无籽沙糖橘泛素结合酶E2基因CrUBC2的克隆及功能分析  

Cloning and Function Analysis of Ubiquitin-conjugating Enzyme Gene CrUBC2 in Citrus reticulata‘Wuzi Shatangju’

作　　者：吴秀兰 李桂花[3] 唐文武[2] WU Xiulan;LI Guihua;TANG Wenwu(College of Food and Pharmaceutical Engineering,Zhaoqing University,Zhaoqing,Guangdong 526061,China;College of Life Sciences,Zhaoqing University,Zhaoqing,Guangdong 526061,China;Vegetable Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China)

机构地区：[1]肇庆学院食品与制药工程学院,广东肇庆526061 [2]肇庆学院生命科学学院,广东肇庆526061 [3]广东省农业科学院蔬菜研究所,广州510640

出　　处：《园艺学报》2023年第10期2069-2078,共10页Acta Horticulturae Sinica

基　　金：国家外专局项目(QN2022030025L);肇庆市科技计划资助项目(2015B010201001);肇庆学院博士科研启动基金项目(611-612281)。

摘　　要：泛素结合酶E2是底物泛素化的关键酶,通过泛素—蛋白酶体系统对雌蕊S-RNase进行泛素化和降解,在自交不亲和机制中发挥重要作用。以‘无籽沙糖橘’和‘沙糖橘’的花蕾为试材,以甜橙EST序列EY715921为依据克隆到965bp的cDNA全长序列,其编码152个氨基酸的UBC2蛋白,命名为CrUBC2。在‘无籽沙糖橘’和‘沙糖橘’的编码区发现1个C/T替换,并引起苏氨酸(Thr)变成异亮氨酸(Ile)。多序列比对表明‘沙糖橘’与其他8种植物UBC2仅有1个氨基酸差异,表现出进化中的高度保守性。qRT-PCR分析表明CrUBC2在花药中的表达量最高,异交授粉后的基因表达量是自交授粉的2.22倍~5.36倍,呈现明显的特异表达。体外花粉萌发试验表明,‘无籽沙糖橘’CrUBC2蛋白对自交花粉管生长有明显抑制作用,但对异交花粉管生长无影响,这可能与该蛋白参与S-RNase泛素化并导致异交亲和有关。Ubiquitin-conjugating enzyme E2 is a key enzyme for substrate ubiquitination reaction,which plays an important role in the mechanism of self-incompatible by S-RNase degradation via ubiquitin-proteasome system.An E2 gene was cloned based on EST sequence(EY715921)of Citrus sinensis using the flower buds from‘Wuzi Shatangju’and‘Shatangju’,named as CrUBC2.The full-length cDNA sequence of the CrUBC2 was 965 bp,encoding 152 amino acids.There was one base difference(C/T)in the coding sequence of CrUBC2 between‘Wuzi Shatangju’and‘Shatangju’,which resulted in the conversion of threonine to isoleucine.Multiple sequence alignment showed that only one amino acid difference(Thr/Ile)was found between CrUBC2 and orthologs.Therefore,CrUBC2 protein was highly conservative during the evolution process.qRT-PCR showed that the highest expression level of the CrUBC2 was detected in anthers,and the expression level of the CrUBC2 after cross-pollination was 2.22–5.36 fold higher than that of self-pollination,indicating obvious expression specificity in different pollination.Pollen germination experiment in vitro showed that CrUBC2 protein of‘Wuzi Shatangju’inhibited significantly the growth of pollen tubes of self-pollination,but it had no effect on the growth of cross-pollination pollen tubes,which may be related to its involvement in S-RNase ubiquitination and resulting in cross-compatibility.

关 键 词：沙糖橘 CrUBC2 自交不亲和 基因克隆 功能分析 

分 类 号：S666[农业科学—果树学]