大白菜几丁质酶基因CHB4的克隆及序列分析

根据已知的几种十字花科植物几丁质酶基因保守序列设计特异性引物, 以大白菜叶片基因组DNA为模板, 通过PCR反应扩增出约1 kb的DNA片段和几丁质酶基因5′端序列片段(150 bp) 。利用不同浓度的水杨酸处理苗龄7 d的大白菜, 选择酶活力较高的处理植株叶片提取RNA, 采用RACE技术扩增。研究结果表明, CHB 4基因的DNA序列长度为1 517 bp (DDBJ 登录号: AB257452) , 包含有1个长度为508 bp内含子。该基因编码的产物是由268个氨基酸残基组成的蛋白质, 氨基酸序列与油菜ClassⅣ类几丁质酶的同源性达99% , 与其它几种高等植物的同源性达50%以上。

Cloning and Sequence Analysis of Chinese Cabbage Chitinase Gene CHB4

Based on the conserved sequence of the chitinase of crucifers,about 1 kb DNA fragment and 5'-end fragment(150 bp)were amplified by PCR from genomic DNA of Chinese cabbage.Experiments were performed on 7-day-old Chinese cabbage seedlings after germination with salicylic acid of different concentrations.Total RNA was prepared from the plant with higher chitinase activity,and 3'-end fragment was amplified by 3' RACE.Comparing Chinese cabbage chitinase sequence cloned in this experiment with that reported in GenBank composed of 1 094 bp nucleotide of Brassica napus,the experiment showed that the chitinase sequence of Chinese cabbage is 1 517 bp in size with one intron of 508 bp(DDBJ accession number:AB257452),and shares as high as 99% identity with B.napus and over 50% with other high plants in coding region.