important realistic meaning, especially the genetical linkage maps, the whole-genome sequence. As a result, the research on functional genome is underway. There are many excellent methods for the study of functional genome, and one of them is the transgenic technology. In our study, we carried out research based on this technology and the results obtained are as follows:
1. The relationship between the internal sequence of piggyBac and its transgenic efficiency
Since the transgenic silkworm based on the piggyBac transposon was reported, more and more researchers highlight its use in bioreactor of silkworm and functional analysis on genes of economical value. But this technology is time consuming and laborious, and the transgenic efficiency is relatively low, inhibiting its extensive application. To improve its transgenic efficiency, we have optimized the internal sequences. A series of piggyBac ID synthetic deletion plasmids containing A3EGFP are compared for germline transformation in BmN cells and silkworm. Our analyses have identified a better combination of ID sequence, by which we got a higher transgenic efficiency,2-7 times the original vector. This optimized system can be a potential tool for our future transgenic study.
2. Clonning and functional analysis of the Bombyx mori hsp70 promoter
We have got two PCR fragments named hsp70-538 (538bp) and hsp70-305 (305bp) directly upstream of Bombyx mori hsp 70 gene. By comparing analysis, these two fragments possess the consensus of HSE (Heat Shock Element) CTnGAAnnTTCnAG in the upstream sequence. Applying the transient expression assay in BmN cells using the dual-luciferase vector, these two fragments exhibited the heat shock ability. We also validated it in silkworm by transgenic analysis. So these two fragments can be recognized as the heat-induced promoter. This information can be used for biological analysis of gene function.
3. The application of IRES on expression system in silkworm
IRES can induce a cap-independent translation and has been greatly used in genetic engineering. To investigate its possible use in genetic research of silkworm, we have got four fragments of IRES by chemical synthesis or PCR amplification and reconstructed them into pFastBac donor plasmid containing dual luciferase genes. Through Bac-to-Bac we got these four kinds of recombinant baculoviruses. Quantization by fluorescence spectrophotometry of the luciferase proteins produced in Sf9 cells and silkworm indicated that the IRES593 has the higher activity than others'and highest in the middle silk gland, higher in the fat body and high in the post silk gland. These results suggested that the IRES can be used in the development of expression vectors for production of multiprotein complexes in silkworm.
4. The promoter trap studies of gene function in the silkworm
Promoter trap system is a highly throughput method. It can creat mutants and also be a tag for the disrupted gene. Combined the above results, we constructed two kinds of transgenic silkworm. One is supposed to provide the transposase steadily. Owing to the low remobilization rates observed with hsp70 inducible transposase, we reengineered the transposase with a constitutive promoter A3. With this construct, we observed somatic excision. The other kind of transgenic silkworm is designed to harbor the non-autonomous transposon with a promoterless EGFP gene. There may exist a piggyBac transposase promoter in the left hand, so we choose another vector, whose direction is opposite to the transposase gene as the potential promoter trap.
5. Insertion site analysis of piggyBac transposon in silkworm
For the better use of piggyBac transposon, we investigated the insertion site in the transgenic silkworm by amplifying the flanking sequences through inverse PCR method. The results showed that 6% of the insertion sites happened in the intron,12% in the exon,8% in the 5'end, 3% in the 3'end, 18% in the repetitive sequence, and 53% in the intergenic region.
All of the results described above including an optimized transposon, a heat-induced promoter, the application of IRES in expression system and the relevant technology about the promoter trap system have been useful for promoting the transgenic research, and elucidating functional genome based on promoter trap analysis.
1、家蚕转基因效率与转座子内部序列的相关分析
自从基于piggyBac转座子方法的家蚕转基因成功问世以来,越来越多的研究学者利用该项技术来进行家蚕生物反应器和家蚕具有潜在经济性能的功能基因研究。然而该项技术的显微注射费时费力,而且成功率仅在3%～5%之间,不利于它的大规模推广与应用。为了提高家蚕转基因成功率,我们对piggyBac转座子载体进行了改造,敲除了载体中的冗余序列,构建得到了四种不同左右臂长度组合的转座子,分别进行了细胞水平和转基因家蚕水平的检测,其中左臂长333 bp、右臂长179 bp的转座子效率最高,经多次转基因实验证实,此种载体能将家蚕转基因效率提高2-7倍,更有利于开展家蚕转基因的研究。
2、家蚕hsp70启动子的克隆及功能研究
通过PCR的方法克隆得到家蚕热激蛋白70基因(Bombyx mori hsp70)的5'侧翼的两个长度分别为538 bp和305 bp的序列hsp70-538和hsp70-305。生物信息分析结果表明这两段序列在TATA序列的上游存在保守的热激元件(Heat Shock Element, HSE) CTnGAAnnTTCnAG。采用双荧光报告基因技术研究表明这两段序列在BmN细胞中都表现出热激活性,转基因家蚕实验证明hsp70-305在家蚕个体中也具有热激活性,可以认为这两个片段具有hsp70热激启动子特性,便于在家蚕中进行条件诱导的研究。
3. IRES在家蚕表达系统中的应用
通过化学合成的方法得到了来源于猪脑心肌炎病毒(Encephalomyocarditisvirus, EMCV)和禾谷缢管蚜虫病毒(Rhopalosiphum padi virus,RhPV)的四种不同长度的的内部核糖体进入序列(Internal Ribosome Entry Site, IRES) IRES,并将它们构建到pFastBac双荧光载体中,通过Bac-to-Bac的方法得到了相应的重组病毒后,在细胞及家蚕的3个组织中比较了它们的活性,结果显示IRES593的活性最高,家蚕组织中以中部丝腺的活性最高,其次是脂肪体和后部丝腺。
4、启动子陷阱在家蚕基因功能研究应用中的技术探讨
