Cloning and Expression of Cyclophilin Gene PhCyP from Phalaenopsis

doi:10.3969/j.issn.1000-2561.2020.02.015

Abstract

Abstract:

To study the molecular regulation mechanism of Phalaenopsis in response to low temperature stress, a cyclophilin gene PhCyP (GenBank accession number: MH992514) was cloned from the leaf of Phalaenopsis through the RT-PCR and RACE method. The full-length cDNA sequence was 829 bp with an open reading frame (ORF) of 522 bp encoding 173 amino acids. PhCyP contained a cyclophilin-like domain (CLD) with alkaline and hydrophilic properties. The phylogenetic tree showed that PhCyP was closely related to the cyclophilin from Phalaenopsis equestris, Vanda hybrid cultivar, Apostasia shenzhenica and Dendrobium catenatum in Orchidaceae. The expression analysis showed that PhCyP was highly expressed in different tissues including root, leaf, flower, ovary and seed in different development stage. Under 13 ℃/8 ℃ day/night temperature, the expression level of PhCyP reduced gradually with treatment time and was the lowest at 6 d, then increased gradually from 9 d to restoring. Under 4 ℃ treatment, the expression level of PhCyP increased gradually, and achieved the highest level at 4 h, then its expression level decreased gradually with a lower level than that before treatment at 48 h. The results suggested that PhCyP was involved in the response to low temperature stress with different molecular regulation mechanism to different low temperature stress.

Key words: Phalaenopsis spp., low temperature, cyclophilin, gene expression

CLC Number:

S682.31
Q786

YUAN Xiuyun,XU Shenping,ZHANG Yan,WANG Mofei,JIANG Suhua,LIANG Fang,CUI Bo. Cloning and Expression of Cyclophilin Gene PhCyP from Phalaenopsis[J]. Chinese Journal of Tropical Crops, 2020, 41(2): 315-322.

Figures/Tables 9

Tab. 1 Primers and sequence

引物 Primer	序列(5'-3') Sequence(5'-3')
CyP-F	TKATCCCTCAGTTYATGTGCC
CyP-R	CGATRCAGAACGGAAACCAACAC
3'-inner	CTTGGCTCGATGGAAAGCATG
3'-outer	GAACGGTTCCCAGTTCTTTATC
5'-inner	TAAGAGATCTGGCCACAGTCAGC
5'-outer	GCCAAATACAACATGCTTTCCATC
ORFF	GGACTGCAAGGCAAGCAAAC
ORFR	CGATACAGAACGGAAACCAACAC
qRT-F	ATCCCTCAGTTCATGTGCCAG
qRT-R	GAATTGCCTTCACAACATCCAG
Actin-F	GCAGCATGAAGATCA AGGTGG
Actin-R	GCCTTAGAAATCCACATCTGTTG

Fig. 1 Cloning of PhCyP

Fig. 2 Nucleotide acid sequence and deduced amino sequence of PhCyP gene ATG: Start codon; TAA: Stop codon; Underline: PPIase motif; Boxes: Active sites of PPIase.

Fig. 3 Conserved domain analysis of predicted protein of PhCyP gene

Fig. 4 Characteristics of predicted protein of PhCyP gene A: Secondary structure prediction; B: Hydrophobicity analysis; C: Three-dimensional structure prediction.

Fig. 5 Phylogenetic analysis of PhCyP protein

Fig. 6 Expression level of PhCyP gene in different tissues 1: Root at seedling stage; 2: Leaf at seedling stage; 3: Root at pre-flowering stage;4: Leaf at pre-flowering stage; 5: Pedicel at pre-flowering stage; 6: Root at full-blossom stage;7: Leaf at full-blossom stage; 8: Pedicel at full-blossom stage; 9: Sepal; 10: Petal; 11: Lip; 12: Column;13: Ovary before pollination; 14: Developing ovary; 15: Seed.

