Cloning and Expression Analysis of Ring-type E3 Ubiquitin Ligase Gene in Camellia sinensis cv. Tieguanyin Under Drought Stress
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摘要: 以铁观音茶树为试材，对其中E3泛素连接酶基因进行克隆和生物信息学分析，并采用qPCR进行不同干旱条件下的定量表达分析。研究结果表明，该序列全长为1 138 bp，开放阅读框（ORF）为810 bp，编码269个氨基酸（GenBank登录号KR819177）。生物信息学分析发现该铁观音茶树E3泛素连接酶基因不含跨膜结构以及信号肽，具有多个磷酸化位点，亚细胞定位于叶绿体中。经BLAST比对，该基因编码的氨基酸序列与烟草、亚麻荠、葡萄、醉蝶花、芜菁中的E3泛素连接酶基因编码的氨基酸序列分别有51%、50%、50%、50%和49%的同源性，且相关保守功能结构域翻译的蛋白质序列具有RING-finger结构，初步确定该基因为铁观音茶树的E3泛素连接酶基因。qPCR分析结果显示在不同干旱胁迫处理下铁观音茶树的E3泛素连接酶基因的表达量，与对照组相比显著增加。本研究认为铁观音茶树RING型E3泛素连接酶基因参与茶树抗旱响应机制。
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关键词:
- 铁观音 /
- RING型E3泛素连接酶 /
- 基因克隆 /
- 干旱胁迫 /
- qPCR
Abstract: The E3 ubiquitin ligase gene from Camellia sinensis cv.Tieguanyin tea plants were cloned and subjected to a bioinformatics analysis. Under varied drought conditions on the plants, the quantitative expressions of the gene was determined using qPCR. The gene sequence (GenBank accession number, KR819177) was found to be 1 138 bp long containing an open reading frame (ORF) of 810 bp and encoding 269 amino acids. The bioinformatics revealed that the ligase gene did not have a transmembrane or signal peptide but multiple phosphorylation sites, and that its subcellular cells located in the chloroplasts. Its nucleotide and deduced amino acid sequences showed 51%, 50%, 50%, 50%, and 49% homology with the E3 ubiquitin ligase genes from Nicotiana sylvestris, Camelina sativa, Vitis vinifera, Tarenaya hassleriana, and Brassica rapa, respectively; while its conserved functional domains related to the translated protein sequences had a RING-finger structure. Consequently, it was preliminarily identified as the E3 ubiquitin ligase gene of C. sinensis cv. Tieguanyin. Subsequently, the qPCR analysis indicated that the transcript of this gene, when the tea plant was under varied drought stresses, was significantly more up-regulated than control. Hence, it was concluded that the previously identified RING-type E3 ubiquitin ligase gene found in C.sinensis cv. Tieguanyin indeed involved in the drought response mechanism of the plant.-
Key words:
- Camellia sinensis cv. tieguanyin /
- RING-type E3 ubiquitin ligase /
- gene cloning /
- drought stress /
- qPCR
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图 1 铁观音茶树RING型E3泛素连接酶基因全长ORF序列扩增
注：A为2 000 bp Marker；B为铁观音茶树RING型E3泛素连接酶基因。
Figure 1. Amplified full-length sequences of RING-type E3 ubiquitin ligase gene and ORF in C. sinensis cv. Tieguanyin
图 2 铁观音茶树RING型E3基因序列及其编码的氨基酸序列
注：双下划线表示RING-finger保守结构域，ATG和TGA分别为翻译起始密码子和终止密码子。
Figure 2. Gene and deduced amino acid sequences of RING-type E3 gene in C. sinensis cv.Tieguanyin
图 3 铁观音茶树RING型E3蛋白的结构功能域预测结果
Figure 3. Predicted functional domains of RING-type E3 protein in C. sinensis cv.Tieguanyin
图 4 铁观音茶树RING型E3蛋白质跨膜区预测结果
Figure 4. Predicted results of RING-type E3 protein in C. sinensis cv.Tieguanyin
图 5 铁观音茶树RING型E3蛋白质三维结构预测结果
Figure 5. Predicted 3-D structure of RING-type E3 protein in C. sinensis cv.Tieguanyin
图 6 铁观音茶树与其他物种RING型E3蛋白质序列比对结果
注：表示相似性为100%；表示相似性≥75%；表示相似性≥50%；表示相似性≥33%。
Figure 6. Alignment of RING-type E3 protein sequence of C. sinensis cv.Tieguanyin compared to those of other species
图 7 铁观音茶树RING型E3蛋白系统进化树构建
Figure 7. Phylogenetic tree of RING-type E3 proteinin C. sinensis cv.Tieguanyin
图 8 铁观音茶树RING型E3泛素连接酶基因在不同干旱胁迫程度的表达量分析
注：CK为空白对照，T1为轻度干旱胁迫处理，T2为中度干旱胁迫处理，T3为重度干旱胁迫处理。
Figure 8. Expression analysison RING-type E3 ubiquitin ligase gene of C. sinensis cv.Tieguanyin under varied drought stresses
表 1 干旱胁迫试验处理
Table 1. Treatments of drought stress
编号 处理水平 土壤含水量:田间持水量
/%CK 正常供水 80±5 T1 轻度干旱胁迫 60±5 T2 中度干旱胁迫 40±5 T3 重度干旱胁迫 20±5 
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