小球藻亲环蛋白A的酶活性及过表达拟南芥抗盐性分析

doi:10.7525/j.issn.1673-5102.2017.05.012

植物研究 ›› 2017, Vol. 37 ›› Issue (5): 722-729.doi: 10.7525/j.issn.1673-5102.2017.05.012

小球藻亲环蛋白A的酶活性及过表达拟南芥抗盐性分析

廖栩¹, 何明良¹, 张素艳², 马海燕¹, 王旭辉¹, 罗秋香¹, 管清杰¹

1. 东北林业大学盐碱地生物资源环境研究中心, 东北盐碱植被恢复与重建教育部重点实验室, 哈尔滨 150040;
2. 吉林省德惠市第九中学, 德惠 130300

收稿日期:2017-04-17 出版日期:2017-09-15 发布日期:2017-09-15
通讯作者: 管清杰,E-mail:guanqingjie@nefu.edu.cn E-mail:guanqingjie@nefu.edu.cn
作者简介:廖栩(1993-),男,硕士研究生,主要从事耐盐碱相关基因挖掘与功能研究。
基金资助:
国家重点研发计划（2016YFC0501203）；黑龙江省博士后科研启动资助金（LBH-Q15004）；国家青年基金（31500317）资助

Prokaryotic Expression,Activity Assay of Chlorella Cyclophilin A and Salt Tolerance Analysis of Arabidopsis Over-expressing CsCyp1A

LIAO Xu¹, HE Ming-Liang¹, ZHANG Su-Yan², MA Hai-Yan¹, WANG Xu-Hui¹, LUO Qiu-Xiang¹, GUAN Qing-Jie¹

1. Northeast Forestry University, Alkali Soil Natural Environmental Science Center, Harbin 150040;
2. Dehui No.9 Middle School in Jilin Province, Dehui 130300

Received:2017-04-17 Online:2017-09-15 Published:2017-09-15
Supported by:
The National Key Research and Development Program of China(2016YFC0501203);Heilongjiang province start postdoctoral scientific research grants(LBH-Q15004);The national youth fund(31500317)

摘要/Abstract

摘要： 在前期研究中分离到噬碳酸盐小球藻（Chlorella sp.X1）的亲环蛋白基因CsCyp1A（登录号：KY207381），编码CsCyp1A蛋白是否具有肽酰脯氨酰顺反异构酶（PPIase）活性功能是进一步研究它参与小球藻耐盐生物学过程的基础。通过His标签的pQE-30-CsCyp1A原核蛋白表达载体，经IPTG诱导它的E.coli M15菌株表达融合蛋白，Nitra-柱纯化后获得纯化的30 kDa左右的His-CsCyp1A融合蛋白质，Western杂交检测到HisCsCyp1A蛋白的杂交信号。酶活性分析显示，HisCsCyp1A融合蛋白中生色基团的产生速度明显快于对照，表明CsCyp1A蛋白可以催化底物N-succinyl-Xaa-Pro-Phe-p-nitroanilide中Xaa-Pro肽键的顺反折叠，说明纯化的CsCyp1A蛋白具有PPIase活性。典型的亲环蛋白家族成员具有肽酰脯氨酰顺反异构酶（PPIase）活性，参与折叠和转运、信号转导、免疫调节、细胞凋亡等生物学过程。真空渗入法侵染转化的拟南芥在35S启动子驱动下超表达CsCyp1A基因，耐盐性研究表明它提高了过表达株系对NaCl胁迫的耐性，揭示小球藻CsCyp1A基因的抗盐性功能，它将成为抗逆育种的基因资源。本研究也为探索小球藻亲环蛋白A的抗盐碱胁迫生物学作用和分子育种奠定了基础。

关键词: 噬碳酸盐小球藻, 盐碱地, 亲环蛋白A, 肽脯氨酰顺反异构酶, 原核表达蛋白

Abstract: In order to test the activity of peptidyl-prolyl cis-trans isomerase of cyclophilin encoded by CsCyp1A, which was isolated from Chlorella sp. X1 in previous research(Accession No:KY207381), the primers were designed by using pMD18-T-CsCyp1A plasmid DNA as template and the target gene fragment that included Kpn Ⅰ and Sal Ⅰ digestion sites were obtained by cloning. After enzyme digestion, ligation and sequence analysis, the His-tagged pQE-30-CsCyp1A prokaryotic expression vector was obtained. IPTG induced E.coli M15 strain which contained its plasmid to express the fusion protein. Purified soluble HisCsCypA fusion protein was obtained by purification on a Nitra-column and the relative molecular mass of CsCyp1A was about 29 kDa. HisCsCyp1A protein hybridization signal was detected by Western blot. By enzyme activity analysis, the production rate of chromogenic groups in HisCsCyp1A fusion protein was significantly faster than in control, and the CsCyp1A protein could catalyze the cis-trans folding of Xaa-Pro peptide bond in N-succinyl-Xaa-Pro-Phe-p-nitroanilide and accelerate the cleavage of blocking groups. The purified CsCyp1A protein had PPIase activity. By overexpression of CsCyp1A gene driven by 35S promoter in Arabidopsis thaliana under vacuum infiltration, the results showed that it increased the tolerance of overexpression lines to NaCl stress, and revealed the salt tolerance of Chlorella CsCyp1A gene. It will become a genetic resource for resistance breeding. The study established the foundation for exploring the biological role of chlorella cyclophilin CsCyp1A in the anti-carbonate stress of algae.

