小麦K型细胞质雄性不育系和保持系蛋白质组差异研究
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摘要
细胞质雄性不育(cytoplasmic male sterility,CMS)是一种广泛存在于高等植物中的生物学现象,因其雄性生殖系统不能产生有功能的花粉,而雌性生殖系统发育和营养生长完全正常的特点,使其在杂交制种中免去了“去雄”的繁琐步骤,已成为作物杂种优势利用的重要途径。关于细胞质雄性不育形成的内在机理仍需更进一步的研究。迄今为止,已从生态学、遗传学、分子生物学等方面细胞质雄性不育开展了相关研究,但蛋白质表达方面的研究还较少。
本研究利用改进的蛋白质双向电泳技术,从发育遗传学的角度,对小麦K型细胞质雄性不育系及其保持系的不同器官及不同发育时期表达相关的蛋白产物进行了差异分析。结果如下:
1.以小麦处于二核-三核期的花药为材料,比较了三种不同蛋白质提取方法三氯乙酸/丙酮法、尿素/硫脲法、KCl-醋酸铵甲醇法对双向电泳结果的影响,并在蛋白质裂解液组成、蛋白质上样量等方面进行了探索与优化。结果表明,与尿素/硫脲法及KCl-醋酸铵甲醇法相比,采用三氯乙酸/丙酮法提取蛋白质操作简便,所得的双向电泳图谱蛋白质点数较多,蛋白点形状规则、清晰,且重复性好,图谱背景也比较清晰。样品溶解于含有硫脲的蛋白质裂解液Ⅱ中,可显著提高蛋白质的溶解性。以三氯乙酸/丙酮提取法提取小麦花药中的蛋白,用蛋白质裂解液[7mol/L尿素,2mol/L硫脲,4%(W/V)CHAPS,1% (V /V) pH=3～10的固相pH梯度( immobilized pH gradients, IPG ) buffer]溶解蛋白,以pH3~7线性24cm的IPG胶条进行双向凝胶电泳,在上样量为1000μg,12%SDS-PAGE胶浓度下,蛋白质得到了更好的分离,图谱效果最好。
2.利用蛋白质双向电泳技术,构建小麦K型不育系豫麦21和豫农43及其保持系的苗期叶片和二核-三核期旗叶的蛋白图谱。发现不育系与保持系凝胶上的蛋白质双向电泳图谱基本相同,只有豫农43不育系与保持系有3个蛋白点存在差异。
3.通过对花药蛋白的双向电泳分析,构建小麦K型不育系豫麦21和豫农43与其保持系处于二核-三核期花药的蛋白图谱。结果表明,大部分蛋白质属于酸性蛋白,主要集中分布在pH 4～7区域内,有12个蛋白质点在不同亲和阶段表现差异,其中在豫农43不育系与保持系之间发现5个差异点:A4、A5、A6、A7、A8。A5是仅在豫农43保持系花药中表达的点;点A4、A6、A8仅在豫农43不育系花药中表达;点A7在豫农43保持系花药中上调表达,而在不育系中下调表达。在豫麦21不育系与保持系之间发现B1、B2、B3、B4、B5、B6、B7,一共7个差异点,从图中可以看出这些差异表达的蛋白质点大部分为量上面的差别。其中,B1和B7在豫麦21不育系花药中表达上调,而在保持系中下调表达;点B2、B3、B4、B5、B6在豫麦21不育系花药中表达下调,在保持系中表达上调。
4.利用二级质谱肽(LC/ESI-MS/MS)和蛋白数据库分析鉴定差异蛋白,共鉴定15个蛋白点,其中10个得到成功鉴定。分别代表6种不同的蛋白,即:OEC33kD蛋白、细胞质型苹果酸脱氢酶、磷酸丙糖异构酶、S-腺苷甲硫氨酸合成酶、细胞壁关联的蛋白激酶、谷氨酰胺合成酶。推测这些差异表达蛋白与植物生理生化代谢的酶活性、能量代谢等存在密切关系,是小麦细胞质雄性不育差异表达的关键蛋白。
CMS (cytoplasmic male sterility, CMS) is a common biological phenomena in higher plants. Because the male reproductive system cannot produce a function of pollen, and the female reproductive system development and vegetative growth is fully functional, so the solution of the tedious step "emasculation" in hybrid seed production is an important way of crop heterosis utilization. The inner mechanism of CMS needs further investigation. So far, the research of cytoplasmic male sterility (CMS) has been carried out from ecology, genetics, molecular biology, but the protein expression of the research is less.
