拟南芥RPS14 基因的克隆表达及功能初步研究

生物技术通报 ›› 2013, Vol. 0 ›› Issue (1): 73-77.

拟南芥RPS14 基因的克隆表达及功能初步研究

范艳荣 ,刘莹, 张飞云

首都师范大学生命科学学院，北京 100048

收稿日期:2012-06-28 修回日期:2013-01-31 出版日期:2013-01-30 发布日期:2013-01-30
作者简介:范艳荣，女，硕士研究生，研究方向：生物化学与分子生物学；E-mail: fyr888888@126.com
基金资助:
国家自然科学基金项目（31071075）

Cloning, Expression and Function Preliminary Research of RPS14 in Arabidopsis thaliana

Fan Yanrong ,Liu Ying ,Zhang Feiyun

College of Life Science，Capital Normal University，Beijing 100048

Received:2012-06-28 Revised:2013-01-31 Published:2013-01-30 Online:2013-01-30

摘要/Abstract

摘要： 拟南芥RPS14（Ribosomal protein S14）是从拟南芥中分离出的一种核糖体蛋白，是核糖体40S 亚基的组成成分，属于S11P 核糖体蛋白家族，主要负责RNA 转录后加工。提取拟南芥总RNA，用RT-PCR 技术得到RPS14 全长基因，经T-A 克隆插入到pEASY-T3 载体上。利用亚克隆法成功构建pGEX-6P-1-RPS14 原核表达质粒。GST-RPS14 融合蛋白在大肠杆菌BL21（DE3）中表达，分子量约为43 kD。运用同样方法构建pET-28a-AK6 原核表达质粒，获得大小约为26 kD 的HIS-AK6 融合蛋白。运用体外pull-down 技术，并用SDS-PAGE 和Western blot 进行验证，证明拟南芥RPS14 蛋白与AK6 蛋白之间存在相互作用。因此，推测拟南芥的AK6 与RPS14 蛋白共同参与核酸代谢过程，影响RNA 转录后加工，进而抑制拟南芥茎细胞的生长，使植株表现矮小特征。

关键词: 拟南芥 , RPS14 , AK6 , Pull-down

Abstract: RPS14(Ribosomal protein S14)is a ribosomal protein of Arabidopsis thaliana. It is a structural component of 40S ribosomalsubunit and belongs to the S11P ribosomal protein family. It is responsible for RNA post-transcriptional processing. We extracted the total RNAof Arabidopsis thaliana. The RPS14 full-length gene was obtained by RT-PCR. Through inserting the gene fragment into pEASY-T3 vector byT-A cloning, we successfully constructed a recombinant vector pGEX-6P-1-RPS14. The GST-RPS14 protein was expressed in BL21(DE3)strain and molecular weight was approximately 43 kD. We constructed the recombinant vector pET-28a-AK6 in the same way and obtained theapproximately 26 kD HIS-AK6 protein. We demonstrated the interaction between RPS14 and AK6 using pull-down, SDS-PAGE and Westernblot. Therefore, we speculate that they together participate in the nucleic acid metabolism and play a role in the RNA post-transcriptionalprocessing, thereby inhibiting growth of stem cells and making plants show dwarf phenotype.

Key words: Arabidopsis thaliana , RPS14 , AK6 , Pull-down

范艳荣, 刘莹, 张飞云. 拟南芥RPS14 基因的克隆表达及功能初步研究[J]. 生物技术通报, 2013, 0(1): 73-77.

Fan Yanrong, Liu Ying, Zhang Feiyun. Cloning, Expression and Function Preliminary Research of RPS14 in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2013, 0(1): 73-77.

