PiggyBac转座子介导的转E.coli半胱氨酸合成酶基因绒山羊研究
摘要
我国是世界上最大羊绒生产国，所产山羊绒在国际上享有“软黄金”的盛誉。但是，不断提高绒山羊的产绒量、改善绒山羊的羊绒品质，一直是人们的努力方向。目前，应用转基因技术是提高绒山羊产绒量和羊绒品质的重要手段之一。半胱氨酸是山羊绒的主要成分，通过转基因技术，借助piggyBac转座子向绒山羊体内导入半胱氨酸合成基因有助于提高羊绒产量，改善羊绒品质，从而为培育高产绒绒山羊新品种奠定理论基础。本研究的对象、技术、方法、研究目的及结果如下：
1．提取大肠杆菌基因组DNA，克隆获得半胱氨酸合成酶基因cysE（serineacetyltransferase，丝氨酸乙酰基转移酶）、cysM（O-acetylserine sulfhydrylase，氧-乙酰丝氨酸巯解酶）完整序列。其中cysE序列全长822bp，对应编码274个氨基酸；cysM序列全长912bp，对应编码304个氨基酸。之后，构建原核表达载体pET-cysE和pET-cysM并在大肠杆菌BL21(DE3)进行了cysE和cysM蛋白表达，建立了cysE和cysM基因原核表达系统。
2．采用基因克隆等分子生物学技术构建了分别包含cysE和cysM基因的红色、绿色荧光蛋白真核表达载体pIRES2-Red2-cysE和pIRES2-EGFP-cysM。利用脂质体LipofectamineTM2000共转染绒山羊胎儿成纤维细胞，经G418筛选获得共表达外源基因cysE和cysM的转基因细胞，随后，通过核移植注射获得了同时携带cysE和cysM基因的绒山羊重构胚。
3．为了研究piggyBac转座子在绒山羊基因组中的整合位点，构建了pB-CMV-EGFP-NEO转座载体和pcDNA-Trans辅助载体，利用lipofectamine2000介导共转染绒山羊胎儿成纤维细胞，经G418筛选获得稳定转染的转基因细胞系。同时，提取转基因细胞基因组DNA，利用反向PCR检测了piggyBac转座子的整合位点。结果显示在绒山羊基因组中有21个piggyBac转座子整合位点；整合位点TTAA毗邻一侧正向的5个碱基组成中，piggyBac转座子3′端倾向于插入到富含AT（58.35%）碱基区域，piggyBac转座子5′端倾向于插入到富含GC（57.8%）碱基区域。本研究获得的整合位点信息可为进一步利用piggyBac转座子开展转基因绒山羊研究提供理论参考。
4．参照绵羊瘤胃特异表达基因小脯氨酸丰富蛋白II（PRD-SPRRII）序列，设计特异引物，克隆获得了绒山羊PRD-SPRRII启动子区序列3400bp。通过密码子优化，合成了更适合在绒山羊体内表达的cysE和cysM基因新序列。之后，成功构建了包含密码子优化后的cysE和cysM基因的绒山羊瘤胃特异表达piggyBac转座子主载体，即pB-EGFP-NEO-CMV-PRD-cysE-cysM。
5．用piggyBac转座子主载体pB-EGFP-NEO-CMV-PRD-cysE-cysM单独转染和与辅助载体pcDNA-Trans共转染绒山羊胎儿成纤维细胞，经G418筛选获得了不同转染方式下的转基因细胞。经实时荧光定量PCR检测发现，piggyBac介导的外源基因EGFP和cysM表达水平分别比对照组提高了7.78倍和6.23倍，表明piggyBac转座子在绒山羊基因组中具有高效转座活性。利用Splinkerette PCR在绒山羊基因组中获得了29个整合位点。分析整合位点TTAA毗邻一侧的5个碱基组成发现，piggyBac转座子倾向于插入到绒山羊基因组富含AT（67.95%）碱基区域。
6．利用piggyBac转座子介导成功获得一只体内携带E.coli半胱氨酸合成酶基因cysE和cysM的转基因陕北白绒山羊个体。Splinkerette PCR检测获得了23个piggyBac转座子在转基因绒山羊基因组中的整合位点，发现piggyBac转座子倾向于整合到转基因绒山羊基因组富含AT（62.70%）碱基区域。
以上结果表明，piggyBac转座子可实现胎儿成纤维细胞高效转座和外源基因高效表达，从而为生产piggyBac转座子介导转E.coli半胱氨酸合成酶基因的高产绒绒山羊新品种奠定了前期研究基础。
China is the world’s largest producer of cashmere. Chinese cashmere enjoys a “softgold” reputation in the international community, but its output is low. Thus, it is of greatinterest to maximize the quantity of the cashmere fiber without affecting the fiber quality.Manipulation of the cashmere goat genome provides opportunities to improve cashmere woolproducts. Cysteine is one of the main ingredients of cashmere. Based on transgenictechnology, this study import cysteine synthesis genes to goats via piggyBac transposon, inorder to provide a more efficient pathway to cysteine synthesis in the cashmere goat andprovide the groundwork for cultivating cashmere goats with high cashmere production. Theresearch object, technology, methods, research purpose and results of this study were asfollows:
1. The cysE （serine acetyltransferase）and cysM（O-acetylserine sulfhydrylase） genesare amplified by PCR from Escherichia coli genomic DNA. The cysE gene is822bp, whichencoding274amino acids, and cysM gene is912bp, which encoding304amino acids.Prokaryotic expression vectors pET-cysE and pET-cysM are successfully constructed andefficiently express in BL21(DE3) after induced.
