利用酵母双杂交系统鉴定羽衣甘蓝ARC1与结球甘蓝SRK激酶结构域的相互作用
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摘要
自交不亲和性(Self—incompatibility, SI)是一种“钥匙和锁”形式防止开花植物自交的机制,研究比较深入的甘蓝等芸薹属植物的自交不亲和反应从S基因座的两个基因相互识别开始,雄性决定因子SCR (S locus cysteine-rich protein)与雌性决定因子SRK (S locus protein kinase)两个因子的相互识别,相同单倍型的花粉的信号由柱头乳突细胞外传至胞内,THL与SRK相分离并释放至细胞质中,SRK的激酶域随后激活ARC1 (Arm repeat containing),进一步将自交不亲和信号传至EX070A1,再通过后续的级联反应,最终导致自交不亲和。
本研究利用酵母双杂交系统研究甘蓝自交不亲和反应中S位点受体激酶(SRK)的激酶域与ARC1间的相互作用。以羽衣甘蓝2003a和结球甘蓝E1为材料,利用PCR技术分别分离出羽衣甘蓝的ARC1和结球甘蓝的SRKJ (S位点受体激酶激酶结构域)；构建以pGBKT7为载体的ARCla、ARClb、ARClc和ARC1d的重组诱饵质粒；以pGADT7为载体的SRKJ的重组猎物载体,进行了相互作用验证,研究结果为芸薹属植物自交不亲和反应的分子机理提供新内容。
1.序列的克隆分析
①所克隆的羽衣甘蓝ARC1的基因序列长度为1992 bp且不含内含子,与已报道甘蓝的ARCl的cDNA序列具有99.7%的相似度,二者编码的蛋白质序列仅有4个位点的氨基酸差异；②所得的羽衣甘蓝的ARC1基因编码的蛋白质没有信号肽,氨基酸序列存在6个磷酸化位点；；⑧所得的ARC1基因与甘蓝型油菜ARCl的cDNA序列相似度达到99.4%,二者编码的蛋白质在一级结构上的一致性高达到92.9%,存在45个位点的氨基酸差异。④对蛋白的序列分析发现ARC1包含U-box和5个连续重复ARM两个重要的区域,经Blast与Vector NTI分析发现：羽衣甘蓝与甘蓝型油菜U-box区存在5个位点的氨基酸差异,ARM区域有14个位点的氨基酸差异。结球甘蓝的SRK激酶域与羽衣甘蓝SRK激酶域的氨基酸序列的相似性达到91.0%,376个氨基酸序列中存在46个差异氨基酸,而这46个差异氨基酸中有12个为相似性氨基酸。SRK激酶域氨基酸序列存在一个高变区,高变区位于序列第250个氨基酸与270个氨基酸之间。
2.酵母双杂交重组诱饵质粒的毒性及自激活检测
Y2HGold [pGBKT7-ARCla]、Y2HGold [pGBKT7-ARClb]、Y2HGold [pGBKT7-ARClc]、Y2HGold [pGBKT7-ARCld]、Y2HGold [pGBKT7-ARCla]和Y2HGold [pGBKT7]转化株在缺陷型SD/-Trp平板上生长状态良好,并且菌斑大小无明显差异；由此表明构建的4组重组诱饵质粒的表达对酵母细胞无毒性作用。另外,pGBKT7-ARC1四个实验组在缺陷型SD/-Trp、SD/-Trp/x-a-gal平板上均能生长、在SD/-Trp/x-a-gal/AbA平板上不生长,其中在SD/-Trp/x-a-gal平板上生长的菌落无明显蓝色出现,说明四个实验组在酵母细胞中没有自激活现象。
3.酵母双杂交重组猎物质粒的毒性检测
将pGADT7空载和pGADT7-SRKJ转化Y187,发现空载和重组表达载体在SD/-Leu平板上生长状态良好,由此表明重组表达质粒pGADT7-ARCl成功转入酵母细胞Y187且无毒性作用。
4.相互作用检测
三个实验组Y2HGold [pGBKT7-ARCla]xY187 [pGADT7-SRKJ]、Y2HGold [pGBKT7-ARClc]×Y187 [pGADT7-SRKJ]、Y2HGold [pGBKT7-ARCld]×Y187 [pGADT7-SRKJ]同时激活了4种报告基因AURl-C、MELl、HIS3、ADE2;表明羽衣甘蓝ARC1与结球甘蓝的SRK激酶结构域能够发生相互作用,且互作的区域位于连续的臂重复区,其与结球甘蓝ARC1的氨基酸差异不足以引起互作区的正确构象改变。研究结果为芸薹属植物自交不亲和反应的分子机理提供新内容。
Self-incompatibility is fascinating molecular "lock-and-key" mechanisms preventing self-fertilization in flowering plants, cross-incompatibility occurs between different individuals that have the same incompatibility type when Self-incompatibility occurs between the same haploid pollen and pistil. Members of the Brassicaceae family have a SSI (sporophytic SI) system, the SSI response of Brassica oleracea results from Recognition of SRK and self-SCR which are encoded by two genes at S locus, then the response of recognition between stigma and self pollen is transmitted to internal cells. THL is separated from SRK and is released into cytoplasm. At the same time, ARC1 is activated by the kinase domain of SRK. Then the response of ARC1 and SRK is passed to EXO70A1, then the subsequent cascade of reactions ultimately lead to self-incompatibility.