参考文献

[1] Granneman S, Nandineni MR, Baserga SJ. The Putative NTPaseFap7 mediates cytoplasmic 20S pre-rRNA processing through adirect interaction with RpS14[J]. Mol Cell Biol, 2005, 25（23）：10352-10364.
[2] Whitford PC, Miyashita O, Levy Y, Onuchic JN. Conformationaltransitions of adenylate kinase ：Switching by cracking[J]. J MolBiol, 2007, 366（5）：1661-1671.
[3] Van Rompay AR, Johansson M, Karlsson A. Identifucation of a novelhuman adenylate kinase. cDNA cloning, expression analysis, chromosomelocalization and characterization of the recombinant protein[J]. Eur J Biochem, 1999, 261 ：509-517.
[4] Zancani M, CasoloV, VianelloA, Macri F. Involvement of apyrasein the regulation of the adenylate pool by adenylate kinase in plantmitochondria[J]. Plant Sci, 2001, 161（5）：927-933.
[5] Zhai R, Meng G, Zhao Y, et al. A novel nuclear-localized proteinwith special adenylate kinase properties from Caenorhabditiselegans[J]. FEBS Lett, 2006, 580（16）：3811-3817.
[6] Ren H, Wang L, Bennett M, et al. The crystal structure of humanadenylate kinase 6 ：An adenylate kinase localized to the cell nucleus[J]. Proc Natl Acad Sci, 2005, 102（2）：303-308.
[7] Lange PR, Geserick C, Tischendorf G, Zrenner R. Functions of chloroplasticadenylate kinases in Arabidopsis[J]. Plant Physiology,2007, 146（2）：492-504.
[8] Juhnke H, Charizanis C, Latifi F, et al. The essential protein fap7is involved in the oxidative stress response of Saccharomycescerevisiae[J]. Mol Microbiol, 2000, 35（4）：936-948.
[9] Carrari F, Coll-Garcia D, Schauer N, et al. Deficiency of a plastidialadenylate kinase in Arabidopsis results in elevated photosyntheticamino acid biosynthesis and enhanced growth[J]. PlantPhysiologl, 2005, 137（1）：70-82.
[10] Zhang J, Zhang F, Zheng X. Depletion of hCINAP by RNAinterference causes defects in Cajal body formation, histonetranscription, and cell viability[J]. Cellular and Molecular LifeSciences, 2010, 67（11）：1907-1918.
[11] Meng G, Zhai R, Chen B, et al. Identification and characterization ofhuman adenylate kinase 6 homologues from Drosophila melanogaster[J]. Biochemistry（Moscow）, 2008, 73（1）：38-43.

[1]	李宇, 李素贞, 陈茹梅, 卢海强. 植物bHLH转录因子调控铁稳态的研究进展[J]. 生物技术通报, 2023, 39(7): 26-36.
[2]	李帜奇, 袁月, 苗荣庆, 庞秋颖, 张爱琴. 盐胁迫盐芥和拟南芥褪黑素含量及合成相关基因表达模式分析[J]. 生物技术通报, 2023, 39(5): 142-151.
[3]	崔吉洁, 蔡文波, 庄庆辉, 高爱平, 黄建峰, 陈亚辉, 宋志忠. 杧果Fe-S簇装配基因MiISU1的生物学功能[J]. 生物技术通报, 2023, 39(2): 139-146.
[4]	鄢梦雨, 韦晓薇, 曹婧, 兰海燕. 异子蓬SabHLH169基因的克隆及抗旱功能分析[J]. 生物技术通报, 2023, 39(11): 328-339.
[5]	阮航, 多浩源, 范文艳, 吕清晗, 姜述君, 朱生伟. AtERF49在拟南芥应答盐碱胁迫中的作用[J]. 生物技术通报, 2023, 39(1): 150-156.
[6]	林蓉, 郑月萍, 徐雪珍, 李丹丹, 郑志富. 拟南芥ACOL8基因在乙烯合成与响应中的功能分析[J]. 生物技术通报, 2023, 39(1): 157-165.
[7]	高聪, 萧楚健, 鲁帅, 王苏蓉, 袁卉华, 曹云英. 氧化石墨烯对拟南芥生长的促进作用[J]. 生物技术通报, 2022, 38(6): 120-128.
[8]	徐红云, 张明意. GRAS转录因子AtSCL4负调控拟南芥应答渗透胁迫[J]. 生物技术通报, 2022, 38(6): 129-135.
[9]	古盼, 齐学影, 李莉, 张曦, 单晓昳. AtRGS1胞吞动态调控G蛋白参与拟南芥生长发育和抗性反应[J]. 生物技术通报, 2022, 38(6): 34-42.
[10]	周娟, 阎晋东, 李新梅, 刘雪晴, 赵强, 赵小英. 拟南芥F-box蛋白FKF1与转录因子FUL互作调控开花研究[J]. 生物技术通报, 2022, 38(3): 1-8.
[11]	杨佳慧, 孙玉萍, 陆雅宁, 刘欢, 卢存福, 陈玉珍. 拟南芥AtTERT对大肠杆菌非生物胁迫抗性的影响[J]. 生物技术通报, 2022, 38(2): 1-9.
[12]	李兵娟, 郑璐, 沈仁芳, 兰平. 拟南芥RPP1A参与幼苗生长的蛋白质组学分析[J]. 生物技术通报, 2022, 38(2): 10-20.
[13]	徐子涵, 刘倩, 苗大鹏, 陈跃, 胡凤荣. 春兰miR396过表达对拟南芥叶片生长、光合及叶绿素荧光特性的影响[J]. 生物技术通报, 2021, 37(5): 28-37.
[14]	杨华杰, 周玉萍, 范甜, 吕天晓, 谢楚萍, 田长恩. 拟南芥IQM4互作蛋白的筛选和鉴定[J]. 生物技术通报, 2021, 37(11): 190-196.
[15]	方丹丹, 张婷, 文晓鹏. 超表达马尾松PmPT3基因提高拟南芥耐低磷能力[J]. 生物技术通报, 2021, 37(10): 1-8.