启动子陷阱是一种高通量、既能制造突变体同时又能得到被突变基因序列的研究方法,在陷阱系统的设计上我们综合了如上所述的3点成果,构建得到了两种转基因家蚕,一种是分别由hsp70、A3启动子控制表达转座酶的家蚕pBhsp70transposase3 xP3EGFP和pB A3 transpo sase3 xP3EGFP,另一种是带有检测基因EGFP的启动子陷阱家蚕。根据我们的初步研究结果表明：第一,由组成型启动子A3控制表达的转座酶能更有效地启动家蚕中转座子的再转座；第二,由于转座臂内可能残留有piggyBac转座酶启动子,因此陷阱中报告基因的方向应与原始转座酶的方向相反。
piggyBac转座子插入位点特性的分析
为更好的利用piggyBac转座子,本文还分析了piggyBac转座子在转基因家蚕基因组中的整合位点特征,得知有6%插入到内含子,12%插入到外显子,8%插入到基因的5'末端,3%插入到基因的3'末端,18%插入到了重复序列,53%插入到了基因间序列。
本论文所取得的成果为基因功能研究提供了参考,为家蚕转基因效率的提高提供了技术储备,同时也为基因陷阱研究奠定了一定的研究基础。
The silkworm, Bombyx mori, is not only the biological foundation of the sericulture, but also an important model for Lepidoptera. Moreover, it has been already explored as a new bioreactor and used as the alternative material for new type of insectical industry. Until recently, the research about the silkworm has achieved a higher scholarship value and important realistic meaning, especially the genetical linkage maps, the whole-genome sequence. As a result, the research on functional genome is underway. There are many excellent methods for the study of functional genome, and one of them is the transgenic technology. In our study, we carried out research based on this technology and the results obtained are as follows:
1. The relationship between the internal sequence of piggyBac and its transgenic efficiency
Since the transgenic silkworm based on the piggyBac transposon was reported, more and more researchers highlight its use in bioreactor of silkworm and functional analysis on genes of economical value. But this technology is time consuming and laborious, and the transgenic efficiency is relatively low, inhibiting its extensive application. To improve its transgenic efficiency, we have optimized the internal sequences. A series of piggyBac ID synthetic deletion plasmids containing A3EGFP are compared for germline transformation in BmN cells and silkworm. Our analyses have identified a better combination of ID sequence, by which we got a higher transgenic efficiency,2-7 times the original vector. This optimized system can be a potential tool for our future transgenic study.
2. Clonning and functional analysis of the Bombyx mori hsp70 promoter
We have got two PCR fragments named hsp70-538 (538bp) and hsp70-305 (305bp) directly upstream of Bombyx mori hsp 70 gene. By comparing analysis, these two fragments possess the consensus of HSE (Heat Shock Element) CTnGAAnnTTCnAG in the upstream sequence. Applying the transient expression assay in BmN cells using the dual-luciferase vector, these two fragments exhibited the heat shock ability. We also validated it in silkworm by transgenic analysis. So these two fragments can be recognized as the heat-induced promoter. This information can be used for biological analysis of gene function.
3. The application of IRES on expression system in silkworm
IRES can induce a cap-independent translation and has been greatly used in genetic engineering. To investigate its possible use in genetic research of silkworm, we have got four fragments of IRES by chemical synthesis or PCR amplification and reconstructed them into pFastBac donor plasmid containing dual luciferase genes. Through Bac-to-Bac we got these four kinds of recombinant baculoviruses. Quantization by fluorescence spectrophotometry of the luciferase proteins produced in Sf9 cells and silkworm indicated that the IRES593 has the higher activity than others'and highest in the middle silk gland, higher in the fat body and high in the post silk gland. These results suggested that the IRES can be used in the development of expression vectors for production of multiprotein complexes in silkworm.
4. The promoter trap studies of gene function in the silkworm
Promoter trap system is a highly throughput method. It can creat mutants and also be a tag for the disrupted gene. Combined the above results, we constructed two kinds of transgenic silkworm. One is supposed to provide the transposase steadily. Owing to the low remobilization rates observed with hsp70 inducible transposase, we reengineered the transposase with a constitutive promoter A3. With this construct, we observed somatic excision. The other kind of transgenic silkworm is designed to harbor the non-autonomous transposon with a promoterless EGFP gene. There may exist a piggyBac transposase promoter in the left hand, so we choose another vector, whose direction is opposite to the transposase gene as the potential promoter trap.
5. Insertion site analysis of piggyBac transposon in silkworm
For the better use of piggyBac transposon, we investigated the insertion site in the transgenic silkworm by amplifying the flanking sequences through inverse PCR method. The results showed that 6% of the insertion sites happened in the intron,12% in the exon,8% in the 5'end, 3% in the 3'end, 18% in the repetitive sequence, and 53% in the intergenic region.
All of the results described above including an optimized transposon, a heat-induced promoter, the application of IRES in expression system and the relevant technology about the promoter trap system have been useful for promoting the transgenic research, and elucidating functional genome based on promoter trap analysis.
引文
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