Construction of the Recombinant Expression Plasmid and Expression Pattern of Chitinase Gene MDCII from Musca domestica

doi:10.13560/j.cnki.biotech.bull.1985.2017.02.015

Abstract

Abstract: The aims of this study are to isolate the chitinase gene MDCII from Musca domestica EST sequencing database,to clone the gene and analyze its molecular characteristic,and to explore the temporal-spatial expression patterns of the gene in different tissues and different developmental stages as well as after induced by different microorganism. Firstly,EST sequencing technology was employed to screen the chitinase gene MDCII from the constructed cDNA plasmid library of M. domestica larvae,the bioinformatics method to analyze the gene sequence and physicochemical properties of the encoded protein,and the neighbor joining to build phylogenetic tree. Then,the target gene amplified by PCR then was constructed into the recombinant plasmid pEASY-E1-MDCII,the recombinant plasmid was transformed in clonal cell Trans1-T1,and the real-time PCR technology was to detect the expression difference of the MDCII gene in different developmental stages and different tissues. Finally,injection method was used to induce different microorganisms into the 3 instar larvae of M. domestica,and real-time PCR to detect the changes of expression level in different time points after inducing. The results indicated that the ORF length of the MDCII gene was 1 374 bp,encoding 457 amino acids,and the molecular weight 51.6 kD. The alignment analysis of the phylogenetic tree revealed that the genetic distance of MDCII was close to the adult growth factor of Drosophila. The recombinant plasmid pEASY-E1-MDCII with correct gene sequence was successfully constructed. The MDCII gene expressed in different developmental stages of M. domesticaat different expression levels,and the highest in salivary glands and fat body of 3 instar larva. The MDCII gene presented up-regulated expression at 3 h after induction of Candida albicans,Staphylococcus aureus,and Escherichia coli. In conclusion,the MDCII gene is the adult growth factor in chitinase,participating in growth and development of M. domestica,and also playing a certain role in immune defense process.

Key words: Musca domestica, chitinase, clone, expression pattern

YANG Yu-jin, GUO Guo, WU Qin-yi, LI Yan, FU Ping, ZHANG Yong. Construction of the Recombinant Expression Plasmid and Expression Pattern of Chitinase Gene MDCII from Musca domestica[J]. Biotechnology Bulletin, 2017, 33(2): 102-108.

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