叶绿体基因编码蛋白的体外翻译系统构筑
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摘要
以高等植物豌豆和烟草叶绿体基因编码的蛋白D1,细胞色素f和细胞色素b_6为研究对象,采用分子生物学基因克隆,体外转录,蛋白质放射自显影分析技术,构建具有翻译活性的同源叶绿体体外翻译系统,以系统深入地研究叶绿体基因编码蛋白的类囊体膜插入合成组装调控的分子机制。所得结果摘要如下:
(1)以完整的叶绿体DNA为模板,采用定向克隆策略,构建了具有高保真性能的叶绿体基因编码光系统蛋白D1,Cyt f和Cytb_6全长基因psbA,petA,petB。采用嵌套PCR方法体外连接烟草psbA 85bp翻译强启动子和petB全长基因,构建至体外转录载体pBluescript Ⅱ SK/KS(+/-)上;设计双酶切位点引物,克隆了D1(5跨膜区)和Cytb_6(4跨膜区)不同跨膜区截断片段基因(10种),新引入5'overhangs酶切位点消除了体外转录中的3'overhangs,避免了多转录物的产生。这为系统研究叶绿体基因编码蛋白何时何阶段稳定插入类囊体膜并组装成功能复合物提供了研究平台。
(2)构建了稳定的,高产,特异的体外转录系统。通过延长蛋白酶K消化时间,增加震荡次数,简化纯化步骤,降低了RNase污染转录体系的几率;向目的基因末端统一引入5'overhangs(Hind Ⅲ)的酶切位点,使得特异性地转录目的基因mRNA成为可能。对10种基因片段的体外转录实验显示,mRNA转录物产量从2.5-11.6mg/ml之间(纯度oD_(260/280)=1.75-1.85之间)。可作为外源添加的mRNA转录物来源,用于之后的体外翻译体系。
(3)制备出具有体外翻译活性的豌豆叶绿体30,000g基质上清(S30)提取液,通过大量种植高代谢活性,低核酸降解活性的幼龄期豌豆,Pcrcoll梯度方法提取完整的叶绿体,缩短提取S30时间,添加蛋白酶抑制剂等保护物质,有效提高S30蛋白质浓度(16-25mg/ml),获得一批具有很强翻译活性的S30提取液,体外翻译实验显示利用该S30提取液,翻译出含Cytb_6第一跨膜区(包含烟草psbA的启动子)的多肽蛋白。证实该系统具有翻译外源基因的能力。
A homologous chloroplast in vitro translation system of chloroplast encoded proteins D_1, cytochrome f (Cytf), cytochrome b_6 (Cytb_6) was established to systematically study the molecular mechanism of chloroplast encoded proteins' assembly and regulatory processes. The results obtained here were summarized as follows:Using Site-Directed PCR cloning strategy and chloroplast DNA template, full length genes of D_1(psbA), cytochrome f (petA), cytochrome b6 (petB) with high fidelity were constructed. We cloned full length petB gene with tobacco psbA 85bp promoter to enhance translation efficiency. To avoid 3' overhangs in the following in vitro transcription, double enzyme sites were introduced into the end of different transmembrane regions (TMs)of D_1 (5TMs) and cytochrome b_6 (4TMs). All these genes were constructed into in vitro transcription vector pBluescript II SK/KS(+/-) and verified by sequencing. These provide research basis for studying when and how the chloroplast encoded proteins insert and assembly into the thylakoid membrane.A stable and specific in vitro transcription system with high productivity was constructed. The risk of RNase contamination was reduced by increased time of protease K incubation, increased vortex frequency and short isolation time. Enzyme sites of 5' overhangs were introduced into the end of each gene fragment to make the possibility of specific transcripts. The in vitro transcription results of 10 gene
fragments showed that its mRNA production was 2.5-11.6mg/ml (purity oD_(260/280) =1.75-1.85) which is suitable for the following in vitro translation as exogenous mRNA resources.Chloroplast stoma supernatant 30,000g extracts (S30)with translation activity were prepared by these pathways: Pea seedlings with high protein synthesis and low nucleic degradation activity were grown in a large scale;the isolation time of S30 extracts preparation was reduced;protease inhibitors were added to avoid proteins degradation;S30 protein concentrations were increased (16-25mg/ml). The in vitro translation results showed that using these S30 fractions, the first transmembrane peptide of Cytb6 with tobacco psbA promoter were translated tested through autoradiography. This verifies the ability of this constructed in vitro translation system to express exogenous genes.