Key words: phytococcal chlorella, saline-sodic soil, cyclophilin A, peptide prolyl cis-trans isomerase, prokaryotic expression protein

中图分类号:

Q812

廖栩, 何明良, 张素艳, 马海燕, 王旭辉, 罗秋香, 管清杰. 小球藻亲环蛋白A的酶活性及过表达拟南芥抗盐性分析[J]. 植物研究, 2017, 37(5): 722-729.

LIAO Xu, HE Ming-Liang, ZHANG Su-Yan, MA Hai-Yan, WANG Xu-Hui, LUO Qiu-Xiang, GUAN Qing-Jie. Prokaryotic Expression,Activity Assay of Chlorella Cyclophilin A and Salt Tolerance Analysis of Arabidopsis Over-expressing CsCyp1A[J]. Bulletin of Botanical Research, 2017, 37(5): 722-729.

参考文献

1. Liu J,Farmer J D Jr,Lane W S,et al. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes[J]. Cell,1991,66(4):807-815.
2. Handschumacher R E,Harding M W,Rice J,et al. Cyclophilin:a specific cytosolic binding protein for cyclosporin A[J]. Science,1984,226(4674):544-547.
3. Fischer G,Wittmann-liebold B,Lang K,et al. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins[J]. Nature,1989,337(6206):476-484.
4. Takahashi N,Hayano T,Suzuki M. Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin[J]. Nature,1989,337(6206):473-475.
5. 罗春,徐灵,何静,等. 池蝶蚌CyPA的应激表达及对Hela细胞生长的影响[J]. 水生生物学报,2015,39(3):475-481. Luo C,Xu L,He J,et al. The expression of cyclophilin a (CyPA) in Hyriopsis schlegelii in response to pathogens and its effect on the growth of Hela cells[J]. Acta Hydrobiologica Sinica,2015,39(3):475-481.
6. Gullerova M,Barta A,Lorkovic Z J. AtCyp59 is a multidomain cyclophilin from Arabidopsis thaliana that interacts with SR proteins and the C-terminal domain of the RNA polymerase Ⅱ[J]. RNA,2006,12(4):631-643.
7. Umezawa T,Okamoto M,Kushiro T,et al. CYP707A3,a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana[J]. The Plant Journal,2006,46(2):171-182.
8. Matilla A J,Carrillo-barral N,Del Carmen Rodríguez-Gacio M. An update on the role of NCED and CYP707A ABA metabolism genes in seed dormancy induction and the response to after-ripening and nitrate[J]. Journal of Plant Growth Regulation,2015,34(2):274-293.
9. Chen A P,Wang G L,Qu Z L,et al. Ectopic expression of ThCYP1,a stress-responsive cyclophilin gene from Thellungiella halophila,confers salt tolerance in fission yeast and tobacco cells[J]. Plant Cell Reports,2007,26(2):237-245.
10. Kim S K,You Y N,Park J C,et al. The rice thylakoid lumenal cyclophilin OsCYP20-2 confers enhanced environmental stress tolerance in tobacco and Arabidopsis[J]. Plant Cell Reports,2012,31(2):417-426.
11. Zhu C F,Wang Y X,Li Y B,et al. Overexpression of a cotton cyclophilin gene (GhCyp1) in transgenic tobacco plants confers dual tolerance to salt stress and Pseudomonas syringae pv. tabaci infection[J]. Plant Physiology and Biochemistry,2011,49(11):1264-1271.
12. 梁凌星. 抗咪鲜胺杧果胶孢炭疽菌突变的CYP51基因功能探究[D]. 福州:福建农林大学,2015. Liang L X. The functional study of mutated CYP51 in Colletotrichum gloeosporioides for resistance to prochloraz[D]. Fuzhou:Fujian Agriculture Forestry University,2015.
13. Sekhar K,Priyanka B,Reddy V D,et al. Isolation and characterization of a pigeonpea cyclophilin (CcCYP) gene,and its over-expression in Arabidopsis confers multiple abiotic stress tolerance[J]. Plant Cell&Environment,2010,33(8):1324-1338.
14. Qiao K,Takano T,Liu S K. Discovery of two novel highly tolerant NaHCO₃ trebouxiophytes:identification and characterization of microalgae from extreme saline-alkali soil[J]. Algal Research,2015,9:245-253.
15. 王旭辉,乔坤,汪振娟,等. 小球藻亲环蛋白A (CyPA)基因克隆及表达特性分析[J]. 植物研究,2014,34(2):245-251. Wang X H,Qiao K,Wang Z J,et al. Cloning and expression analysis of cyclophilins A (CyPA) gene form chlorella[J]. Bulletin of Botanical Research,2014,34(2):245-251.
