收稿日期: 2017-05-31
网络出版日期: 2019-09-04
Cloning and expression vector construction of E3 ubiquitin ligase PpARI1 gene from juicy peaches
Received date: 2017-05-31
Online published: 2019-09-04
以新鲜水蜜桃为材料, 根据NCBI中预测的桃E3泛素连接酶ARI1基因序列设计引物, 通过聚合酶链式反应(polymerase chain reaction, PCR)克隆该序列. 利用双酶切定向连接的方法将PpARI1基因开放阅读框(open reading frame, ORF)正向重组到表达载体pCAMBIAy1300的XbaⅠ和BamHⅠ酶切位点之间, 并用热激法转化到农杆菌感受态细胞中. 成功克隆出的长度为1 764 bp的PpARI1基因CDS序列, 与预测基因序列的一致性高达99.83${\%}$. 对重组子测序结果表明, PpARI1基因ORF准确插入pCAMBIAy1300载体的启动子和终止子之间, 表明载体构建成功. PCR检测结果也表明, 该重组载体已成功转入农杆菌中. 这为下一步更深入地研究水蜜桃PpARI1基因的功能奠定了实验基础.
苑婕, 马方玮, 李梦云, 曹庆, 郑伟尉, 万嗣宝 . 水蜜桃E3泛素连接酶PpARI1基因的克隆及表达载体构建[J]. 上海大学学报(自然科学版), 2019 , 25(4) : 604 -611 . DOI: 10.12066/j.issn.1007-2861.1949
Fresh juicy peaches were used as materials, and a novel sequence of E3 ubiquitin ligase PpARI1 was obtained using a polymerase chain reaction (PCR) method with the primers designed according to the predicted gene sequence of ARI1 in NCBI. The open reading frame (ORF) box of PpARI1 gene was forward recombined into pCAMBIAy1300, an efficient expression vector, between Xba I site and BamH I site. The sequencing results showed that the obtained PpARI1 was 1 764 bp with 99.83${\%}$ identity to that of the predicted gene sequence, and the ORF of PpARI1 gene was correctly inserted between the promoter and the terminator. The recombined plasmid was transformed to competent Agrobacterium cells with a heat shock method. The PCR result showed that the vector pCAMBIAy1300-PpARI1 was successfully transferred into Agrobacterium. The results would lay a foundation for further function researches of PpARI1 gene in juicy peaches.
Key words： juicy peach; E3 ubiquitin ligase; cloning; expression vector construction
| [1] | Gupta N, Rathore M, Goyary D, et al. Marker-free transgenic cucumber expressing Arabidopsis CBF1 gene confers chilling stress tolerance[J]. Biologia Plantarum, 2012,56(1):57-63. |
| [2] | Smalle J, Vierstra R D . The ubiquitin 26S proteasome proteolytic pathway[J]. Annual Review Plant Biology, 2004,55:555-590. |
| [3] | 黄海杰, 陈雄庭 . 植物泛素/26S蛋白酶体途径研究进展[J]. 中国生物工程杂志, 2008,28(7):127-132. |
| [4] | Craig A, Ewan R, Mesmar J , et al. E3 ubiquitin ligases and plant innate immunity[J]. Journal of Experimental Botany, 2009,60(4):1123-1132. |
| [5] | Glickman M H, Ciechanover A . The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction[J]. Physiological Reviews, 2002,82(2):373-428. |
| [6] | Vierstra R D . The ubiquitin-26S proteasome system at the nexus of plant biology[J]. Nature Reviews Molecular Cell Biology, 2009,10(6):385-397. |
| [7] | Lim C W, Park C, Kim J H , et al. Pepper CaREL1, a ubiquitin E3 ligase, regulates drought tolerance via the ABA-signalling pathway[J]. Scientific Reports, 2017,7(1):477. |
| [8] | Suber T, Wei J, Jacko A M , et al. SCFFBXO17 E3 ligase modulates inflammation by regulating proteasomal degradation of glycogen synthase kinase-3$\beta $ in lung epithelia[J]. Journal of Biological Chemistry, 2017,292(18):7452-7461. |
| [9] | Pavicic M, Mouhu K, Wang F, et al. Genomic and phenomic screens for flower related RING type ubiquitin E3 ligases in Arabidopsis[J]. Frontiers in Plant Science, 2017,8:416. |
