小麦冰重结晶抑制蛋白基因TaIRI5的克隆及分析

为了明确小麦冰重结晶抑制蛋白基因(TaIRI5)在小麦生长发育过程中的作用,该研究以小麦品种‘北京841’为材料,利用RT-PCR方法克隆TaIRI5基因,并对该基因进行生物信息学分析、组织特异性表达分析、启动子活性分析以及亚细胞定位分析。结果显示:(1)成功克隆到TaIRI5基因,该基因全长1203 bp,开放阅读框为858 bp,编码285个氨基酸,蛋白质分子量为70.7 kD,等电点为5.07,属于疏水性蛋白。(2)实时荧光定量PCR结果显示,TaIRI5基因在小麦的根、茎、叶片、雌蕊、雄蕊、护颖、种子中均有表达,其中根部的相对表达量最高,在雌蕊中表达量最低,表明该基因在小麦的生长发育过程中起重要作用。(3)TaIRI5基因的启动子分析表明,该区域除CAAT-box和TATA-box启动子核心元件外,该序列还包含9个光响应元件和6个激素应答元件及其他元件;利用TaIRI5基因不同长度(498 bp、999 bp、1500 bp)的3个候选启动子,构建了含有GUS基因的pCAMBIA1301融合表达载体;烟草转化实验表明,3个候选启动子都能启动该基因的表达,但表达模式略有差异。(4)成功构建含有GFP基因的融合载体pCAMBIA1300-TaIRI5-GFP,亚细胞定位结果显示,TaIRI5定位于细胞膜上。该研究结果为进一步研究小麦TaIRI5基因功能奠定了基础。

Cloning and Analysis of Ice Recrystallization Inhibition Protein Gene TaIRI5 in Wheat

In order to study the effect of ice recrystallization inhibition protein gene(TaIRI5)on wheat growth and development,we cloned the TaIRI5 gene from wheat cultivar‘Beijing841’by RT-PCR technique.The gene was studied by bioinformatics analysis,tissue-specificity analysis,promoter activity analysis,subcellular localization analysis.The results showed that:(1)the TaIRI5 gene was successfully cloned,and the full length of TaIRI5 gene is 1203 bp.The open reading frame was 858 bp,encoding 285 amino acids.The size of TaIRI5 protein is about 70.7 kD and the isoelectric point is 5.07.TaIRI5 is a hydrophobic protein.(2)Quantitative real time PCR analysis showed that TaIRI5 gene was expressed in roots,stems,leaves,stamens,pistils,glumes,seeds and the relative expression level of roots was the highest and stamens was the lowest.It is speculated that TaIRI5 gene plays an important role in the growth and development of wheat.(3)The promoter of TaIRI5 analysis showed that in addition to the core elements of CAAT-box and TATA-box,the sequence also contains 9 light response elements and 6 hormone response elements as well as other elements.We have successfully constructed pCAMBIA1301 fusion expression vector containing GUS gene by three candidate promoters of TaIRI5 gene with different length of 498 bp,999 bp and 1500 bp.Tobacco transformation experiments showed that the three promoter candidates had strong activity with different expression patterns.(4)We have successfully constructed pCAMBIA1300-TaIRI5-GFP fusion expression vector containing GFP gene,subcellular localization analysis showed that TaIRI5 was located on the cell membrane.The results laid a foundation for the further study of TaIRI5 gene function in wheat.