|Table of Contents|

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

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

Issue:
2019 03
Page:
672-678
Research Field:
Articles
Publishing date:

Info

Title:
Over-expression and RNAi expression vector construction for the ChiI2 gene of banana
Author(s):
HUANG Yuji1 ＆ LAI Zhongxiong2**
1 College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China 2 Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
Keywords:
banana;  chitinase;  RNAi expression vector;  over-expression vector;  seamless cloning
CLC:
S668.1: Q78
PACS:
DOI:
10.19675/j.cnki.1006-687x.201809019
DocumentCode:

Abstract:
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 beta-folding, 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 than 70% 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-ChiI2i 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.

References

1. 张慧坚, 曾小红, 刘晓光, 刘海清, 卢琨, 李晓娜, 董定超, 濮文辉, 麦雄俊, 谢龙莲, 秦斌华. 国内外热带作物生产与科技发展研究综述[J]. 农学学报, 2018, 8 (3): 69-77 [Zhang HJ, Zeng XH, Liu XG, Liu HQ, Lu K, Li XN, Dong DC, Pu WH, Mai XJ, Xie LL, Qin BH. Development and research on tropical agricultural science and technology at home and abroad: a review [J]. J Agric, 2018, 8 (3): 69-77]
2. 张静, 孙秀秀, 徐碧玉, 金志强, 刘菊华. 香蕉分子育种研究进展[J]. 分子植物育种, 2018, 16 (3): 914-923 [Zhang J, Sun XX, Xu BY, Jin ZQ, Liu JH. Research advances in molecular breeding of banana [J]. Mol Plant Breed, 2018, 16 (3): 914-923]
3. 吴志刚, 朱旭芬. 几丁质酶的分子生物学特性及其在转基因植物中的应用[J]. 生命科学, 2002, 14 (2): 117-121 [Wu ZG, Zhu XF. Molecular biology characterization and application in transgenic plant for chitinase [J]. Chin Bull Life Sci, 2002, 14 (2): 117-121]
4. Vellicce GR, Ricci JCD, Hernández L, Castagnaro AP. Enhanced resistance to Botrytis cinerea, mediated by the transgenic expression of the chitinase gene ch5b in strawberry [J]. Transgenic Res, 2006, 15 (1): 57-68
5. Peumans WJ, Proost P, Swennen RL, Damme V. The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins [J]. Plant Physiol, 2002, 130 (2): 1063-1072
6. Arimori T, Tamada T. Structure and function of a novel chitinase belonging to GH family 23 [J]. X-RAYS, 2014, 56 (3): 201-206
7. Clendennen SK, May GD. Differential gene expression in ripening banana fruit [J]. Plant Physiol, 1997, 115 (2): 463-469
8. Peumans WJ, Proost P, Swennen RL, Van Damme EJM. The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins [J]. Plant Physiol, 2002, 130 (2): 1063-1072
9. Clendennen SK, López-Gómez R, Gómez-Lim M, Arntzen CJ, May GD. The abundant 31-kilodalton banana pulp protein is homologous to class-III acidic chitinases [J]. Phytochemistry, 1998, 47 (4): 613-619
10. Rihs HP, Dumont B, Rozynek P, Lundberg M, Cremer R, Brüning T, Raulf-Heimsoth M. Molecular cloning, purification, and IgE-binding of a recombinant class I chitinase from Hevea brasiliensis leaves (rHev b 11.0102) [J]. Allergy, 2015, 58 (3): 246-251
11. 张妙霞, 赖钟雄. 野生香蕉几丁质酶基因的克隆与序列分析[J]. 热带作物学报, 2010, 31 (8): 1244-1252 [Zhang MX, Lai ZX. Cloning and sequence analyzing of a chitinase gene from the wild banana [J]. Chin J Trop Crops, 2010, 31 (8): 1244-1252]