From the developmental genetics angle,the protein comparisons from different developing stages of different organs of K-cytoplasmic male sterile(CMS)line and maintainer line in common wheat were carried on by means of two-dimesional gel electrophoresis(2-DE). The results are as follows:
1.The effects of different protein extraction methods (TCA/acetone precipitation method,urea/thiourea extraction method and KCl-methanol/ammonium-acetate method) on two-dimensional electrophoresis (2-DE) gels were compared, and different lysis buffer and loading quantity of sample were also optimized for the analysis of 2-DE system on the binucleus-trinucleate stage anthers of wheat. The results showed that, more stable repetitiveness, higher clarity and more regular shape protein spots had observed in the samples prepared by TCA/acetone precipitation than by phenol extraction method, and clearer background and protein spots were obtained in the 2-DE map. The sample dissolved in the lysis bufferⅡwhich contained thiourea, could enhanced the protein solubility. Extracted proteins by the TCA/acetone method, dissolved proteins with the lysis bufferⅡ,with pH 3-7 17cm IPG scip , 1000μg loading quantity and 12% concentrations of the SDS-PAGE gel , the proteins were well separated .
2.Two-dimensional electrophoresis(2-DE)was performed to generate the images of proteins of leaves and flag leaf of K-cytoplasmic male sterile(CMS) line and maintainer line at seeding and binucleus-trinucleate stages, respectively. The results showed that, almost no difference protein spots between K-cytoplasmic male sterile(CMS)line and maintainer line in the 2-DE map. Only Cytoplasmic Male Sterile(CMS)line and maintainer line of yunong 43 had slight differences in the 2-DE map.
3.Two-dimensional electrophoresis was performed to generate the images of proteins of anthers at binucleus-trinucleate stage. The results indicated that most of the wheat proteins were acid proteins and laid in pH4-7 position of the gel. The protein expression pattern showed that 12 protein spots with differential expression were found.There were 5 five different spots between CMS line and maintainer line of yunong 43 ,namely A4,A5,A6,A7 and A8.Among them, A5 appeared only in anthers of the maintainer line, and A4, A6 and A8 only in those of the CMS line. A7 had high expression in anthers of the maintainer line.There were 7 five different spots between CMS line and maintainer line of yumai 21, namely B1,B2,B3,B4,B5,B6,B7.Among them, B1and B7 had high expression in anthers of the CMS line, and B2, B3, B4, B5and B6 had lower expression.
4.According to LC/ESI-MS/MS analysis and protein database searching, 15 different protein spots were identified from the 2D protein maps, and 10 protein spots among them were identified succesfully.They represented 6 types of proteins: cytosolic malate dehydrogenase, triosephosphat-isomerase, S-adenosylmethionine synthetase, wall-associated kinase-like 1, glutamine synthetase isoform and OEC33kDa. This study speculated that these differentially expressed proteins were connected intensivly with plant physiological and bio-chemical activity metabolism and energy metabolism, which were key proteins of CMS.
本研究利用改进的蛋白质双向电泳技术,从发育遗传学的角度,对小麦K型细胞质雄性不育系及其保持系的不同器官及不同发育时期表达相关的蛋白产物进行了差异分析。结果如下:
1.以小麦处于二核-三核期的花药为材料,比较了三种不同蛋白质提取方法三氯乙酸/丙酮法、尿素/硫脲法、KCl-醋酸铵甲醇法对双向电泳结果的影响,并在蛋白质裂解液组成、蛋白质上样量等方面进行了探索与优化。结果表明,与尿素/硫脲法及KCl-醋酸铵甲醇法相比,采用三氯乙酸/丙酮法提取蛋白质操作简便,所得的双向电泳图谱蛋白质点数较多,蛋白点形状规则、清晰,且重复性好,图谱背景也比较清晰。样品溶解于含有硫脲的蛋白质裂解液Ⅱ中,可显著提高蛋白质的溶解性。以三氯乙酸/丙酮提取法提取小麦花药中的蛋白,用蛋白质裂解液[7mol/L尿素,2mol/L硫脲,4%(W/V)CHAPS,1% (V /V) pH=3～10的固相pH梯度( immobilized pH gradients, IPG ) buffer]溶解蛋白,以pH3~7线性24cm的IPG胶条进行双向凝胶电泳,在上样量为1000μg,12%SDS-PAGE胶浓度下,蛋白质得到了更好的分离,图谱效果最好。
2.利用蛋白质双向电泳技术,构建小麦K型不育系豫麦21和豫农43及其保持系的苗期叶片和二核-三核期旗叶的蛋白图谱。发现不育系与保持系凝胶上的蛋白质双向电泳图谱基本相同,只有豫农43不育系与保持系有3个蛋白点存在差异。
3.通过对花药蛋白的双向电泳分析,构建小麦K型不育系豫麦21和豫农43与其保持系处于二核-三核期花药的蛋白图谱。结果表明,大部分蛋白质属于酸性蛋白,主要集中分布在pH 4～7区域内,有12个蛋白质点在不同亲和阶段表现差异,其中在豫农43不育系与保持系之间发现5个差异点:A4、A5、A6、A7、A8。A5是仅在豫农43保持系花药中表达的点;点A4、A6、A8仅在豫农43不育系花药中表达;点A7在豫农43保持系花药中上调表达,而在不育系中下调表达。在豫麦21不育系与保持系之间发现B1、B2、B3、B4、B5、B6、B7,一共7个差异点,从图中可以看出这些差异表达的蛋白质点大部分为量上面的差别。其中,B1和B7在豫麦21不育系花药中表达上调,而在保持系中下调表达;点B2、B3、B4、B5、B6在豫麦21不育系花药中表达下调,在保持系中表达上调。