Fig. 7 Effects of 13 ℃/8 ℃ day/night temperature and restoring on the expression level of PhCyP gene

Fig. 8 Effects of 4 ℃ low temperature stress on the expression level of PhCyP gene

References 37

[1]	李冬梅, 梁炫强, 朱根发 , 等. 利用抑制消减杂交法筛选低夜温诱导的蝴蝶兰生殖发育相关基因[J]. 农业生物技术学报, 2013,21(8):883-895.
[2]	刘学庆, 王秀峰, 朴永吉 . 蝴蝶兰不同品种耐冷特性的研究[J]. 园艺学报, 2007,34(2):425-430.
[3]	郝平安, 梁芳, 张燕 , 等. 低温胁迫对蝴蝶兰光合及生理特性的影响[J]. 热带作物学报, 2018,39(10):1955-1962.
[4]	Yuan X Y, Liang F, Jiang S H , et a1. Differential protein expression in phalaenopsis under low temperature[J]. Applied Biochemistry Biotechnology, 2015,175(2):909-924.
[5]	袁秀云, 许申平, 王洁琼 , 等. 蝴蝶兰Rubisco活化酶基因 PhRCAα 的序列特征及在低温胁迫下的表达分析[J]. 分子植物育种, 2016,14(4):835-843.
[6]	袁秀云, 许申平, 王默霏 , 等. 蝴蝶兰PhTSJT1基因的克隆及在低温胁迫下的表达分析[J]. 基因组学与应用生物学, 2019,38(2):707-713.
[7]	Galat A . Variations of sequences and amino acid compositions of proteins that sustaintheir biological functions: an analysis of the cyclophilin family of proteins[J]. Archives of Biochemistry and Biophysics, 1999,371(2):149-162.
[8]	Wang P, Heitman J . The cyclophilins[J]. Genome Biology, 2005,6(7):226.
[9]	白宇杰, 马华升, 王火旭 , 等. 植物细胞亲环素研究进展[J]. 植物生理通讯, 2010,46(9):881-889.
[10]	童惠姗, 汪珊珊, 朱馨妮 , 等. 植物亲环素基因功能研究进展[J]. 西北植物学报, 2017,37(4):830-838.
[11]	Li H, Luan S . The cyclophilin AtCYP71 Interacts with CAF-1 and LHP1 and functions in multiple chromatin remodeling processes[J]. Molecular Plant, 2011,4(4):748-758.
[12]	Wu H, Wensley E, Bhave M . Identification and analysis of genes encoding a novel ER-localised cyclophilin B, in wheat potentially involved in storage protein folding[J]. Plant Science, 2009,176(3):420-432.
[13]	Cheong H, Santos I B D, Liu W . Cyclophilin 20-3 is positioned as a regulatory hub between light-dependent redox and 12-oxo-phytodienoic acid signaling[J]. Plant Signaling & Behavior, 2017,12(9):e1362520.
[14]	Spiegelman Z, Omer S, Mansfeld B N , et a1. Function of Cyclophilin1 as a long-distance signal molecule in the phloem of tomato plants[J]. Journal of Experimental Botany, 2017,68(5):953-964.
[15]	Zhang H, Wang J, Li S , et a1. Molecular cloning, expression, purification and functional characterization of an antifungal cyclophilin protein from Panax ginseng[J]. Biomedical Reports, 2017,7(6):527-531.
[16]	Mainali H R, Vadivel A K A, Li X , et a1. Soybean cyclophilin GmCYP1 interacts with an isoflavonoid regulator GmMYB176[J]. Scientific Reports, 2017,7(7):39550.
[17]	Singh K, Winter M, Zouhar M , et a1. Cyclophilins: less studied proteins with critical roles in pathogenesis[J]. Phytopathology, 2018,108(1):4-16.
[18]	Romano P G, Horton P, Gray J E . The Arabidopsis cyclophilin gene family[J]. Plant Physiology, 2004,134(4):1268-1282.
[19]	张玉芳, 赵丽娟, 曾幼玲 . 基因表达研究中内参基因的选择与应用[J]. 植物生理学报, 2014,50(8):1119-1125.
[20]	Liang C, Hao J, Meng Y , et a1. Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress[J]. PloS One, 2018,13(3):e0194436.
