香蕉几丁质酶基因ChiI2超表达载体和干涉表达载体的构建

Over-expression and RNAi expression vector construction for the ChiI2 gene of banana

分离克隆栽培香蕉中的几丁质酶基因ChiI2,构建超表达载体和干涉表达载体,可为进一步研究ChiI2基因响应抗逆胁迫的功能提供基础.从栽培香蕉天宝蕉(Musa spp., AAA)中克隆几丁质酶基因ChiI2,利用生物信息软件对该基因进行分析,并用无缝克隆技术分别构建ChiI2的超表达载体和干涉表达载体.从香蕉果皮中克隆到一个几丁质酶基因ChiI2,该基因全长942 bp,蛋白编码313个氨基酸,其编码的蛋白质理论分子量(Mr)为32.96,等电点(pI)为6.77.ChiI2蛋白的α-螺旋结构有6个,β-折叠结构有5个,转角结构有33个.蛋白质疏水性预测分析值为-0.233,属于亲水性蛋白.功能保守域分析表明,该蛋白是糖苷水解酶家族几丁质酶家族的一员.该基因核苷酸序列推导的氨基酸与野生香蕉、玉米、水稻、小麦、莲等植物的同源性都在70%以上.对ChiI2基因进行荧光定量PCR检测,发现4℃处理6 h的表达显著高于对照和38℃处理6 h,表明ChiI2基因可能与低温响应有关,诱导香蕉苗产生抗冷性.利用无缝克隆技术成功构建了pGreenII-ChiI2超表达载体和pGreenII-ChiI2i干涉表达载体,并转化到农杆菌EHA105菌株中.本研究成功地从栽培香蕉天宝蕉中分离克隆到了ChiI2基因,并对其基因特点和蛋白功能进行了预测分析,成功构建了超表达载体和干涉表达载体,可为进一步研究其功能奠定基础,也为采用基因工程方法改良选育香蕉抗寒品种提供了新尝试.

This study aimed to clone the full-length cDNA of the chitinase gene ChiI2 of the cultivated banana and construct over-expression and RNA interference(RNAi) expression vectors of it, which could form the basis of further studies on the functions of the ChiI2 gene in stress responses. According to the ChiI sequences obtained from the NCBI database, the ChiI2 gene was first isolated from the Tianbao banana(Musa spp., AAA). The gene characteristics, protein sequence, and phylogeny of the ChiI2 gene were analyzed using Blast, Expasy, and other types of bioinformatics software. Over-expression and RNA interference expression vectors were constructed using seamless cloning technology. A chitinase gene named ChiI2 was cloned from banana by homologous cloning. Using the relevant bioinformatics software for analyses, the open reading frame of the ChiI2 gene was found to be 942 bp in length and encode 313 amino acids. The theoretical molecular weight of the putative protein produced was 32.96 ku, and its isoelectric point(pI) was predicted to be 6.77. After Blast analysis, the nucleotide sequence of ChiI2 was found to share 99 and 97% identity with the chitinase isoform 2(AJ277279) and chitinase isoform 1(AJ277278) genes of dwarf banana, respectively. The ChiI2 protein was predicted to have six alpha-helical, five betafolding, and 33 corner motifs. The protein's predicted hydrophobicity value was-0.233, which means it belongs to the group of hydrophilic proteins. Functional conserved domain analysis showed that the protein was a member of the chitinase gene subfamily of the glycoside hydrolase family. The amino acid sequence deduced from the nucleotide sequence was more than70% homologous to those of this gene in wild banana, maize, rice, wheat, and lotus. Real-time quantitative polymerase chain reaction(PCR) analysis indicated that a temperature of 4 ℃ promoted the expression of ChiI2 after plantlets were cooled to this temperature. These results indicated that ChiI2 may play a role in promoting the cold tolerance of banana. The over-expression vector pGreenII-ChiI2 and the RNAi expression vector pGreenII-ChiI2 i were successfully constructed using seamless cloning techniques and transformed into Agrobacterium strain EHA105. The ChiI2 gene was successfully isolated and cloned from the cultivated Tianbao banana, and this gene's characteristics and protein function were predicted and analyzed. Over-expression and RNAi expression vectors for this gene were successfully constructed, which laid the foundation for the further study of its function, and could be used to improve and breed cold-resistant banana varieties through genetic engineering.