12. Kovács G, Sági L, Jacon G, Arinaitwe G, Busogoro JP, Thiry E, Strosse H, Swennen R, Remy S. Expression of a rice chitinase gene in transgenic banana (‘Gros Michel’, AAA genome group) confers resistance to black leaf streak disease [J]. Transgenic Res, 2013, 22 (1): 117-130
13. Ma BC, Tang WL, Ma LY, Li LL, Zhang LB, Zhu SJ. The role of chitinase gene expression in the defense of harvested banana against anthracnose disease [J]. J Am Soc Hortic Sci, 2009, 134 (3): 379-386
14. 张妙霞, 赖钟雄, 蔡英卿. 野生香蕉几丁质酶抗冻蛋白基因(ChiI2)的克隆及分析[J]. 龙岩学院学报, 2016, 34 (5): 120-124 [Zhang MX, Lai ZX, Cai YQ. Cloning and sequence analyzing of a chitinase anti-freeze protein gene (ChiI2) from the wild banana [J]. J Longyan Univ, 2016, 34 (5): 120-124]
15. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM. pGreen: a versatile and flexible binary Ti vector for agrobacterium-mediated plant transformation [J]. Plant Mol Biol, 2000, 42 (6): 819-832
16. 周海涛, 付永平, 王丕武. 大豆Kunitz型胰蛋白酶抑制剂基因RNAi载体的构建[J]. 大豆科学, 2010, 29 (5): 742-746 [Zhou HT, Fu YP, Wang PW. Construction of RNAi expression vector of Kunitz trypsin inhibitor gene from soybean [J]. Soybean Sci, 2010, 29 (5): 742-746]
17. Suzuki HI, Spengler RM, Grigelioniene G, Kobayashi T, Sharp PA. Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics [J]. Nat Genet, 2018, 50: 657-661
18. Liu N, Xie K, Jia Q, Zhao JP, Chen TY, Li HG, Wei X, Diao XM, Hong YG, Liu YL. Foxtail mosaic virus-induced gene silencing in monocot plants [J]. Plant Physiol, 2016, 171 (3): 1801-1807
19. 冯媛媛, 李颖楠, 侯佩, 张景荣, 汪松虎, 刘永胜. 番茄ARF4基因果实特异RNAi载体的构建及遗传转化[J]. 应用与环境生物学报, 2012, 18 (2): 206-211 [Feng YY, Li YN, Hou P, Zhang JR, Wang SH, Liu YS. Construction and genetic transformation of tomato ARF4 RNA interference expression vector with fruit specific promoter [J]. Chin J Appl Environ Biol, 2012, 18 (2): 206-211]
20. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM. Total silencing by intron-spliced hairpin RNAs [J]. Nature, 2000, 407 (6802): 319-320
21. Bally J, Mcintyre GJ, Doran RL, Lee K, Perez A, Jung H, Naim F, Larrinua IM, Narva KE, Waterhouse PM. In-plant protection against Helicoverpa armigera by production of long hpRNA in chloroplasts [J]. Front Plant Sci, 2016, 7 (e47534): 1453
22. Liu H, Zhao HX, Wu LH, Liu AN, Zhao FJ, Xu WZ. Heavy metal ATPase 3 (HMA3) confers cadmium hyper tolerance on the cadmium/zinc hyper accumulator Sedum plumbizincicola [J]. New Phytol, 2017, 215 (2): 687-698
23. 李静辉, 孟宇, 李玉恒, 李昌盛, 杨焕民, 李士泽. 冷诱导RNA结合蛋白(CIRP)过表达载体的构建及鉴定[J]. 应用与环境生物学报, 2015, 21 (2): 381-384 [Li JH, Meng Y, Li YH, Li CS, Yang HM, Li SZ. Construction and identification of over-expressing vector of cold inducible RNA-binding protein (CIRP) [J]. Chin J Appl Environ Biol, 2015, 21 (2): 381-384]
24. 邝翡婷, 袁定阳, 李莉, 李新奇. 一种载体构建的新方法: 重组融合PCR法[J]. 基因组学与应用生物学, 2012, 31 (6): 634-639 [Kuang FT, Yuan DY, Li L, Li XQ. A novel method for vector making: recombination-fusion PCR [J]. Genomics Appl Biol, 2012, 31 (6): 634-639]
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Last Update: 2019-06-25