无籽沙糖橘泛素结合酶E2基因CrUBC2的克隆及功能分析

doi:10.16420/j.issn.0513-353x.2022-1185

摘要/Abstract

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965 bp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。

关键词: 沙糖橘, CrUBC2, 自交不亲和, 基因克隆, 功能分析

Abstract:

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.

Key words: Citrus reticulata, CrUBC2, self-incompatible, gene cloning, function analysis

吴秀兰, 李桂花, 唐文武. 无籽沙糖橘泛素结合酶E2基因CrUBC2的克隆及功能分析[J]. 园艺学报, 2023, 50(10): 2069-2078.

WU Xiulan, LI Guihua, TANG Wenwu. Cloning and Function Analysis of Ubiquitin-conjugating Enzyme Gene CrUBC2 in Citrus reticulata‘Wuzi Shatangju’[J]. Acta Horticulturae Sinica, 2023, 50(10): 2069-2078.

图/表 8

表1 本试验中所用的引物序列

Table 1 Primers for experiment

引物名称Primer	引物序列（5′-3′）Primer sequence
CrUBC2-F1	TAGGCTCGACTCAACACACGCAA
CrUBC2-R1	CGAGCTCCACCTTTAATAGAGGTG
CrUBC2-F2	CATGTCGACACCTGCAAGGA
CrUBC2-R2	CCTTAATCTGCTGTCCAGCTC
qCrUBC2-F	GCAAGGAAGAGGCTGATGAGAGA
qCrUBC2-R	CAAATCGCACAGTTGGCGGTTTG
Actin-F	CCAATTCTCTCTTGAACCTGTCCTT
Actin-R	TGACTGATGAGAACTGCCAGAAG
GFPCrUBC2-F	CAAATTCGCGACCGGTATGTCGACACCTGCAAGGAAGAG
GFPCrUBC2-R	TGCTAGCCATACCGGTATCTGCTGTCCAGCTCTGTTCCA
pET32a-CrUBC2-F	CGGAATTCATGTCGACACCTGCAAG
pET32a-CrUBC2-R	CGCGTCGAATCTGCTGTCCAGCTCT
GFP-UBE2-F	CAAATTCGCGACCGGTATGTCGACACCTGCAAGGAAGAG
GFP-UBE2-R	TGCTAGCCATACCGGTATCTGCTGTCCAGCTCTGTTCCA

图1 沙糖橘和无籽沙糖橘CrUBC2部分序列比对

Fig. 1 Sequence alignment of CrUBC2 gene between‘Shatangju’and‘Wuzi Shatangju’

图2 CrUBC2与其他物种的UBC2蛋白的多序列比对

Fig. 2 Alignments of protein sequences of CrUBC2 and other UBC2

图3 CrUBC2在‘无籽沙糖橘’和‘沙糖橘’不同组织的表达量 **或*表示两个材料间的差异达到极显著（P < 0.01）或显著水平（P < 0.05）。

Fig. 3 Expression analyses of CrUBC2 gene in different tissues of‘Wuzi Shatangju’and‘Shatangju’ ** or * on the bars indicate significant differences between‘Wuzi Shatangju’and‘Shatangju’at 0.01 or 0.05 level.

图4 无籽沙糖橘自交或与沙糖橘异交授粉后CrUBC2的不同时间表达量不同小写字母表示异交授粉后各时间段的差异显著（P <0.05）。** 表示自交和异交间差异达到极显著水平（P < 0.01）。

Fig. 4 Expression analyses of CrUBC2 genes in different stages after self-pollination and cross-pollination Different lowercase letters on the bars indicate significant differences among different times（P < 0.05）. ** indicate significant differences between self-pollination and cross-pollination（P < 0.01）.

图5 CrUBC2蛋白的亚细胞定位分析

Fig. 5 Subcellular localization analysis of CrUBC2 in tobacco

图6 无籽沙糖橘（A）和沙糖橘（B）原核诱导表达CrUBC2蛋白

Fig. 6 Heterologous expression protein of CrUBC2 using E. coli expression system in‘Wuzi Shatangju’（A）and‘Shatangju’（B）

图7 ‘无籽沙糖橘’CrUBC2蛋白对‘无籽沙糖橘’和‘沙糖橘’花粉管生长的影响不同小写字母表示‘无籽沙糖橘’各浓度处理下的差异显著性（P < 0.05），**表示两材料间差异达到极显著水平（P < 0.01）。

Fig. 7 Effects of CrUBC2 protein from‘Wuzi Shatangju’on pollen tube length of‘Wuzi Shatangju’and‘Shatangju’ Different lowercase letters indicate significant differences between treatments with‘Wuzi Shatangju’（P < 0.05），** indicates significant differences between‘Wuzi Shatangju’and‘Shatangju’（P < 0.01）.

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