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[1]黄玉吉 赖钟雄**.香蕉几丁质酶基因ChiI2超表达载体和干涉表达载体的构建*[J].应用与环境生物学报,2019,25(03):1-10.[doi:10.19675/j.cnki.1006-687x.2018.09019]
 HUANG Yuji,LAI Zhongxiong**.Over-expression and RNAi expression vectors construction of ChiI2 gene of banana *[J].Chinese Journal of Applied & Environmental Biology,2019,25(03):1-10.[doi:10.19675/j.cnki.1006-687x.2018.09019]
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香蕉几丁质酶基因ChiI2超表达载体和干涉表达载体的构建*()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年03期
页码:
1-10
栏目:
研究论文
出版日期:
2019-06-25

文章信息/Info

Title:
Over-expression and RNAi expression vectors construction of ChiI2 gene of banana *
文章编号:
201809019
作者:
黄玉吉1 赖钟雄2**
1福建农林大学园艺学院 福州 3500022福建农林大学园艺植物生物工程研究所 福州 350002
Author(s):
HUANG Yuji1 LAI Zhongxiong2**?
1College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China 2Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
关键词:
香蕉几丁质酶干涉表达载体超表达载体无缝克隆
Keywords:
banana chitinase RNAi expression vector over- expression vector seamless cloning
DOI:
10.19675/j.cnki.1006-687x.2018.09019
摘要:
为了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,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 )中克隆了几丁质酶基因ChiI2,利用生物信息软件对该基因进行了分析,并用无缝克隆技术分别构建了ChiI2的超表达载体和干涉表达载体。从香蕉果皮中克隆到了一个几丁质酶基因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为32.96 ku,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。为了进一步研究ChiI2基因的功能,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,为进一步研究其功能奠定了基础,可为采用基因工程方法改良选育香蕉抗寒品种提供新尝试。(图8表1参24)
Abstract:
This study aims to clone the full-length cDNA of chitinase gene ChiI2 in cultivated banana and construct the over-expression and RNA interference (RNAi) expression vectors, which can provide the basis for further study on the function of ChiI2 gene in response to stress resistance. According to the previous results of ChiI sequences from NCBI database, the ChiI2 gene was isolated from Tianbao banana (Musa spp., AAA ). The gene character , protein sequence and phylogenetic tree of ChiI2 gene were analyzed by Blast, Expasy and other bioinformatics software. Over- expression and RNA interference expression vector were constructed by seamless cloning technology. A chitinase gene named ChiI2 was cloned from banana by homologous cloning. Using the relevant bioinformatics software for analysis, the open reading frame of ChiI2 gene was 942 bp in length and encoded 313 amino acids. The theoretical molecular weight of the putative was 32.96 ku. The isoelectric point ( pI) was 6.77. After blast analysis, the nucleotide sequence of ChiI2 shared 99% and 97% identity with chitinase isoform 2 (AJ277279) and chitinase isoform 1 (AJ277278) of dwarf banana, respectively. ChiI2 protein had six alpha-helical structures, five beta-folding structures and 33 corner structures. .The predicted value of protein hydrophobicity is -0.233, which belongs to hydrophilic protein. Functional conserved domain analysis showed that the protein was a member of the chitinase gene of glycoside hydrolase family. The amino acids deduced from the nucleotide sequence were more than 70% homologous to wild banana, maize, rice, wheat and lotus. Real-time quantitative PCR analysis indicated that 4℃ promoted the expression of ChiI2 after cooling the plantlets to be 4 ℃. The results indicated that ChiI2 may play a positive role in cold tolerant in banana. The over-expression vector pGreenII-ChiI2 and RNAi expression vector pGreenII-ChiI2i were successfully constructed by using seamless cloning technique and transformed into Agrobacterium strain EHA105. ChiI2 gene was successfully isolated and cloned from cultivated banana Tianbao, and its gene characteristics and protein function were predicted and analyzed. Over-expression and RNAi expression vector were successfully constructed, which laid a foundation for further study of its function and could be used to improve and breed banana cold-resistant varieties by g enetic engineering.? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

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备注/Memo

备注/Memo:
收稿日期: 2018-09-12 接受日期 Accepted: 2018-10-15*福建省教育厅中青年教师教育科研项目(JA15189)、福建省自然科学基金面上项目(2016J01704)、国家自然科学基金青年基金项目(31701900)和福建农林大学博士后启动基金(61201400723)资助 **通讯作者(E-mail: laizx01@163.com)点击摘要页题目后的“PDF”可下载阅读全文;本文为已录用的作者修定稿,尚未经编辑全面修改。引用本文请注明出处本刊;发表刊期和页码将以正式出版时的安排为准,但DOI确定不变。
更新日期/Last Update: 2018-12-28