不结球白菜Ty1-copia类逆转座子序列的克隆及表达

Cloning and Expression Analysis of Ty1-copia-like Retrotransposons in Brassica campestris ssp.chinensis

参照Tyl-copia类逆转座子逆转录酶的保守区设计简并引物，分别从10个不结球白菜（Brassica campestris ssp．chinensis）品种的全基因组中均扩增出260bp左右的目标条带。将目的条带回收、克隆和测序后进行分析，DNAstar分析发现，这些序列存在高度的异质性，28个核苷酸序列变化范围为224～278bp，同源性范围为16．7％～83．0％。28条序列通过核苷酸聚类分为8个家族。推导氨基酸序列有移框突变、终止子突变或二者兼有；与已登录的不同物种同一类型逆转录酶氨基酸系统进化树分析表明，不结球白菜Tyl-copia类逆转座子与芥菜型油菜、拟南芥、芜菁、甜菜可能有共同的起源。半定量和实时定量PCR检测表明，水杨酸（salicylicacid）和Peronospora parasitica均能激活不结球白菜Tyl-copia类逆转座子，逆转座子在不结球白菜叶片中的表达特征说明它可能参与寄主对病原菌的抗性。

Using degenerate oligoncleotide primers corresponding to conserved domains of the Tyl-copialike retrotransposon reverse transcriptase, a fragment of 260 bp was amplified by PCR from 10 Special Brassica carnpestris ssp. chinensis. Twenty-eight clones isolated from the Brassica campestris ssp. chinensis were sequenced and showed high heterogeneity. The length of the nucleotide sequences varied from 224 bp ～278 bp,amino acid ranging from 16.7% to 83.0% by DNAstar analysis. Eight families were distinguished after cluster and alignment analyses of their nucleotide sequences. The deduced amino acid sequences of the 28 clones,had frameshift,stop codon or both by BioXM software;Cluster and alignment analyses of the 28 sequences and other published sequences of the reverse transcriptase from other species showed that the Tyl-copia-like retrotransposon reverse transcriptase in Brassica carnpestris ssp. chinensis have a closer relationship with those in Brassica juncea , Arabidopsis thaliana , Brassica rapa subsp, olei f era and Beta vulgaris subsp, vulgaris. They may share the common origin. Semi-quantitative RT-PCR and Q-PCR anal- ysis revealed that Tyl-copia-like retrotransposon transcript was activated expression after inoculated with salicylic acid and Peronospora parasitica. Expression characterization of Tyl-copia-like retrotransposon in Brassica campestris ssp. chinensis indicated that it might be involved in host resistance against pathogens.