The Cloning And Expression Analysis On Encoding Chitinase ChB4 Gene Of Chinese Cabbage

Chitin is the major ingredient of most fungal cell walls. Chitinase, widely existing in higher plant, could catalyze the hydrolysis of chitin and played a very important role in biophylaxis. With PCR and RACE techniques, the full and the mature gene encoding chitinase were isolated from Chinese cabbage genome DNA and cDNA, which was analyzed by bioinformatics method. Prokaryote expression vector pET-CHB4 was constructed and expressed in BL21. The products were analyzed by SDS-PAGE. Biological activity and the function identification were carried out. The results were as follows.1. A specific DNA fragment about 10kb and N-end fragment about 150bp were amplified from genome DNA of Chinese cabbage, and 3'end fragment was amplified by 3'RACE.2. The chitinase gene CHB4 was amplified from genome DNA by PCR and its mature gene was gained by RT-PCR reaction from cDNA.3. Comparing Chinese cabbage chitinase sequence cloned in this experiment with that reported in GenBank composed of 1094bp nucleotide of Brassica napus. It showed that the chitinase sequence of Chinese cabbage is 1517bp in size and contained 1 intron of 508bp (DDBJ accession No. AB257452), encoding a polypeptide of 268 amino acid residues (pI/Mw: 8.28 / 28701.23, www.expasy.org). Analysis of amino acid sequence of several CHIV from different species indicates that it shares 99% identity with brassica napus and over 50% with other higher plants in coding region (DNAMAN). The result suggests that the chitinase gene may function similarly to chitinase of ClassIV.4. Utilizing vector pET-22b(+) to construct expression vector pET-CHB4,through the expression vector induced to express, the result showed that the protein molecular of this expressive gene was 28kDa by analysis of SDS-PAGE and its expression products were mostly composed of inclusion body.5. Through changing parameters of induced expressing, the result showed that solubility of expression products was not change in 25℃,on the other hand, There were recombinant protein bands in lysis supernatant while pH9.5.6. Take Microzyme as demonstrative bacterium to take test on restrain bacterium circle, the result showed that the change of restrain bacterium circles were comparatively rernarkable through dealing with different densities of IPTG induced expression protein liquid ,and the restrain bacterium effect was remarkable in 0.6mmol/L of IPTG density. 7. Through mensurating chitinase activity under induced expression by a series of IPTG, the result showed, at the 0.6mmol/L IPTG , chitinase activity reached maximum 2.8U which was higher than that of original bacterium liquid 1.2U.In sum, the paper had succeeded to clone chitinase gene CHB4 and its mature gene from Chinese cabbage, and construct high-efficient prokaryotic expression vector pET-CHB4, optimize the induced expressive parameter of project bacterium, analyze the function of the chitinase. These results had provided theoretical foundation for the more research of transforming other plants and obtaining anti-disease transgenic plants.