In this study, a yeast two-hybrid system was used to investigate the interaction between ARC1 and the Kinase Domain of SRK. The sequences of SRK Kinase Domain (SRKJ) and different-length ARCla, ARClb, ARClc and ARC1d of Brassica oleracea were amplified by PCR, then SRKJ was subcloned into pGADT7 and different longth ARC1 were subcloned into pGBKT7 vectors. After sequencing, the plasmids were transformed into the yeast cells, and the interaction between the truncated fragments of ARC1 from Brassica oleracea var. acephala and kinase domain of SRK from Brassica oleracea var. capitata L were tested by Yeast Two-Hybrid System provides some insights into the molecular mechanism of self-incompatibility in Brassica.The results showed:
1 Cloning and analysis of sequence
The results indicate that the gDNA, consisting of 1992 bp without introns, encodes a 663 amino acid ORF which has 99.7%similarity to Brassica oleracea var acephala (sequence accession:EU344909), containing a total of four amino acid polymorphisms. It has 45 amino acid differences when compared to B. napus (AF024025), with which it shares a 92.9%similarity. The ARC1 has no signal peptide, but has six phosphorylation sites; Sequence analysis also demonstrated that the ARC1 protein coding region contains U-box and ARM areas. Vector NTITM analysis indicated that there were 5 amino acid differences contained in the U-box region and 14 amino acid differences in the ARM region between the cloned Brassica oleracea var. acephala and Brassica napus. The sequences of Kinase Domain of SRK (SRKJ) was amplified by PCR from Brassica oleracea var. apitata L. the length of SRKJ is 1131bp and encodes 376 amino acid ORF which has 91.0%similarity to Kinase Domain of SRK from Brassica oleracea var acephala, and has 12 similarityamino acid in46 differences amino acid; there is a hypervariable region in Kinase Domain of SRK and located between the sequence of 250 amino acids and 270 amino acids.
2 The toxicity and Autoactivation detection of recombinant bait plasmids
The yeast which were transformed by Y2HGold [pGBKT7-ARC1a], Y2HGold [pGBKT7-ARClb]、Y2HGold [pGBKT7-ARClc]、Y2HGold [pGBKT7-ARCld]、Y2HGold [pGBKT7-ARCla] Y2HGold [pGBKT7] were cultured on SD/-Trp plates at 30℃. After three days, there were white clones in the plates. The result showed that the recombinant bait plasmids pGBKT7-ARC1 were confirmed not toxic to yeast. In addition, four experimental groups could grow on SD/-Trp, SD/-Trp/x-a-gal plates but not grow on SD/-Trp/x-a-gal/AbA plates, the colonies did not turn blue on SD/-Trp/x-a-gal plates. The results indicates that the baits were confirmed not activate the expression of reporter genes by the test for autoactivation.
3 The toxicity detection of recombinant prey plasmid pGADT7-SRKJ
The empty vector pGADT7 and recombinant prey plasmid pGADT7-SRKJ were transformed into Y187, the yeast of Y187 [pGADT7] and Y187 [pGADT7-SRKJ] grew well on the SD/-Leu plates. The results suggested that recombinant plasmid pGADT7-SRKJwas successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187.
4 The interaction detection between different truncated fragments of ARCl and SRKJ
Three experimental groups Y2HGold [pGBKT7-ARCla]×Y187 [pGADT7-SRKJ], Y2HGold [pGBKT7-ARClc]×Y187 [pGADT7-SRKJ] and Y2HGold [pGBKT7-ARCld]×Y187 [pGADT7-SRKJ] could grow on QDO/x/A nutritional media with transcription activation of the reporter gene AUR1-C, MEL1, HIS3, ADE2, it indicated that there exists interaction between ARC1 from Brassica oleracea var. acephala L and SRK from Brassica oleracea var. capitata L and the interaction domain was located on ARM repeats, and the fifference at amino acid level with the ARC1 of Brassica oleracea var. capitata L is not enough to change the conformation in the interaction region. All above mentioned provides some insights into the molecular mechanism of self-incompatibility in Brassica.