| [10] | Bachmair A, Novatchkova M, Potuschak T , et al. Ubiquitylation in plants: a post-genomic look at a post-translational modification[J]. Trends in Plant Science, 2001,6(10):463-470. |
| [11] | Bates P W, Vierstra R D . UPL1 and $2$, two 405 kDa ubiquitin-protein ligases from $\textit{Arabidopsis thaliana}$ related to the HECT-domain protein family[J]. The Plant Journal, 1999,20(2):183-195. |
| [12] | Mazzucotelli E, Belloni S, Marone D , et al. The E3 ubiquitin ligase gene family in plants: regulation by degradation[J]. Current Genomics, 2006,7(8):509-522. |
| [13] | Weissman A M . Themes and variations on ubiquitylation[J]. Nature Reviews Molecular Cell Biology, 2001,2(3):169-178. |
| [14] | Silvia C, Brancaccio A, Dall'agnese A , et al. {Praja1} E3 ubiquitin ligase promotes skeletal myogenesis through degradation of EZH2 upon p38$\alpha $ activation[J]. Nature Communications, 2017,8:13956. |
| [15] | Liu J, Li X, Zhang H , et al. Ubiquitin E3 ligase itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins[J]. Bone and Joint Research, 2017,6(3):154-161. |
| [16] | Wei C, Wang Y, Du Z , et al. The yersinia type Ⅲ secretion effector YopM is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3[J]. Cell Death & Disease, 2016,7(12):e2519. |
| [17] | Dong H, Dumenil J, Lu F H, et al. Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis[J]. Genes & Development, 2017,31(2):197-208. |
| [18] | Li M, Li Y, Zhao J, et al. GpDSR7, a novel E3 ubiquitin ligase gene in Grimmiapilifera is involved in tolerance to drought stress in Arabidopsis[J]. PLoS ONE, 2016,11(5):e0155455. |
| [19] | Liu Y, Zhang X, Zhu S, et al. Overexpression of GhSARP1 encoding a E3 ligase from cotton reduce the tolerance to salt in transgenic Arabidopsis[J]. Biochem Biophys Res Commun, 2016,478(4):1491-1496. |
| [20] | Suh J Y, Kim W T. Arabidopsis RING E3 ubiquitin ligase AtATL80 is negatively involved in phosphate mobilization and cold stress response in sufficient phosphate growth conditions[J]. Biochem Biophys Res Commun, 2015,463(4):793-799. |
| [21] | Lourenço T, Sapeta H, Figueiredo D D , et al. Isolation and characterization of rice ($\textit{Oryza sativa}$ L.) E3-ubiquitin ligase ${OsCTR1}$ gene in the modulation of cold stress response[J]. Plant Molecular Biology Reporter, 2013,83(4/5):351-363. |
| [22] | 倪晓光, 赵平 . 泛素-蛋白酶体途径的组成和功能[J]. 生理科学进展, 2006,37(3):255-258. |
| [23] | Qin X, Huang S, Liu Y , et al. Overexpression of a RING finger ubiquitin ligase gene AtATRF1 enhances aluminium tolerance in $\textit{Arabidopsis thaliana}$[J]. Journal of Plant Biology, 2017,60(1):66-74. |
| [24] | Qi S, Lin Q, Zhu H , et al. The RING finger E3 ligase SpRing is a positive regulator of salt stress signaling in salt-tolerant wild tomato species[J]. Plant and Cell Physiology, 2016,57(3):528-539. |
| [25] | Lim S D, Lee C, Jang C S . The rice RING E3 ligase, ${OsCTR1}$, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in $\textit{Arabidopsis}$[J]. Plant, cell & Environment, 2014,37(5):1097-1113. |
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