小桐子JcCIPK2基因的克隆与表达分析
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引用本文:杨 宇,陈永坤,孔春艳,龚 明.小桐子JcCIPK2基因的克隆与表达分析[J].西北植物学报,2019,39(12):2123~2131
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作者单位
杨 宇,陈永坤,孔春艳,龚 明* (云南师范大学 生命科学学院生物能源持续开发利用教育部工程研究中心云南省生物质能与环境生物技术重点实验室昆明 650500) 
基金项目:国家自然科学基金(31860062,31460059)
中文摘要:钙调磷酸酶B类似蛋白互作蛋白激酶(CBL interacting protein kinase, CIPK)是一类植物中特有的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,参与多种生物和非生物胁迫响应过程。该实验通过RT PCR克隆了小桐子(Jatropha curcas L.) JcCIPK2基因cDNA全长序列,采用荧光定量qRT PCR分析JcCIPK2基因在不同组织及不同处理(12 ℃和1 ℃低温、42 ℃高温、30% PEG、250 mmol/L NaCl、150 μmol/L ABA )下的表达模式。结果显示:(1)小桐子JcCIPK2基因开放阅读框全长1 398 bp,编码465个氨基酸,相对分子量为52.95 kD,等电点为8.89。(2)蛋白质结构分析表明,JcCIPK2的N端含有位于第11~265个氨基酸之间的丝氨酸/苏氨酸激酶_蔗糖非发酵 1型相关蛋白激酶3催化域STKc_SnRK3,在激酶结构域内还具有激活环(Activation Loop);C端含有位于第316~430个氨基酸之间的CIPK蛋白激酶调控域CIPK_C,其调控域中含有CIPK家族典型的能与CBL特异性结合的NAF结构域,位于第314~369个氨基酸之间。(3)系统进化分析显示,小桐子JcCIPK2蛋白与同属于大戟科的木薯(Manihot esculenta Crantz.)同源关系最近,序列一致性达87%。(4)qRT PCR分析表明,JcCIPK2基因在小桐子根、茎、叶中均有表达,经12 ℃和1 ℃低温处理后,叶片中JcCIPK2基因的表达都呈现先上调后下调表达的趋势,且都在低温处理24 h的表达量最高,与对照相比分别上调了6.0倍和16.72倍;小桐子JcCIPK2基因在42 ℃高温、30% PEG、150 μmol/L ABA、250 mmol/L NaCl处理下也受到不同程度的诱导表达。研究推测,JcCIPK2基因在小桐子对逆境的响应与适应中起重要作用。
中文关键词:小桐子  JcCIPK2  基因克隆  非生物胁迫  表达分析
 
Cloning and Expression Analysis of JcCIPK2 Gene in Jatropha curcas L.
Abstract:CBL interacting protein kinase (CIPK) is a family of specific serine/threonine (Ser/Thr) protein kinases in plants, which participates in response to various biotic and abiotic stresses. In this study, the full length cDNA of Jatropha curcas JcCIPK2 gene was cloned by RT PCR, and the expression patterns of JcCIPK2 in different tissues and treatments (low temperature at 12 ℃ and 1 ℃, high temperature at 42 ℃, 30% PEG, 250 mmol/L NaCl, 150 μmol/L ABA) were analyzed by fluorescence quantitative qRT PCR. The results showed that: (1) The open reading frame of JcCIPK2 gene was 1 398 bp, encoding 465 amino acids, the relative molecular weight was 52.95 kD, and the isoelectric point was 8.89. (2) Protein structure analysis indicated that the N terminus of JcCIPK2 contains serine/threonine kinase_sucrose non fermenting 1 related protein kinase 3 catalytic domain STKc_SnRK3, which located between the 11th and 265th amino acids, and with an activation loop in the kinase domain; C terminal contains the CIPK protein kinase regulated domain CIPK_C, between the 316th and 430th amino acids, and its regulatory domain also contains a NAF domain which is specifically associated with CBL, and is typical of the CIPK family, between the 314th and 369th amino acids. (3) Phylogenetic analysis showed that the JcCIPK2 protein has the highest homology with Manihot esculenta belonging to the Euphorbiaceae, and the sequence identity is up to 87%. (4) JcCIPK2 gene was expressed in roots, stems and leaves of J. curcas. After treatment at 12 ℃ and 1 ℃ the expression of JcCIPK2 gene in leaves was up regulated and then down regulated. The highest expression level was observed at 24 h under the low temperature treatment, and was increased by 6.0 and 16.72 folds, respectively, compared with the control. In addition, JcCIPK2 gene was induced to different levels by treatments with NaCl (250 mmol/L), high temperature (42 ℃), ABA (150 μmol/L), and drought stress (30% PEG). These results indicated that JcCIPK2 gene plays important roles in the response and adaptation of J. curcas to various stresses.
keywords:Jatropha curcas L.  JcCIPK2  gene cloning  abiotic stress  expression analysis
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