摘要
以普通六倍体小麦品种德抗961为材料,根据野大麦HbCIPK2的mRNA序列(GenBank序列号JN831652)设计引物,采用同源克隆方法从小麦中克隆TaCIPK2基因(GenBank序列号KU640381),测序结果显示该基因序列全长1421bp,开放阅读框(ORF)1359bp,编码452个氨基酸残基,预测分子量为50.91kD,pI为9.07。氨基酸序列比对结果表明:该基因与HvCIPK2(KP638475.1)、TaCIPK2(KJ561791.1)、HbCIPK2(JN831652.1)的相似度分别为94.69%、96.93%、95.80%,具有高度同源性;系统进化分析表明它们位于相同进化支上,亲缘关系最近。采用Real-timePCR方法分析不同逆境胁迫处理下TaCIPK2基因表达的特异性,200mmol/LNaCl高盐胁迫处理小麦幼苗后根和叶部TaCIPK2基因均上调表达;4℃低温胁迫处理后根部TaCIPK2基因上调表达,而叶部下调表达;100μmol/LABA胁迫处理后根和叶中TaCIPK2均上调表达。为检测TaCIPK2与TaCBL1、TaCBL2、TaCBL6、TaCBL7的相互作用,构建酵母表达载体pGADT7-TaCIPK2,转化酵母Y187感受态细胞;同理pGBKT7-TaCBL1、pGBKT7-TaCBL2、pGBKT7-TaCBL6、pGBKT7-TaCBL7转化到酵母细胞Y2Hgold中,都没有出现自激活活性和毒性现象。共转化的二倍体酵母,只有pGADT7-TaCIPK2×pGBKT7-TaCBL2和pGBKT7-53×pGADT7-T在SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA培养基板上长出的菌落呈现蓝色,说明TaCIPK2能够与TaCBL2相互作用,激活下游报告基因HIS3、AUR1-C、MEL1和ADE2的表达,该研究结果对进一步研究TaCIPK2的功能有一定的指导意义。
Wheat cultivar De Kang 961 was used to obtain total RNA for first-strain c DNA synthesis. Based on primers according to m RNA sequence of Hb CIPK2(Gen Bank No.JN831652) in hordeum brevisubulatum, Ta CIPK2 gene fragment(Gen Bank No.KU640381) was cloned from wheat by homologous cloning method. The sequence analysis indicated that the total length of gene sequence was 1 421 bp and the ORF was 1 359 bp which encoded452 amino acid residues. The predicted molecular weight of Ta CIPK2 protein was 50.91 k D with p I of 9.07. The alignment results of amino acid sequence showed that Ta CIPK2(KU640381) shared the similarity of 94.69% 、96.93%、95.80% with the CIPK2 protein from Hv CIPK2(KP638475.1), Ta CIPK2(KJ561791.1), Hb CIPK2(JN83-1652.1), respectively, which had high homology. Phylogenetic tree analysis revealed that they located in the same phylogenetic branch and had closer relationships. Using Real-time PCR to analyze the specificities of Ta CIPK2 gene expression under different stress condition, we found that Ta CIPK2 had a higher expression both in the root and shoot. Ta CIPK2 had a higher expression in root under 200 mmol/L Na Cl salt stress condition. Ta CIPK2 gene had a higher expression in root while a lower expression in shoot under 4℃ stress. Under 100 μmol/L ABA stress condition, Ta CIPK2 had a higher expression both in the root and shoot. To verify the interactions between Ta CIPK2 and Ta CBL1、Ta CBL2、Ta CBL6、Ta CBL7, we constructed yeast expression vector p GADT7-Ta CIPK2 and transformed it into yeast strain Y187. The yeast expression vectors of p GBKT7-Ta CBL1, p GBKT7-Ta CBL2, p GBKT7-Ta CBL6, p GBKT7-Ta CBL7 were constructed and transformed into yeast of Y2 Hgold strains. Finally, diploid yeast p GADT7-Ta CIPK2×p GBKT7-Ta CBL2 and p GBKT7-53×p GADT7-T showed positive results in SD/-AdeTrp-Leu-His/X-α-gal/Ab A synthetic dropout. It demonstrated that interaction between Ta CIPK2 and Ta CBL2 can activate downstream reporter genes HIS3, AUR1-C, MEL1 and ADE2. The result will be helpful for un
出处
《分子植物育种》
CAS
CSCD
北大核心
2016年第7期1655-1665,共11页
Molecular Plant Breeding
基金
973计划(2014CB138105)
自然科学基金项目(31101207)共同资助