16. 李秀军,李取生,王志春,等. 松嫩平原西部盐碱地特点及合理利用研究[J]. 农业现代化研究,2002,23(5):361-364. Li X J,Li Q S,Wang Z C,et al. A research on characteristics and rational exploitation of soda saline land in the western Songnen Plain[J]. Research of Agricultural Modernization,2002,23(5):361-364.
17. 林栖凤,李冠一. 植物耐盐性研究进展[J]. 生物工程进展,2000,20(2):20-25. Lin Q F,LI G Y. Research progress in salt tolerance in plants[J]. Progress in Biotechnology,2000,20(2):20-25.
18. Fischer G,Bang H,Mech C. Determination of enzymatic catalysis for the cis-trans-isomerization of peptide binding in proline-containing peptides[J]. Biomedica Biochimica Acta,1984,43(10):1101-1111.
19. Clough S J,Bent A F. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal,1998,16(6):735-743.
20. Hodges D M,Delong J M,Forney C F,et al. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds[J]. Planta,1999,207(4):604-611.
21. 冯玉杰,陈桥. 盐碱地生态体系构成及研究进展[C]. //第八次全国环境微生物学术研讨会论文集. 哈尔滨:中国微生物学会,2005. Feng Y J,CHEN Q. Composition and research progress of Saline-alkali land ecological system[C]. //Proceedings of the eighth national conference on environmental microbiology. Harbin:Chinese Society for Microbiology,2005.
22. Ivery M T. Immunophilins:switched on protein binding domains?[J]. Medicinal Research Reviews,2000,20(6):452-484.
23. Bauer K,Kretzschmar A K,Cvijic H,et al. Cyclophilins contribute to Stat3 signaling and survival of multiple myeloma cells[J]. Oncogene,2009,28(31):2784-2795.
24. Sun S,Wang Q W,Giang A,et al. Knockdown of CyPA inhibits interleukin-8(IL-8) and IL-8-mediated proliferation and tumor growth of glioblastoma cells through down-regulated NF-κB[J]. Journal of Neuro-Oncology,2011,101(1):1-14.
25. Fan T J,Han L H,Cong R S,et al. Caspase family proteases and apoptosis[J]. Acta Biochimica et Biophysica Sinica,2005,37(11):719-727.
26. 刘沛,徐兆师,晏月明,等. 小麦TaMAPK2激酶基因的原核表达及多克隆抗体制备[J]. 植物遗传资源学报,2011,12(1):92-95,102. Liu P,Xu Z S,Yan Y M,et al. Prokaryotic expression and polyclonal antibody preparation of the wheat TaMAPK2 gene[J]. Journal of Plant Genetic Resources,2011,12(1):92-95,102.
27. 王镜岩,朱圣庚,徐长发. 生物化学[M]. 北京:高等教育出版社,2002:164-165. Wang J Y,Zhu S G,Xu C F. Biochemistry[M]. Beijing:Higher Education Press,2002:164-165.
28. He Z Y,Li L G,Luan S. Immunophilins and parvulins. Superfamily of peptidyl prolyl isomerases in Arabidopsis[J]. Plant Physiology,2004,134(4):1248-1267.
29. Vallon O. Chlamydomonas immunophilins and parvulins:survey and critical assessment of gene models[J]. Eukaryotic Cell,2005,4(2):230-241.
30. 张安红,王志安,肖娟丽,等. 亲环素基因GhCYP1在陆地棉中的过量表达及耐盐性分析[J]. 山西农业科学,2014,42(2):107-109,122. Zhang A H,Wang Z A,Xiao J L,et al. Overexpression of cyclophilin gene (GhCYP1) in trangentic cotton and its salt tolerance analysis[J]. Journal of Shanxi Agricultural Sciences,2014,42(2):107-109,122.
31. Trivedi D K,Bhatt H,Pal R K,et al. Structure of RNA-interacting cyclophilin A-like protein from Piriformospora indica that provides salinity-stress tolerance in plants[J]. Scientific Reports,2013,3:3001.
32. Trivedi D K,Ansari M W,Bhavesh N S,et al. Response of PiCypA tobacco T2 transgenic matured plant to potential tolerance to salinity stress[J]. Plant Signaling&Behavior,2014,9(1):e27538.

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小球藻亲环蛋白A的酶活性及过表达拟南芥抗盐性分析

Prokaryotic Expression,Activity Assay of Chlorella Cyclophilin A and Salt Tolerance Analysis of Arabidopsis Over-expressing CsCyp1A

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