本研究利用酵母双杂交系统研究甘蓝自交不亲和反应中S位点受体激酶(SRK)的激酶域与ARC1间的相互作用。以羽衣甘蓝2003a和结球甘蓝E1为材料,利用PCR技术分别分离出羽衣甘蓝的ARC1和结球甘蓝的SRKJ (S位点受体激酶激酶结构域)；构建以pGBKT7为载体的ARCla、ARClb、ARClc和ARC1d的重组诱饵质粒；以pGADT7为载体的SRKJ的重组猎物载体,进行了相互作用验证,研究结果为芸薹属植物自交不亲和反应的分子机理提供新内容。
1.序列的克隆分析
①所克隆的羽衣甘蓝ARC1的基因序列长度为1992 bp且不含内含子,与已报道甘蓝的ARCl的cDNA序列具有99.7%的相似度,二者编码的蛋白质序列仅有4个位点的氨基酸差异；②所得的羽衣甘蓝的ARC1基因编码的蛋白质没有信号肽,氨基酸序列存在6个磷酸化位点；；⑧所得的ARC1基因与甘蓝型油菜ARCl的cDNA序列相似度达到99.4%,二者编码的蛋白质在一级结构上的一致性高达到92.9%,存在45个位点的氨基酸差异。④对蛋白的序列分析发现ARC1包含U-box和5个连续重复ARM两个重要的区域,经Blast与Vector NTI分析发现：羽衣甘蓝与甘蓝型油菜U-box区存在5个位点的氨基酸差异,ARM区域有14个位点的氨基酸差异。结球甘蓝的SRK激酶域与羽衣甘蓝SRK激酶域的氨基酸序列的相似性达到91.0%,376个氨基酸序列中存在46个差异氨基酸,而这46个差异氨基酸中有12个为相似性氨基酸。SRK激酶域氨基酸序列存在一个高变区,高变区位于序列第250个氨基酸与270个氨基酸之间。
2.酵母双杂交重组诱饵质粒的毒性及自激活检测
Y2HGold [pGBKT7-ARCla]、Y2HGold [pGBKT7-ARClb]、Y2HGold [pGBKT7-ARClc]、Y2HGold [pGBKT7-ARCld]、Y2HGold [pGBKT7-ARCla]和Y2HGold [pGBKT7]转化株在缺陷型SD/-Trp平板上生长状态良好,并且菌斑大小无明显差异；由此表明构建的4组重组诱饵质粒的表达对酵母细胞无毒性作用。另外,pGBKT7-ARC1四个实验组在缺陷型SD/-Trp、SD/-Trp/x-a-gal平板上均能生长、在SD/-Trp/x-a-gal/AbA平板上不生长,其中在SD/-Trp/x-a-gal平板上生长的菌落无明显蓝色出现,说明四个实验组在酵母细胞中没有自激活现象。
3.酵母双杂交重组猎物质粒的毒性检测
将pGADT7空载和pGADT7-SRKJ转化Y187,发现空载和重组表达载体在SD/-Leu平板上生长状态良好,由此表明重组表达质粒pGADT7-ARCl成功转入酵母细胞Y187且无毒性作用。
4.相互作用检测
三个实验组Y2HGold [pGBKT7-ARCla]xY187 [pGADT7-SRKJ]、Y2HGold [pGBKT7-ARClc]×Y187 [pGADT7-SRKJ]、Y2HGold [pGBKT7-ARCld]×Y187 [pGADT7-SRKJ]同时激活了4种报告基因AURl-C、MELl、HIS3、ADE2;表明羽衣甘蓝ARC1与结球甘蓝的SRK激酶结构域能够发生相互作用,且互作的区域位于连续的臂重复区,其与结球甘蓝ARC1的氨基酸差异不足以引起互作区的正确构象改变。研究结果为芸薹属植物自交不亲和反应的分子机理提供新内容。
Self-incompatibility is fascinating molecular "lock-and-key" mechanisms preventing self-fertilization in flowering plants, cross-incompatibility occurs between different individuals that have the same incompatibility type when Self-incompatibility occurs between the same haploid pollen and pistil. Members of the Brassicaceae family have a SSI (sporophytic SI) system, the SSI response of Brassica oleracea results from Recognition of SRK and self-SCR which are encoded by two genes at S locus, then the response of recognition between stigma and self pollen is transmitted to internal cells. THL is separated from SRK and is released into cytoplasm. At the same time, ARC1 is activated by the kinase domain of SRK. Then the response of ARC1 and SRK is passed to EXO70A1, then the subsequent cascade of reactions ultimately lead to self-incompatibility.
