大豆半胱氨酸蛋白酶基因GmCASc的克隆及初步功能分析
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摘要
质核互作雄性不育在作物杂种优势利用中起重要作用,研究其遗传基础和机制具有重要的理论和实践意义。本研究在大豆质核互作雄性不育系和保持系的差异蛋白质组学研究基础上,进一步对差异表达蛋白半胱氨酸蛋白酶进行基因克隆并对其功能进行初步分析,获得如下结果：
1.根据半胱氨酸蛋白酶的质谱数据,在大豆基因组数据库中搜索,发现一个相似性很高的基因,采用PCR技术扩增得到其全长cDNA,命名为GmCASc,该基因包含一个1101bp的阅读框,编码366个氨基酸,推导的GmCASc分子量为40.8kD、等电点为7.49；同源性分析表明GmCASc与毛果杨(Populus trichocarpa,XP 002323761)、葡萄(Vitis vinifera, XP_002264984)、蓖麻(Ricinus communis、XP 002525857)和水稻(Oryza saliva Japonica Group, EAZ27173)的CASPASE同源性分别是54%、52%、56%和44%；生物信息学分析表明,GmCASc具有CASPASE结构域,属于胱天蛋白酶亚家族,该蛋白是一个非跨膜的亲水蛋白。
2、利用实时荧光定量PCR方法检测大豆不同组织器官和不同胁迫条件下GmCASc的表达情况,结果显示GmCASc在质核互作雄性不育系NJCMS1A、NJCMS2A和其相应保持系NJCMS1B、NJCMS2B的根、茎、叶、花中都有表达,以茎中基因的相对表达量最高；不育系NJCMS1A花中GmCASc的相对表达量低于保持系NJCMS1B,而不育系NJCMS2A花中GmCASc的相对表达量高于保持系NJCMS2B; GmCASc在叶片中响应生物和非生物胁迫的应答各不相同,低温胁迫下,GmCASc表达下调,盐胁迫、SMV及机械损伤均引起GmCASc mRNA的累积。
3、应用酵母双杂交系统研究大豆半胱氨酸蛋白酶、V型H+-ATP酶亚基、腺苷酸脱氨酶、寡尿苷酸结合酶、Cullin、β-香树素合成酶、MADS-box蛋白、淀粉分枝酶间的相互作用,结果发现只有转化pGBKT-7-GmCASc与pGADT-7-MADS-box的酵母阳性克隆能够在SD/-Ade/-Leu/-Trp/-His平板上生长并显示β-gal活性,表明GmCASc与MADS-box之间存在相互作用,推测GmCASc与MADS-box可能在细胞凋亡信号转导通路中协同相互作用,从而引起大豆质核互作雄性不育。
Cytoplasmic-nuclear male sterility (CMS) plays an important role in the utilization of crop heterosis. It is of important significance on the theory and practice to study the genetic base and mechanism of CMS. Based on the differential proteomic studies of soybean cytoplasmic-nuclear male-sterile lines and their maintainer lines, the cloning and preliminary functional analysis of the cysteine protease gene were carried on in this paper.
The main results were as follows:
1. According to the mass spectrum data of the cysteine protease, a gene highly homology with the sequence of the cysteine protease was found through searching the database of soybean genome. Then the cysteine protease gene was cloned in soybean and named GmCASc.The GmCASc had an ORF of 1101 bp and encoded 366 amino acids. The molecular weight of the deduced GmCASc protein was 40.8 KD and the theoretical pi was 7.49. Homologous analysis of the amino acid sequences showed that GmCASc shared 54%, 52%,56%,44% homology with those of the caspases from Populus trichocarpa, Vitis vinifera, Ricinus communis, Oryza sativa respectively. Bioinformatics analysis indicated that GmCASc had a caspase domain and belonged to CASc superfamily. GmCASc was a non-transmembrane hydropathic protein.
2. The expression of GmCASc in different soybean organs and under different stress conditions was analyzed by real time quantitative PCR. GmCASc was found to be expressed in root, stem, leaf and flower of soybean cytoplasmic-nuclear male-sterile lines NJCMS1A, NJCMS2A and their corresponding maintainer lines NJCMS1B, NJCMS2B. The relative expression quantity of GmCASc showed the highest level in the stem. The relative expression quantity of GmCASc in the flower of NJCMS1A was lower than that of NJCMS1B, while the relative expression quantity of GmCASc in the flower of NJCMS2A was higher than that of NJCMS2B. The expression quantity of GmCASc in the leaves acted differently under biotic and abiotic stress conditions. The GmCASc expression was down-regulated under low temperature stress, but accumulated under salt stress, SMV, and mechanical damage treatment.
3. The yeast two-hybrid system was used to study the interations among cysteine protease, V-type H+-ATP enzyme subunit, AMP deaminase, oligouridylate bindin protein, Cullin,β-amyrin synthase, MADS -box protein, and starch branching enzyme. The results showed that only the positive yeast clone transformed with pGBKT-7-GmCASc and pGADT-7-MADS-box could grow on the SD/-Ade/-Leu/-Trp/-His plate and displayed the activity ofβ—gal. The above results showed that there was interaction between GmCASc and MADS-box. It was infered that the synergistic interaction of GmCASc and MADS-box in cell apoptosis and signal transduction might be one reason of soybean cytoplasmic-nuclear male sterility.
