甘蓝SRK的S域酵母双杂交表达载体的构建及其与SCR作用区段的研究
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摘要
芸薹属植物自交不亲和性受单一位点的复等位基因控制,此位点被命名为S位点。它决定柱头表面花粉识别的专一性。S受体激酶基因(S-locus receptor kinase, SRK)和S位点糖蛋白基因(S-locus glycoprotein,SLG)是控制芸薹属植物花柱自交不亲和性的两个关键因子。SRK是自交不亲和专一性的决定因子,SLG仅是辅助因子,而S位点富半胱氨酸蛋白(S-locus cystein-rich protein,SCR)是SRK激酶的配体分子,并且三基因完全连锁。蛋白质是生命功能的物质基础,揭示蛋白与蛋白间的相互作用,已成为揭示蛋白质组复杂体系与蛋白质功能模式的先导。本实验采用酵母双杂交技术检测甘蓝SRK胞外域不同片段与SCR不同片段蛋白间相互作用。通过PCR扩增甘蓝SRK胞外域不同片段序列及SCR不同片段基因编码序列,扩增的片段分别与质粒载体pGADT7和PGBKT7连接,构建pGADT7-eSRKs和pGBKT7-SCRs系列重组载体。检测转化酵母Y2HGold的钓饵质粒是否具有自激活发生以及重组钓饵蛋白的毒性。将pGBKT7和pGADT7的重组质粒分别转化酵母菌株Y2HGold和Y187检测蛋白间是否具有相互作用,通过SD/-Leu-Trp-His-Ade培养基筛选得到pGBKT7-SCRs和pGADT7-eSRKs的阳性克隆,然后用X-a-Gal显色反应鉴定转化到酵母中的两个重组质粒相互作用。主要研究结果总结如下：
1.不同截短长度的S位点受体激酶胞外域(eSRK)片段的克隆及其结构特征
以结球甘蓝B3幼叶的gDNA为模板,采用PCR扩增不同片段长度的eSRKs基因。经1%(w/v)琼脂糖凝胶电泳检测,eSRKs的目的片段大小均与引物的理论扩增值大小相符。利用分子克隆技术构建酵母双杂交pGADT7-eSRKs表达载体,DNA测序结果分析表明,pGADT7-eSRKs载体中包含有eSRKs片段且插入的eSRKs片段序列与eSRK28序列完全一致。BLAST在线分析结果表明,eSRKB3编码421个氨基酸,包含了SRK蛋白B-Lectin结构域、SLG结构域和PAN-APPLE结构域,是SRK胞外域的全长；eSRKl包含了eSRK序列的三个高变区域,是编码第5位至第427位氨基酸；eSRK2包含了eSRK序列的两个高变区域,编码第139位至第427位氨基酸；eSRK3包含了eSRK序列的两个高变区域,编码第5位至第273位氨基酸；eSRK4包含了eSRK序列的一个高变区域,编码第5位至第140位氨基酸。
2.酵母双杂交重组AD质粒的毒性及自激活检测
将pGADT7空载和pGADT7-eSRKs转化Y187,观察得到Y187 [pGADT7]和Y187 [pGADT7-eSRKs]在SD/-Leu平板上生长状态良好,由此表明重组表达质粒pGADT7-eSRKs成功转入酵母细胞Y187且无毒性作用。此外,Y187[pGADT7-eSRKs]在SD/-Leu平板上呈现生长状态良好的白色菌斑；在SD/-Leu/x-a-gal平板上没有明显蓝色克隆出现。由此可说明pGADT7-eSRKs重组载体在酵母细胞中没有激活报告基因MEL1,无自身的转录激活作用发生。
3. SCRs与eSRKs片段间相互作用检测
利用生物信息学分析软件autodocktools对eSRKs与SCRs进行相互作用分析,结果表明eSRKs与SCRs在空间能够相互识别形成融合蛋白。
试验显示eSRKs与SCRs片段间两两组合的9个试验组均能在DDO平板上出现生长状态良好的菌斑,其中有Y187[pGADT7-eSRKl]xY2HGold[pGBKT7-SCR2和Y187[pGADT7-eSRK4]xY2HGold[pGBKT7-SCR2]2个试验组能在QDO/x/A平板上出现蓝色克隆,激活报告基因AUR1-C、MEL1、ADE2和HIS3的表达。结果显示SRK和SCR具有相互作用,且初步显示其相互作用区域为SRK第一个外显子的第16～421 bp的片段和SCR第1876～2068 bp的片段。
The multiple alleles of a single locus control the self-incompatibility of Brassica, which named as S-locus. It determines the specificity of the pollen recognition which locate in the stigma surface, control S receptor kinase gene(S-locus receptor kinase, SRK)and S locus glycoprotein gene (S-locus glycoprotein,SLG) are Two of the key proteins which control the self-incompatibility in Brassica:the SRK determine the self-incompatibility, SLG only the cofactor. The pollen S locus protein (S-locus cystein-rich protein,SCR) is SRK'S ligands, which is completely linked with two other genes.In this study, using yeast two-hybrid system,the pGADT7-eSRKs and pGBKT7-SCR srecombinant vector were constructed and transformed into yeast Y187 and Y2HGold separately. Our results showed that:
