小麦TaCIPK8基因的表达分析及其与TaCBLs的互作
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转基因生物新品种培育重大专项(2016ZX08002003-004);973 计划(2014CB138105)


Expression analysis of TaCIPK8 gene and its interaction with TaCBLs in wheat
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    摘要:

    CIPK是植物钙感受器钙调磷酸酶B类似蛋白特定靶向的一类丝氨酸/苏氨酸蛋白激酶。我们根据拟南芥AtCIPK8基因序列,利用同源克隆的方法从抗逆性较强的小麦品种石4185中克隆了一个编码序列全长为1350 bp的蛋白激酶基因TaCIPK8(GenBank号:KJ561804.1)。序列分析表明该基因编码的蛋白含有449个氨基酸,分子量为52.24 kD,理论等电点为7.16,具有CIPKs家族蛋白所特有的N端激酶域和C端NAF/FISL结构域;与拟南芥AtCIPK8蛋白的相似度为81%。为进一步研究其功能,采用Real-time PCR方法检测该基因在胁迫条件下的响应情况,发现TaCIPK8基因受高盐、外源ABA和低温(4℃)胁迫诱导表达。利用植物启动子数据库PlantCARE和PLACE对TaCIPK8基因启动子序列进行分析,结果显示TaCIPK8基因启动子存在大量应答脱水胁迫、干旱、低温和ABA的顺式作用元件,也存在一些响应激素GA和茉莉酸甲酯的元件。利用酵母双杂交方法研究TaCIPK8和TaCBL家族蛋白的互作,结果显示,共转化含有TaCIPK8和TaCBL3基因载体的酵母菌能够在SD/-Trp/-Leu、SD/-Trp/-Leu/X-α-Gal/AbA和SD/-Trp/-Leu/-Ade/-His/X-α-Gal/AbA三种培养基上生长,且在后两种培养基上长出蓝色菌落。结果说明TaCIPK8与TaCBL3发生了互作,进而引起了报告基因MEL1、AUR1-C、HIS3和ADE2的表达。该研究结果对于研究TaCIPK8基因的功能以及与CBL蛋白的互作调控网络具有一定的参考作用。

    Abstract:

    CIPKs, Calcineurin B-like (CBL) interacting protein kinases, belong to a group of serine/threonine protein kinases in plants. We cloned TaCIPK8 gene (GenBank number: KJ561804) from wheat variety Shi4185 based on the gene sequence of Arabidopsis AtCIPK8, and the length of TaCIPK8 coding sequence is 1350 bp. Sequence analysis indicated that TaCIPK8 protein consists of 449 amino acids, and its molecular mass and theoretical isoelectric point are 52.24 kD and 7.16, respectively. The protein contains an N-terminal kinase domain and a C-terminal NAF/FISL motif specifically possessed by the CIPK family proteins; the similarity betweenTaCIPK8 and AtCIPK8 protein is 81%. In order to investigate the function of TaCIPK8 gene, we detected the responses of TaCIPK8 gene to different stress conditions using Real-time PCR, and found that the expression of TaCIPK8 gene was induced by high salinity, ABA and low temperature (4℃). We analysed the promoter of TaCIPK8 gene using plant promoter databases PlantCARE and PLACE. The results indicated that the promoter harbors not only many cis-acting elements responsive to dehydration, drought, low temperature and ABA, but also DNA elements responsive to GA and jasmonic acid methyl ester. Yeast two hybrid was used to study the interaction between TaCIPK8 and TaCBLs proteins, and we found that the yeast transformed with both TaCIPK8 containing vector and TaCBL3 containing vector could grow on SD/-Trp/-Leu、SD/-Trp/-Leu/X-α-Gal/AbA and SD/-Trp/-Leu/-Ade/-His/X-α-Gal/AbA media, with blue colonies on the latter two media. The results indicate that the TaCIPK8 protein interacted with TaCBL3, which activated the expression of the reporter genes MEL1, AUR1-C, HIS3 and ADE2. Our results provide useful information for revealing the functions of TaCIPK8 gene and the interaction regulatory network between the protein of TaCIPK8 and TaCBLs in wheat.

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毕惠惠,贺亚伟,毛伟伟,等.小麦TaCIPK8基因的表达分析及其与TaCBLs的互作[J].植物遗传资源学报,2018,19(2):296-304.

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  • 收稿日期:2017-09-03
  • 最后修改日期:2018-01-28
  • 录用日期:2017-11-13
  • 在线发布日期: 2018-02-23
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