1.根据半胱氨酸蛋白酶的质谱数据,在大豆基因组数据库中搜索,发现一个相似性很高的基因,采用PCR技术扩增得到其全长cDNA,命名为GmCASc,该基因包含一个1101bp的阅读框,编码366个氨基酸,推导的GmCASc分子量为40.8kD、等电点为7.49；同源性分析表明GmCASc与毛果杨(Populus trichocarpa,XP 002323761)、葡萄(Vitis vinifera, XP_002264984)、蓖麻(Ricinus communis、XP 002525857)和水稻(Oryza saliva Japonica Group, EAZ27173)的CASPASE同源性分别是54%、52%、56%和44%；生物信息学分析表明,GmCASc具有CASPASE结构域,属于胱天蛋白酶亚家族,该蛋白是一个非跨膜的亲水蛋白。
2、利用实时荧光定量PCR方法检测大豆不同组织器官和不同胁迫条件下GmCASc的表达情况,结果显示GmCASc在质核互作雄性不育系NJCMS1A、NJCMS2A和其相应保持系NJCMS1B、NJCMS2B的根、茎、叶、花中都有表达,以茎中基因的相对表达量最高；不育系NJCMS1A花中GmCASc的相对表达量低于保持系NJCMS1B,而不育系NJCMS2A花中GmCASc的相对表达量高于保持系NJCMS2B; GmCASc在叶片中响应生物和非生物胁迫的应答各不相同,低温胁迫下,GmCASc表达下调,盐胁迫、SMV及机械损伤均引起GmCASc mRNA的累积。
3、应用酵母双杂交系统研究大豆半胱氨酸蛋白酶、V型H+-ATP酶亚基、腺苷酸脱氨酶、寡尿苷酸结合酶、Cullin、β-香树素合成酶、MADS-box蛋白、淀粉分枝酶间的相互作用,结果发现只有转化pGBKT-7-GmCASc与pGADT-7-MADS-box的酵母阳性克隆能够在SD/-Ade/-Leu/-Trp/-His平板上生长并显示β-gal活性,表明GmCASc与MADS-box之间存在相互作用,推测GmCASc与MADS-box可能在细胞凋亡信号转导通路中协同相互作用,从而引起大豆质核互作雄性不育。
Cytoplasmic-nuclear male sterility (CMS) plays an important role in the utilization of crop heterosis. It is of important significance on the theory and practice to study the genetic base and mechanism of CMS. Based on the differential proteomic studies of soybean cytoplasmic-nuclear male-sterile lines and their maintainer lines, the cloning and preliminary functional analysis of the cysteine protease gene were carried on in this paper.
The main results were as follows:
1. According to the mass spectrum data of the cysteine protease, a gene highly homology with the sequence of the cysteine protease was found through searching the database of soybean genome. Then the cysteine protease gene was cloned in soybean and named GmCASc.The GmCASc had an ORF of 1101 bp and encoded 366 amino acids. The molecular weight of the deduced GmCASc protein was 40.8 KD and the theoretical pi was 7.49. Homologous analysis of the amino acid sequences showed that GmCASc shared 54%, 52%,56%,44% homology with those of the caspases from Populus trichocarpa, Vitis vinifera, Ricinus communis, Oryza sativa respectively. Bioinformatics analysis indicated that GmCASc had a caspase domain and belonged to CASc superfamily. GmCASc was a non-transmembrane hydropathic protein.
2. The expression of GmCASc in different soybean organs and under different stress conditions was analyzed by real time quantitative PCR. GmCASc was found to be expressed in root, stem, leaf and flower of soybean cytoplasmic-nuclear male-sterile lines NJCMS1A, NJCMS2A and their corresponding maintainer lines NJCMS1B, NJCMS2B. The relative expression quantity of GmCASc showed the highest level in the stem. The relative expression quantity of GmCASc in the flower of NJCMS1A was lower than that of NJCMS1B, while the relative expression quantity of GmCASc in the flower of NJCMS2A was higher than that of NJCMS2B. The expression quantity of GmCASc in the leaves acted differently under biotic and abiotic stress conditions. The GmCASc expression was down-regulated under low temperature stress, but accumulated under salt stress, SMV, and mechanical damage treatment.
3. The yeast two-hybrid system was used to study the interations among cysteine protease, V-type H+-ATP enzyme subunit, AMP deaminase, oligouridylate bindin protein, Cullin,β-amyrin synthase, MADS -box protein, and starch branching enzyme. The results showed that only the positive yeast clone transformed with pGBKT-7-GmCASc and pGADT-7-MADS-box could grow on the SD/-Ade/-Leu/-Trp/-His plate and displayed the activity ofβ—gal. The above results showed that there was interaction between GmCASc and MADS-box. It was infered that the synergistic interaction of GmCASc and MADS-box in cell apoptosis and signal transduction might be one reason of soybean cytoplasmic-nuclear male sterility.
引文
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