In this study, a yeast two-hybrid system was used to investigate the interaction between ARC1 and the Kinase Domain of SRK. The sequences of SRK Kinase Domain (SRKJ) and different-length ARCla, ARClb, ARClc and ARC1d of Brassica oleracea were amplified by PCR, then SRKJ was subcloned into pGADT7 and different longth ARC1 were subcloned into pGBKT7 vectors. After sequencing, the plasmids were transformed into the yeast cells, and the interaction between the truncated fragments of ARC1 from Brassica oleracea var. acephala and kinase domain of SRK from Brassica oleracea var. capitata L were tested by Yeast Two-Hybrid System provides some insights into the molecular mechanism of self-incompatibility in Brassica.The results showed:
1 Cloning and analysis of sequence
The results indicate that the gDNA, consisting of 1992 bp without introns, encodes a 663 amino acid ORF which has 99.7%similarity to Brassica oleracea var acephala (sequence accession:EU344909), containing a total of four amino acid polymorphisms. It has 45 amino acid differences when compared to B. napus (AF024025), with which it shares a 92.9%similarity. The ARC1 has no signal peptide, but has six phosphorylation sites; Sequence analysis also demonstrated that the ARC1 protein coding region contains U-box and ARM areas. Vector NTITM analysis indicated that there were 5 amino acid differences contained in the U-box region and 14 amino acid differences in the ARM region between the cloned Brassica oleracea var. acephala and Brassica napus. The sequences of Kinase Domain of SRK (SRKJ) was amplified by PCR from Brassica oleracea var. apitata L. the length of SRKJ is 1131bp and encodes 376 amino acid ORF which has 91.0%similarity to Kinase Domain of SRK from Brassica oleracea var acephala, and has 12 similarityamino acid in46 differences amino acid; there is a hypervariable region in Kinase Domain of SRK and located between the sequence of 250 amino acids and 270 amino acids.
2 The toxicity and Autoactivation detection of recombinant bait plasmids
The yeast which were transformed by Y2HGold [pGBKT7-ARC1a], Y2HGold [pGBKT7-ARClb]、Y2HGold [pGBKT7-ARClc]、Y2HGold [pGBKT7-ARCld]、Y2HGold [pGBKT7-ARCla] Y2HGold [pGBKT7] were cultured on SD/-Trp plates at 30℃. After three days, there were white clones in the plates. The result showed that the recombinant bait plasmids pGBKT7-ARC1 were confirmed not toxic to yeast. In addition, four experimental groups could grow on SD/-Trp, SD/-Trp/x-a-gal plates but not grow on SD/-Trp/x-a-gal/AbA plates, the colonies did not turn blue on SD/-Trp/x-a-gal plates. The results indicates that the baits were confirmed not activate the expression of reporter genes by the test for autoactivation.
3 The toxicity detection of recombinant prey plasmid pGADT7-SRKJ
The empty vector pGADT7 and recombinant prey plasmid pGADT7-SRKJ were transformed into Y187, the yeast of Y187 [pGADT7] and Y187 [pGADT7-SRKJ] grew well on the SD/-Leu plates. The results suggested that recombinant plasmid pGADT7-SRKJwas successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187.
4 The interaction detection between different truncated fragments of ARCl and SRKJ
Three experimental groups Y2HGold [pGBKT7-ARCla]×Y187 [pGADT7-SRKJ], Y2HGold [pGBKT7-ARClc]×Y187 [pGADT7-SRKJ] and Y2HGold [pGBKT7-ARCld]×Y187 [pGADT7-SRKJ] could grow on QDO/x/A nutritional media with transcription activation of the reporter gene AUR1-C, MEL1, HIS3, ADE2, it indicated that there exists interaction between ARC1 from Brassica oleracea var. acephala L and SRK from Brassica oleracea var. capitata L and the interaction domain was located on ARM repeats, and the fifference at amino acid level with the ARC1 of Brassica oleracea var. capitata L is not enough to change the conformation in the interaction region. All above mentioned provides some insights into the molecular mechanism of self-incompatibility in Brassica.
引文
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