Gene Cloning And Activity Determination Of Wheat Chitinase

As a important PR protein，chitinase(EC3．2．1.14) has been a hot point of genetic engineering of plant resistance to fungal diseases all time. Also chitinase is an inducible enzyme it can be induced by a variety of biotic and abiotic factors. This text based on chitinase gene of wheat Chi1, Chi2, Chi3, Cht2, and Cht4from NCBI, some pairs of primers were designed, to do PCR method with the randomly selected ninety-four wheat Varieties of Mini-core Collection of Chinese Wheat Varieties and two hundred and sixty wheat Varieties from home and abroad. At the same time, some of them were analyzed with the sequences. Then based on the low temperature on230wheat varieties of the260wheat varieties, determine the changes and analyze the effects of wheat chitinase activity before and after treatment, and at last this article shows the prospects of transferring chitinase gene into high quality wheat to obtain much better wheat varieties. The main results are as follows:(1)With the94sample materials, the primers of Cht4have all amplified fragment, most of Chi3and Cht2have also amplified target band. But with the first primer of Chi1and Chi2,only37.2％have amplified fragment, while to the second primer most have amplified fragment. It shows the same tendency with the260wheat varieties, more than85%have amplified fragment of Cht4、Chi3and Cht2,but of Chi1and Chi2the amplification rate is still very low. Although the distribution of each chitinase gene is different, there is at least one gene playing a role in these wheat varieties.(2)The analysis of sequence with eight varieties shows that, the former four materials have high homology with Cht4and it showed extensive similarities to the known ClassⅣ chitinase of some plants. While the last four materials have high homology with Chi3and it belongs to ClassⅢchitinase gene.(3)The determination of wheat chitinase activity indicates that, most chitinase activity of the sample materials are200-600U, and the mean value of chitinase activity is421.26U, the activity of overall is stable. After the low treatment, the activity of chitinase was significantly raised most increased range is between0-200U, and the increased multiple is unequal between0.16%and1.199however the increased range of overall is much smaller. The reason maybe that chitinase could be induced by low temperature, but the influences were not obviously.