小麥TaCIPK2基因克隆及與TaCBLs蛋白互作分析
發(fā)布時間：2019-06-20 07:43
【摘要】：以普通六倍體小麥品種德抗961為材料,根據(jù)野大麥HbCIPK2的mRNA序列(GenBank序列號JN831652)設(shè)計引物,采用同源克隆方法從小麥中克隆TaCIPK2基因(GenBank序列號KU640381),測序結(jié)果顯示該基因序列全長1421bp,開放閱讀框(ORF)1359bp,編碼452個氨基酸殘基,預(yù)測分子量為50.91kD,pI為9.07。氨基酸序列比對結(jié)果表明:該基因與HvCIPK2(KP638475.1)、TaCIPK2(KJ561791.1)、HbCIPK2(JN831652.1)的相似度分別為94.69%、96.93%、95.80%,具有高度同源性;系統(tǒng)進化分析表明它們位于相同進化支上,親緣關(guān)系最近。采用Real-timePCR方法分析不同逆境脅迫處理下TaCIPK2基因表達的特異性,200mmol/LNaCl高鹽脅迫處理小麥幼苗后根和葉部TaCIPK2基因均上調(diào)表達;4℃低溫脅迫處理后根部TaCIPK2基因上調(diào)表達,而葉部下調(diào)表達;100μmol/LABA脅迫處理后根和葉中TaCIPK2均上調(diào)表達。為檢測TaCIPK2與TaCBL1、TaCBL2、TaCBL6、TaCBL7的相互作用,構(gòu)建酵母表達載體pGADT7-TaCIPK2,轉(zhuǎn)化酵母Y187感受態(tài)細胞;同理pGBKT7-TaCBL1、pGBKT7-TaCBL2、pGBKT7-TaCBL6、pGBKT7-TaCBL7轉(zhuǎn)化到酵母細胞Y2Hgold中,都沒有出現(xiàn)自激活活性和毒性現(xiàn)象。共轉(zhuǎn)化的二倍體酵母,只有pGADT7-TaCIPK2×pGBKT7-TaCBL2和pGBKT7-53×pGADT7-T在SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA培養(yǎng)基板上長出的菌落呈現(xiàn)藍色,說明TaCIPK2能夠與TaCBL2相互作用,激活下游報告基因HIS3、AUR1-C、MEL1和ADE2的表達,該研究結(jié)果對進一步研究TaCIPK2的功能有一定的指導(dǎo)意義。
[Abstract]:Using common hexaploid wheat variety Dekang 961 as material, primers were designed according to the mRNA sequence of wild barley HbCIPK2 (GenBank serial number JN831652). The TaCIPK2 gene (GenBank serial number KU640381) was cloned from wheat by homologous cloning method. The sequencing results showed that the full length of the gene sequence was 1421bp, the open reading frame (ORF) 1359bp, encoding 452amino acid residues, the predicted molecular weight was 50.91kD, Pi was 9.07. The results of amino acid sequence alignment showed that the similarity between the gene and HvCIPK2 (KP638475.1), TaCIPK2 (KJ561791.1) and HbCIPK2 (JN831652.1) was 94.69%, 96.93% and 95.80%, respectively, which had high homology, and the phylogenetic analysis showed that they were located on the same evolutionary branch and were closely related to each other. The specificity of TaCIPK2 gene expression under different stress treatments was analyzed by Real-timePCR. The expression of TaCIPK2 gene in roots and leaves was up-regulated after 200mmol/LNaCl high salt stress, the expression of TaCIPK2 gene in roots was up-regulated while the expression in leaves was down-regulated after 4 鈩，
本文編號：2503052
[Abstract]:Using common hexaploid wheat variety Dekang 961 as material, primers were designed according to the mRNA sequence of wild barley HbCIPK2 (GenBank serial number JN831652). The TaCIPK2 gene (GenBank serial number KU640381) was cloned from wheat by homologous cloning method. The sequencing results showed that the full length of the gene sequence was 1421bp, the open reading frame (ORF) 1359bp, encoding 452amino acid residues, the predicted molecular weight was 50.91kD, Pi was 9.07. The results of amino acid sequence alignment showed that the similarity between the gene and HvCIPK2 (KP638475.1), TaCIPK2 (KJ561791.1) and HbCIPK2 (JN831652.1) was 94.69%, 96.93% and 95.80%, respectively, which had high homology, and the phylogenetic analysis showed that they were located on the same evolutionary branch and were closely related to each other. The specificity of TaCIPK2 gene expression under different stress treatments was analyzed by Real-timePCR. The expression of TaCIPK2 gene in roots and leaves was up-regulated after 200mmol/LNaCl high salt stress, the expression of TaCIPK2 gene in roots was up-regulated while the expression in leaves was down-regulated after 4 鈩，
本文編號：2503052
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