2. Two integrative fluorescent protein plasmids pIRES2-Red2-cysE andpIRES2-EGFP-cysM which containing cysteine biosynthesis enzymes gene cysE and cysMrespectively was constructed and co-transfected into cashmere goat fetal fibroblasts byLipofectamineTM2000. Cell clones stably co-expressed of cysE and cysM genes wereobtained after screening by G418. Subsequently, co-expression of the cysE and cysM genes incashmere goats embryos were gained by nuclear transfer oocytes.
3. The objective of the experiment was to study the integration site of piggyBactransposon in Cashmere goat genome and piggyBac donor plasmid of pB-CMV-EGFP-NEOand helper-dependent plasmid of pcDNA-Trans were constructed and transferred intoCashmere goat fetal fibroblasts by lipofectamine2000. Cell clones stably transfected wereobtained after screening by G418. Transgenic cell genome was obtained and the integration sites of piggyBac transposons were detected by r-PCR. There were21integration sites ofpiggyBac transposon in Cashmere goat genome after r-PCR detection; Analysis of a flankingregion of TTAA flanking sequences from position+5showed that piggyBac3′tended toinsert into region rich in AT (58.35%) and the piggyBac5′tended to insert into region rich inGC (57.8%). The integration site information which is acquired from this research willprovide theoretical references for Cashmere goats study by piggyBac transposons.
4. The3400bp of promoter sequences of small proline-rich protein II (PRD-SPRRII) wereobtained according to specifically expressed genes in rumen of sheeps. The trend is thatheterologous expression of some proteins in goat can be improved by altering codonpreference. New sequences of cysE and cysM which were more appropriate to express incashmere goats were synthetized by codons bias in goat. Then, the donor plasmidpB-EGFP-NEO-CMV-PRD-cysE-cysM was constructed with two genes driven by arumen-specific promoter from the goat small proline-rich protein gene (PRD-SPRRII).
5. A piggyBac transposon system including donor vector ofpB-EGFP-NEO-CMV-PRD-cysE-cysM and helper vector of pcDNA-Trans were developed,and transgenosis cells differently transfected were obtained by G418. The expression levels ofpiggyBac-mediated EGFP and cysM increased7.78-fold and6.63-fold respectively comparedto control group, confirming that the transposition of foreign piggyBac transposons incashmere goats was highly efficient.29integration sites were obtained in the goat genome bysplinkerette PCR. Analysis of a flanking region of TTAA flanking sequences from position+5also revealed that piggyBac tended to insert into the region rich in AT (67.95%).
6．A transgenic Shanbei white cashmere goat with E.coli cysteine synthesis gene cysEand cysM in its body was got through piggybac transposon.23integration sites were obtainedin the goat genome by splinkerette PCR and it was found that piggybac transposon tended toinsert into the region rich in AT (62.70%) of transgenic cashmere goat gene.
In summary, this study confirms the function of piggyBac transposon on highly effectivetransposition of GFFs and on highly effective expression of foreign gene, which provides thegroundwork for cultivating cashmere goats with high cashmere production throughpiggyBac-mediated cysteine biosynthesis gene expression.