4.利用二级质谱肽(LC/ESI-MS/MS)和蛋白数据库分析鉴定差异蛋白,共鉴定15个蛋白点,其中10个得到成功鉴定。分别代表6种不同的蛋白,即:OEC33kD蛋白、细胞质型苹果酸脱氢酶、磷酸丙糖异构酶、S-腺苷甲硫氨酸合成酶、细胞壁关联的蛋白激酶、谷氨酰胺合成酶。推测这些差异表达蛋白与植物生理生化代谢的酶活性、能量代谢等存在密切关系,是小麦细胞质雄性不育差异表达的关键蛋白。
CMS (cytoplasmic male sterility, CMS) is a common biological phenomena in higher plants. Because the male reproductive system cannot produce a function of pollen, and the female reproductive system development and vegetative growth is fully functional, so the solution of the tedious step "emasculation" in hybrid seed production is an important way of crop heterosis utilization. The inner mechanism of CMS needs further investigation. So far, the research of cytoplasmic male sterility (CMS) has been carried out from ecology, genetics, molecular biology, but the protein expression of the research is less.
From the developmental genetics angle,the protein comparisons from different developing stages of different organs of K-cytoplasmic male sterile(CMS)line and maintainer line in common wheat were carried on by means of two-dimesional gel electrophoresis(2-DE). The results are as follows:
1.The effects of different protein extraction methods (TCA/acetone precipitation method,urea/thiourea extraction method and KCl-methanol/ammonium-acetate method) on two-dimensional electrophoresis (2-DE) gels were compared, and different lysis buffer and loading quantity of sample were also optimized for the analysis of 2-DE system on the binucleus-trinucleate stage anthers of wheat. The results showed that, more stable repetitiveness, higher clarity and more regular shape protein spots had observed in the samples prepared by TCA/acetone precipitation than by phenol extraction method, and clearer background and protein spots were obtained in the 2-DE map. The sample dissolved in the lysis bufferⅡwhich contained thiourea, could enhanced the protein solubility. Extracted proteins by the TCA/acetone method, dissolved proteins with the lysis bufferⅡ,with pH 3-7 17cm IPG scip , 1000μg loading quantity and 12% concentrations of the SDS-PAGE gel , the proteins were well separated .
2.Two-dimensional electrophoresis(2-DE)was performed to generate the images of proteins of leaves and flag leaf of K-cytoplasmic male sterile(CMS) line and maintainer line at seeding and binucleus-trinucleate stages, respectively. The results showed that, almost no difference protein spots between K-cytoplasmic male sterile(CMS)line and maintainer line in the 2-DE map. Only Cytoplasmic Male Sterile(CMS)line and maintainer line of yunong 43 had slight differences in the 2-DE map.
3.Two-dimensional electrophoresis was performed to generate the images of proteins of anthers at binucleus-trinucleate stage. The results indicated that most of the wheat proteins were acid proteins and laid in pH4-7 position of the gel. The protein expression pattern showed that 12 protein spots with differential expression were found.There were 5 five different spots between CMS line and maintainer line of yunong 43 ,namely A4,A5,A6,A7 and A8.Among them, A5 appeared only in anthers of the maintainer line, and A4, A6 and A8 only in those of the CMS line. A7 had high expression in anthers of the maintainer line.There were 7 five different spots between CMS line and maintainer line of yumai 21, namely B1,B2,B3,B4,B5,B6,B7.Among them, B1and B7 had high expression in anthers of the CMS line, and B2, B3, B4, B5and B6 had lower expression.
4.According to LC/ESI-MS/MS analysis and protein database searching, 15 different protein spots were identified from the 2D protein maps, and 10 protein spots among them were identified succesfully.They represented 6 types of proteins: cytosolic malate dehydrogenase, triosephosphat-isomerase, S-adenosylmethionine synthetase, wall-associated kinase-like 1, glutamine synthetase isoform and OEC33kDa. This study speculated that these differentially expressed proteins were connected intensivly with plant physiological and bio-chemical activity metabolism and energy metabolism, which were key proteins of CMS.
引文
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