1. The amplification of different Truncated fragments eSRKs and Sequence Analysis.
The different Truncated fragments eSRKs were amplified from leaf genomic DNA in Brassica oleracea L.'B3'by PCR and using 1%(w/v) agarose gel electrophoresis, the fragments length are identical with the theory value of primer extension. Then subcloned into pGADT7 and constructed pGADT7-eSRKs plasmids. The DNA sequencing analysis showed that pGADT7-eSRKs plasmids contain eSRKs fragments, and the insertion site and the reading frame are correct.The analysis of BLAS online showed that.the eSRK encodes 421 amino acids, contains B-Lectin domain, SLG domain and PAN-APPLE domain. The eSRKl encodes the 5 to 427 amino acids, which contains three hypervariable region of eSRK sequence; eSRK2 encodes the amino acids between 139 and 427, which contains two hypervariable region of eSRK sequence; eSRK3 encodes the amino acids between 5 and 273, which contains two hypervariable region of eSRK sequence. eSRK2 encodes the amino acids between 5 and 140,which contains one hypervariable region of eSRK sequence.
2.The toxicity and Autoactivation detection of recombinant AD plasmid pGADT7-eSRKs.
The empty vector pGADT7 and pGADT7-eSRKs were transformed into Y187, the ransformants of Y187 [pGADT7] and Y187 [pGADT7-eSRKs] grew well on the SD/-Leu plates, while the yeast strain Y187 which was not transformed recombinant plasmid did not grow on SD/-Leu medium.It is suggested that recombinant plasmid pGADT7-eSRKs was successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187. In addition, the white clones of Y187 [pGADT7-eSRKs] grew well in SD/-Leu plates and the colonies did not turn blue on SD/-Leu/x-a-gal plates. The results indicate that recombinant plasmid pGADT7-eSRKs do not activate the reporter gene MEL1.
3.The interaction detection between different truncated fragments of SCRs and eSRKs.
The analysis of bioinformatics autodocktools software shows that SRKs can mutually recognize with SCRs in the space to form a fusion protein.
The recombinant plasmids were success fully transformed into the diploid yeast mating cell, the two group of Y187 [pGADT7-eSRKl] x Y2HGold [pGBKT7-SCR2] and Y187 [pGADT7-eSRK4] xY2HGold[pGBKT7-SCR2] could emerge significantly blue clones, then remove blue clones on DDO/x/A plates to QDO/x/A plate, after four days, we can find the blue clones on the plate, which may indicate that activate the reporter gene AUR1-C, MEL1, ADE2 and HIS3. The resoults show that the yeast two-hybrid system could be used to study the interaction domain between eSRK and SCR. The results indicated that there existed two kinds of interactions among all interaction assays between SRK and SCR, whose interaction regions span 16-421 bp fragment of the first exon of SRK and 1876～2068 bp fragment of SCR.
1.不同截短长度的S位点受体激酶胞外域(eSRK)片段的克隆及其结构特征
以结球甘蓝B3幼叶的gDNA为模板,采用PCR扩增不同片段长度的eSRKs基因。经1%(w/v)琼脂糖凝胶电泳检测,eSRKs的目的片段大小均与引物的理论扩增值大小相符。利用分子克隆技术构建酵母双杂交pGADT7-eSRKs表达载体,DNA测序结果分析表明,pGADT7-eSRKs载体中包含有eSRKs片段且插入的eSRKs片段序列与eSRK28序列完全一致。BLAST在线分析结果表明,eSRKB3编码421个氨基酸,包含了SRK蛋白B-Lectin结构域、SLG结构域和PAN-APPLE结构域,是SRK胞外域的全长；eSRKl包含了eSRK序列的三个高变区域,是编码第5位至第427位氨基酸；eSRK2包含了eSRK序列的两个高变区域,编码第139位至第427位氨基酸；eSRK3包含了eSRK序列的两个高变区域,编码第5位至第273位氨基酸；eSRK4包含了eSRK序列的一个高变区域,编码第5位至第140位氨基酸。
2.酵母双杂交重组AD质粒的毒性及自激活检测
将pGADT7空载和pGADT7-eSRKs转化Y187,观察得到Y187 [pGADT7]和Y187 [pGADT7-eSRKs]在SD/-Leu平板上生长状态良好,由此表明重组表达质粒pGADT7-eSRKs成功转入酵母细胞Y187且无毒性作用。此外,Y187[pGADT7-eSRKs]在SD/-Leu平板上呈现生长状态良好的白色菌斑；在SD/-Leu/x-a-gal平板上没有明显蓝色克隆出现。由此可说明pGADT7-eSRKs重组载体在酵母细胞中没有激活报告基因MEL1,无自身的转录激活作用发生。
3. SCRs与eSRKs片段间相互作用检测
利用生物信息学分析软件autodocktools对eSRKs与SCRs进行相互作用分析,结果表明eSRKs与SCRs在空间能够相互识别形成融合蛋白。
试验显示eSRKs与SCRs片段间两两组合的9个试验组均能在DDO平板上出现生长状态良好的菌斑,其中有Y187[pGADT7-eSRKl]xY2HGold[pGBKT7-SCR2和Y187[pGADT7-eSRK4]xY2HGold[pGBKT7-SCR2]2个试验组能在QDO/x/A平板上出现蓝色克隆,激活报告基因AUR1-C、MEL1、ADE2和HIS3的表达。结果显示SRK和SCR具有相互作用,且初步显示其相互作用区域为SRK第一个外显子的第16～421 bp的片段和SCR第1876～2068 bp的片段。