1．提取大肠杆菌基因组DNA，克隆获得半胱氨酸合成酶基因cysE（serineacetyltransferase，丝氨酸乙酰基转移酶）、cysM（O-acetylserine sulfhydrylase，氧-乙酰丝氨酸巯解酶）完整序列。其中cysE序列全长822bp，对应编码274个氨基酸；cysM序列全长912bp，对应编码304个氨基酸。之后，构建原核表达载体pET-cysE和pET-cysM并在大肠杆菌BL21(DE3)进行了cysE和cysM蛋白表达，建立了cysE和cysM基因原核表达系统。
2．采用基因克隆等分子生物学技术构建了分别包含cysE和cysM基因的红色、绿色荧光蛋白真核表达载体pIRES2-Red2-cysE和pIRES2-EGFP-cysM。利用脂质体LipofectamineTM2000共转染绒山羊胎儿成纤维细胞，经G418筛选获得共表达外源基因cysE和cysM的转基因细胞，随后，通过核移植注射获得了同时携带cysE和cysM基因的绒山羊重构胚。
3．为了研究piggyBac转座子在绒山羊基因组中的整合位点，构建了pB-CMV-EGFP-NEO转座载体和pcDNA-Trans辅助载体，利用lipofectamine2000介导共转染绒山羊胎儿成纤维细胞，经G418筛选获得稳定转染的转基因细胞系。同时，提取转基因细胞基因组DNA，利用反向PCR检测了piggyBac转座子的整合位点。结果显示在绒山羊基因组中有21个piggyBac转座子整合位点；整合位点TTAA毗邻一侧正向的5个碱基组成中，piggyBac转座子3′端倾向于插入到富含AT（58.35%）碱基区域，piggyBac转座子5′端倾向于插入到富含GC（57.8%）碱基区域。本研究获得的整合位点信息可为进一步利用piggyBac转座子开展转基因绒山羊研究提供理论参考。
4．参照绵羊瘤胃特异表达基因小脯氨酸丰富蛋白II（PRD-SPRRII）序列，设计特异引物，克隆获得了绒山羊PRD-SPRRII启动子区序列3400bp。通过密码子优化，合成了更适合在绒山羊体内表达的cysE和cysM基因新序列。之后，成功构建了包含密码子优化后的cysE和cysM基因的绒山羊瘤胃特异表达piggyBac转座子主载体，即pB-EGFP-NEO-CMV-PRD-cysE-cysM。
5．用piggyBac转座子主载体pB-EGFP-NEO-CMV-PRD-cysE-cysM单独转染和与辅助载体pcDNA-Trans共转染绒山羊胎儿成纤维细胞，经G418筛选获得了不同转染方式下的转基因细胞。经实时荧光定量PCR检测发现，piggyBac介导的外源基因EGFP和cysM表达水平分别比对照组提高了7.78倍和6.23倍，表明piggyBac转座子在绒山羊基因组中具有高效转座活性。利用Splinkerette PCR在绒山羊基因组中获得了29个整合位点。分析整合位点TTAA毗邻一侧的5个碱基组成发现，piggyBac转座子倾向于插入到绒山羊基因组富含AT（67.95%）碱基区域。
6．利用piggyBac转座子介导成功获得一只体内携带E.coli半胱氨酸合成酶基因cysE和cysM的转基因陕北白绒山羊个体。Splinkerette PCR检测获得了23个piggyBac转座子在转基因绒山羊基因组中的整合位点，发现piggyBac转座子倾向于整合到转基因绒山羊基因组富含AT（62.70%）碱基区域。
以上结果表明，piggyBac转座子可实现胎儿成纤维细胞高效转座和外源基因高效表达，从而为生产piggyBac转座子介导转E.coli半胱氨酸合成酶基因的高产绒绒山羊新品种奠定了前期研究基础。
China is the world’s largest producer of cashmere. Chinese cashmere enjoys a “softgold” reputation in the international community, but its output is low. Thus, it is of greatinterest to maximize the quantity of the cashmere fiber without affecting the fiber quality.Manipulation of the cashmere goat genome provides opportunities to improve cashmere woolproducts. Cysteine is one of the main ingredients of cashmere. Based on transgenictechnology, this study import cysteine synthesis genes to goats via piggyBac transposon, inorder to provide a more efficient pathway to cysteine synthesis in the cashmere goat andprovide the groundwork for cultivating cashmere goats with high cashmere production. Theresearch object, technology, methods, research purpose and results of this study were asfollows:
1. The cysE （serine acetyltransferase）and cysM（O-acetylserine sulfhydrylase） genesare amplified by PCR from Escherichia coli genomic DNA. The cysE gene is822bp, whichencoding274amino acids, and cysM gene is912bp, which encoding304amino acids.Prokaryotic expression vectors pET-cysE and pET-cysM are successfully constructed andefficiently express in BL21(DE3) after induced.