(1)以完整的叶绿体DNA为模板,采用定向克隆策略,构建了具有高保真性能的叶绿体基因编码光系统蛋白D1,Cyt f和Cytb_6全长基因psbA,petA,petB。采用嵌套PCR方法体外连接烟草psbA 85bp翻译强启动子和petB全长基因,构建至体外转录载体pBluescript Ⅱ SK/KS(+/-)上;设计双酶切位点引物,克隆了D1(5跨膜区)和Cytb_6(4跨膜区)不同跨膜区截断片段基因(10种),新引入5'overhangs酶切位点消除了体外转录中的3'overhangs,避免了多转录物的产生。这为系统研究叶绿体基因编码蛋白何时何阶段稳定插入类囊体膜并组装成功能复合物提供了研究平台。
(2)构建了稳定的,高产,特异的体外转录系统。通过延长蛋白酶K消化时间,增加震荡次数,简化纯化步骤,降低了RNase污染转录体系的几率;向目的基因末端统一引入5'overhangs(Hind Ⅲ)的酶切位点,使得特异性地转录目的基因mRNA成为可能。对10种基因片段的体外转录实验显示,mRNA转录物产量从2.5-11.6mg/ml之间(纯度oD_(260/280)=1.75-1.85之间)。可作为外源添加的mRNA转录物来源,用于之后的体外翻译体系。
(3)制备出具有体外翻译活性的豌豆叶绿体30,000g基质上清(S30)提取液,通过大量种植高代谢活性,低核酸降解活性的幼龄期豌豆,Pcrcoll梯度方法提取完整的叶绿体,缩短提取S30时间,添加蛋白酶抑制剂等保护物质,有效提高S30蛋白质浓度(16-25mg/ml),获得一批具有很强翻译活性的S30提取液,体外翻译实验显示利用该S30提取液,翻译出含Cytb_6第一跨膜区(包含烟草psbA的启动子)的多肽蛋白。证实该系统具有翻译外源基因的能力。
A homologous chloroplast in vitro translation system of chloroplast encoded proteins D_1, cytochrome f (Cytf), cytochrome b_6 (Cytb_6) was established to systematically study the molecular mechanism of chloroplast encoded proteins' assembly and regulatory processes. The results obtained here were summarized as follows:Using Site-Directed PCR cloning strategy and chloroplast DNA template, full length genes of D_1(psbA), cytochrome f (petA), cytochrome b6 (petB) with high fidelity were constructed. We cloned full length petB gene with tobacco psbA 85bp promoter to enhance translation efficiency. To avoid 3' overhangs in the following in vitro transcription, double enzyme sites were introduced into the end of different transmembrane regions (TMs)of D_1 (5TMs) and cytochrome b_6 (4TMs). All these genes were constructed into in vitro transcription vector pBluescript II SK/KS(+/-) and verified by sequencing. These provide research basis for studying when and how the chloroplast encoded proteins insert and assembly into the thylakoid membrane.A stable and specific in vitro transcription system with high productivity was constructed. The risk of RNase contamination was reduced by increased time of protease K incubation, increased vortex frequency and short isolation time. Enzyme sites of 5' overhangs were introduced into the end of each gene fragment to make the possibility of specific transcripts. The in vitro transcription results of 10 gene
fragments showed that its mRNA production was 2.5-11.6mg/ml (purity oD_(260/280) =1.75-1.85) which is suitable for the following in vitro translation as exogenous mRNA resources.Chloroplast stoma supernatant 30,000g extracts (S30)with translation activity were prepared by these pathways: Pea seedlings with high protein synthesis and low nucleic degradation activity were grown in a large scale;the isolation time of S30 extracts preparation was reduced;protease inhibitors were added to avoid proteins degradation;S30 protein concentrations were increased (16-25mg/ml). The in vitro translation results showed that using these S30 fractions, the first transmembrane peptide of Cytb6 with tobacco psbA promoter were translated tested through autoradiography. This verifies the ability of this constructed in vitro translation system to express exogenous genes.
引文
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