The multiple alleles of a single locus control the self-incompatibility of Brassica, which named as S-locus. It determines the specificity of the pollen recognition which locate in the stigma surface, control S receptor kinase gene(S-locus receptor kinase, SRK)and S locus glycoprotein gene (S-locus glycoprotein,SLG) are Two of the key proteins which control the self-incompatibility in Brassica:the SRK determine the self-incompatibility, SLG only the cofactor. The pollen S locus protein (S-locus cystein-rich protein,SCR) is SRK'S ligands, which is completely linked with two other genes.In this study, using yeast two-hybrid system,the pGADT7-eSRKs and pGBKT7-SCR srecombinant vector were constructed and transformed into yeast Y187 and Y2HGold separately. Our results showed that:
1. The amplification of different Truncated fragments eSRKs and Sequence Analysis.
The different Truncated fragments eSRKs were amplified from leaf genomic DNA in Brassica oleracea L.'B3'by PCR and using 1%(w/v) agarose gel electrophoresis, the fragments length are identical with the theory value of primer extension. Then subcloned into pGADT7 and constructed pGADT7-eSRKs plasmids. The DNA sequencing analysis showed that pGADT7-eSRKs plasmids contain eSRKs fragments, and the insertion site and the reading frame are correct.The analysis of BLAS online showed that.the eSRK encodes 421 amino acids, contains B-Lectin domain, SLG domain and PAN-APPLE domain. The eSRKl encodes the 5 to 427 amino acids, which contains three hypervariable region of eSRK sequence; eSRK2 encodes the amino acids between 139 and 427, which contains two hypervariable region of eSRK sequence; eSRK3 encodes the amino acids between 5 and 273, which contains two hypervariable region of eSRK sequence. eSRK2 encodes the amino acids between 5 and 140,which contains one hypervariable region of eSRK sequence.
2.The toxicity and Autoactivation detection of recombinant AD plasmid pGADT7-eSRKs.
The empty vector pGADT7 and pGADT7-eSRKs were transformed into Y187, the ransformants of Y187 [pGADT7] and Y187 [pGADT7-eSRKs] grew well on the SD/-Leu plates, while the yeast strain Y187 which was not transformed recombinant plasmid did not grow on SD/-Leu medium.It is suggested that recombinant plasmid pGADT7-eSRKs was successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187. In addition, the white clones of Y187 [pGADT7-eSRKs] grew well in SD/-Leu plates and the colonies did not turn blue on SD/-Leu/x-a-gal plates. The results indicate that recombinant plasmid pGADT7-eSRKs do not activate the reporter gene MEL1.
3.The interaction detection between different truncated fragments of SCRs and eSRKs.
The analysis of bioinformatics autodocktools software shows that SRKs can mutually recognize with SCRs in the space to form a fusion protein.
The recombinant plasmids were success fully transformed into the diploid yeast mating cell, the two group of Y187 [pGADT7-eSRKl] x Y2HGold [pGBKT7-SCR2] and Y187 [pGADT7-eSRK4] xY2HGold[pGBKT7-SCR2] could emerge significantly blue clones, then remove blue clones on DDO/x/A plates to QDO/x/A plate, after four days, we can find the blue clones on the plate, which may indicate that activate the reporter gene AUR1-C, MEL1, ADE2 and HIS3. The resoults show that the yeast two-hybrid system could be used to study the interaction domain between eSRK and SCR. The results indicated that there existed two kinds of interactions among all interaction assays between SRK and SCR, whose interaction regions span 16-421 bp fragment of the first exon of SRK and 1876～2068 bp fragment of SCR.
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