无籽沙糖桔E3泛素连接酶U-box基因的克隆及表达分析

Cloning and Expression Analysis of U-boxgene,an E3 Ubiquitin Ligase,in ‘Wuzhishatangju' (Citrus reticulata Blanco)

为探明无籽沙糖桔自交不亲和的分子机制,研究以无籽沙糖桔和普通有籽沙糖桔为试材,克隆了无籽沙糖桔E3泛素连接酶U-box基因,命名为CrU-box基因。该基因的ORF长度为3 150 bp,编码1049aa的蛋白质,无籽沙糖桔和普通有籽沙糖桔的CrU-box基因有4个核苷酸差异,并导致2个位点氨基酸发生改变。植物Ubox蛋白间序列相似性较高,表明其在进化过程中较为保守。基因表达分析表明,CrU-box基因在子房中表达量最高,其授粉后0~96 h表达量逐渐增加,96 h表达量达到最大值,96~168 h表达量逐渐下降。但在授粉后48,72,96 h时间段上,无籽沙糖桔自交授粉后的CrU-box基因表达量远高于异交授粉后表达量。初步推测无籽沙糖桔CrU-box基因突变,以及自交授粉后CrU-box高水平表达可能是导致自交不亲和形成的原因之一。

To study the molecular mechanism of self-incompatibility(SI)of Wuzhishatangju(Citrus reticulata Blanco),the U-box was identified from Wuzishatangju and Putongyouzishatangju,named as CrU-box.The results showed that the CrU-box gene of Wuzhishatangju and Putongyouzishatangju,a common variety,contained a 3 150 bp ORF,which was encoded as a putative 1 049 amino acid polypeptide.4 bases were different in ORF sequence between Wuzhishatangju and Putongyouzishatangju,2 amino acids were different in CrU-box putative protein sequence between Wuzhatangju and Putongyouzishatangju.The U-box protein from different plants had high similarity,which indicated that the U-box gene was very conservative.The results from Real-time PCR showed that the highest expression level of CrU-box was detected in ovary,after pollination the expression level increased from 0 to 96 h,followed by declined from 96 to 168 h,the highest expression was in 96 h.However,from 48,72 to 96 h after self-pollination,the expression level of CrU-box gene was much higher than that after cross-pollination.It can be preliminarily predicted that CrU-box gene mutation and high expression level of CrU-box after self-pollination may be one of the reasons for self-incompatibility.