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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]

2019 03
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Over-expression and RNAi expression vector construction for the ChiI2 gene of banana
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
banana chitinase RNAi expression vector over-expression vector seamless cloning
S668.1: Q78

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.


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Last Update: 2019-06-25