2. Two integrative fluorescent protein plasmids pIRES2-Red2-cysE andpIRES2-EGFP-cysM which containing cysteine biosynthesis enzymes gene cysE and cysMrespectively was constructed and co-transfected into cashmere goat fetal fibroblasts byLipofectamineTM2000. Cell clones stably co-expressed of cysE and cysM genes wereobtained after screening by G418. Subsequently, co-expression of the cysE and cysM genes incashmere goats embryos were gained by nuclear transfer oocytes.
3. The objective of the experiment was to study the integration site of piggyBactransposon in Cashmere goat genome and piggyBac donor plasmid of pB-CMV-EGFP-NEOand helper-dependent plasmid of pcDNA-Trans were constructed and transferred intoCashmere goat fetal fibroblasts by lipofectamine2000. Cell clones stably transfected wereobtained after screening by G418. Transgenic cell genome was obtained and the integration sites of piggyBac transposons were detected by r-PCR. There were21integration sites ofpiggyBac transposon in Cashmere goat genome after r-PCR detection; Analysis of a flankingregion of TTAA flanking sequences from position+5showed that piggyBac3′tended toinsert into region rich in AT (58.35%) and the piggyBac5′tended to insert into region rich inGC (57.8%). The integration site information which is acquired from this research willprovide theoretical references for Cashmere goats study by piggyBac transposons.
4. The3400bp of promoter sequences of small proline-rich protein II (PRD-SPRRII) wereobtained according to specifically expressed genes in rumen of sheeps. The trend is thatheterologous expression of some proteins in goat can be improved by altering codonpreference. New sequences of cysE and cysM which were more appropriate to express incashmere goats were synthetized by codons bias in goat. Then, the donor plasmidpB-EGFP-NEO-CMV-PRD-cysE-cysM was constructed with two genes driven by arumen-specific promoter from the goat small proline-rich protein gene (PRD-SPRRII).
5. A piggyBac transposon system including donor vector ofpB-EGFP-NEO-CMV-PRD-cysE-cysM and helper vector of pcDNA-Trans were developed,and transgenosis cells differently transfected were obtained by G418. The expression levels ofpiggyBac-mediated EGFP and cysM increased7.78-fold and6.63-fold respectively comparedto control group, confirming that the transposition of foreign piggyBac transposons incashmere goats was highly efficient.29integration sites were obtained in the goat genome bysplinkerette PCR. Analysis of a flanking region of TTAA flanking sequences from position+5also revealed that piggyBac tended to insert into the region rich in AT (67.95%).
6．A transgenic Shanbei white cashmere goat with E.coli cysteine synthesis gene cysEand cysM in its body was got through piggybac transposon.23integration sites were obtainedin the goat genome by splinkerette PCR and it was found that piggybac transposon tended toinsert into the region rich in AT (62.70%) of transgenic cashmere goat gene.
In summary, this study confirms the function of piggyBac transposon on highly effectivetransposition of GFFs and on highly effective expression of foreign gene, which provides thegroundwork for cultivating cashmere goats with high cashmere production throughpiggyBac-mediated cysteine biosynthesis gene expression.
引文
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成勇,王玉阁,罗金平等.2002.由成年转基因山羊体细胞而来的克隆山羊.生物工程学报，18(1):79-83
丁昇.2007. piggyBac转座系统-哺乳动物遗传分析的新工具.[博士学位论文].上海:复旦大学.
冯亚杰.2007.山羊BLG调控序列及CMV增强子指导人胰岛素原基因在细胞和小鼠中表达..[硕士学位论文].扬州：扬州大学.
郭丽,周红莉,屈建国等.2006.人诺如病毒衣壳蛋白的密码子优化及在昆虫细胞中的表达.中国病毒学,21(2):121-125
郭旭东,尹俊,杨东山等.2009.IGF-1毛囊特异表达载体的构建以及转染绒山羊胎儿成纤维细胞的研究.畜牧兽医学报,40(10):1460-1467
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