[21]	Ashrafi M, Azimi Moqadam M R, Moradi P , et a1. Evaluation and validation of housekeeping genes in two contrast species of thyme plant to drought stress using real-time PCR[J]. Plant Physiology and Biochemistry, 2018,132:54-60.
[22]	Lee S S, Park H J, Yoon D H , et a1. Rice cyclophilin OsCYP18-2 is translocated to the nucleus by an interaction with SKIP and enhances drought tolerance in rice and Arabidopsis[J]. Plant Cell & Environment, 2015,38(10):2071-2087.
[23]	Kumari S, Joshi R, Singh K , et a1. Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation[J]. Functional & Integrative Genomics, 2015,15(4):395-412.
[24]	廖栩, 何明良, 张素艳 , 等. 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.
[25]	罗朝兵, 鞠丹, 刘中帅 , 等. 青杆CYP1基因的克隆与表达[J]. 东北林业大学学报, 2015,43(10):14-20.
[26]	Yoon D H, Lee S S, Park H J , et a1. Overexpression of OsCYP19-4 increases tolerance to cold stress and enhances grain yield in rice (Oryza sativa)[J]. Journal of Experimental Botany, 2016,67(1):69-82.
[27]	Mainali H R, Chapman P, Dhaubhadel S . Genome-wide analysis of cyclophilin gene family in soybean (Glycine max)[J]. BMC Plant Biology, 2014,14(1):282.
[28]	Hanhart P, Thieß M, Amari K , et a1. Bioinformatic and expression analysis of the Brassica napus L. cyclophilins[J]. Scientific Reports, 2017,7(1):1514.
[29]	He Z, Li L, Luan S . Immunophilins and parvulins. superfamily of peptidyl prolyl isomerases in Arabidopsis [J]. Plant Physiology, 2004,134(4):1248-1267.
[30]	Nicot N, Hausman J F, Hoffmann L , et a1. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 2005,56(421):2907-2914.
[31]	Jian B, Liu B, Bi Y R , et a1. Validation of internal control for gene expression study in soybean by quantitative real-time PCR[J]. BMC Molecular Biology, 2008,9(1):59.
[32]	Coker J S, Davis E . Selection of candidate housekeeping controls in tomato plants using EST data[J]. Biotechniques, 2003,35(4):740-748.
[33]	Yuan X Y, Jiang S H, Wang M F , et a1. Evaluation of internal control for gene expression in phalaenopsis by quantitative Real-Time PCR[J]. Applied Biochemistry and Biotechnology, 2014,173(6):1431-1445.
[34]	Marivet J, Frendo P, Burkard G . Effects of abiotic stresses on cyclophilin gene expression in maize and bean and sequence analysis of bean cyclophilin cDNA[J]. Plant Science, 1992,84(2):171-178.
[35]	Meza-Zepeda L A, Baudo M M, Palva E T , et al. Isolation and characterization of a cDNA corresponding to a stress-activated cyclophilin gene in Solanum commersonii [J]. Journal of Experimental Botany, 1998,49(325):1451-1452.
[36]	Lee A, Sang S L, Jung W Y , et a1. The OsCYP19-4 gene is expressed as multiple alternatively spliced transcripts encoding isoforms with distinct cellular localizations and PPIase activities under cold stress[J]. International Journal of Molecular Sciences, 2016,17(7):1154.
[37]	于树涛, 于洪波, 苏君伟 , 等. 低温胁迫下花生差异表达基因的分离与分析[J]. 核农学报, 2014,28(4):569-576.