欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2012, Vol. 38 ›› Issue (03): 549-555.doi: 10.3724/SP.J.1006.2012.00549

• 研究简报 • 上一篇    下一篇

苎麻Δ1-吡咯啉-5-羧酸合成酶(P5CS)基因的克隆和表达分析

周精华1，邢虎成1,2，揭雨成1,2,*，钟英丽1,3，朱守晶1，蒋杰1，王亮1   

  1. 1 湖南农业大学苎麻研究所，湖南长沙410128；2 湖南省种质资源创新与资源利用重点实验室，湖南长沙410128；3 湖南农业大学生物科学与技术学院，湖南长沙410128
  • 收稿日期:2011-06-25 修回日期:2011-10-12 出版日期:2012-03-12 网络出版日期:2011-12-01
  • 通讯作者: 揭雨成, E-mail: ibfcjyc@vip.sina.com, Tel: 13874940038
  • 基金资助:

    本研究由湖南省科技计划重点项目(2010TP4004-1), 湖南省教育厅重点项目(09A042), 湖南农业大学人才引进科技资助项目(07YT03), 湖南省教育厅优秀人才项目(10B050)和省部共建国家重点实验室培育基地科学基金开放项目(10KFXW09)资助。

Molecular Cloning and Express Analysis of Δ1-Pyrroline-5-Carboxylate Synthetase (P5CS) Gene in Ramie

ZHOU Jing-Hua1,XING Hu-Cheng1,2, JIE Yu-Cheng1,2,ZHONG Yin-Li1,3,ZHU Shou-Jing1,JIANG Jie1,WANG Liang1   

  1. 1 Institute of Ramie, Hunan Agriculture University, Changsha 410128, China; 2 Hunan Provincial Key Laboratory of Corp Germplasm Innovation and Utilization, Changsha 410128, China; 3 College of Bioscience and Biotechnology, Changsha 410128, China
  • Received:2011-06-25 Revised:2011-10-12 Published:2012-03-12 Published online:2011-12-01
  • Contact: 揭雨成, E-mail: ibfcjyc@vip.sina.com, Tel: 13874940038

PDF

1675

被引次数

9

摘要： 以耐旱性较强的湘苎3号为材料，从苎麻转录组测序结果中获得了1个与P5CS基因高度相似的Unigene，该片段长1 448 bp，根据该序列设计5′RACE和3′RACE槽式PCR引物，利用RACE结合RT-PCR技术分别克隆得到590 bp的5′端和293 bp的3′端，拼接获得该基因全长cDNA序列。对该序列进行生物信息学分析表明，该基因全长为2 318 bp，其中开放读码框为2 154 bp，编码717个氨基酸，其编码蛋白质的等电点和分子量分别为6.57 kD和77.56 kD，与亚洲棉、麻风树、拟南芥和水稻的P5CS基因的核苷酸序列相似性分别为81%、81%、75%和72%，蛋白质序列的相似性分别为86%、85%、78%和77%，说明该基因与P5CS为同源，被命名为BnP5CS1。半定量RT-PCR分析表明，该基因在苎麻的茎尖中表达量最高，在根中其次, 且受干旱胁迫的诱导。P5CS基因是植物体内合成脯氨酸的关键酶基因，BnP5CS1基因的克隆将为苎麻的抗逆分子育种和进一步的功能分析鉴定基础。

关键词: 苎麻, P5CS, 脯氨酸, 干旱, 表达分析

Abstract: Proline is one of osmotic regulators in plant, which plays an important role in regulating ramie in response to drought stress. The synthesis of proline has been reported to be restricted by the enzyme of Δ1-pyrroline-5-carboxylate synthetase (P5CS). In this study, taking Xiangzhu 3 with drought tolerance as an experimental material, we obtained a ramie unigene highly identified with P5CS gene from the ramie transcription group sequencing, and the gene length was 1 448 bp. According to the known fragment, the 5' and 3' RACE PCR nest primers were designed, and a 590 bp 5′ terminal sequence and a 293 bp 3′ terminal sequence were amplified by RACE and RT-PCR, separately. The results of bioinformatics analysis showed that the full length of the P5CS gene was 2 318 bp, the open reading frame was 2 154 bp, coding 717 amino acids, the isoelectric point and the molecular weight of coded protein were 6.57 and 77.56 kD, respectively. Nucleotide sequence Blast indicated that the unigene of ramie shared an identity of 81%, 81%, 75% and 72% with P5CS genes of cotton, leprosy tree,Arabidopsis thaliana and rice, respectively, and the similarity in protein Blast was 86%, 85%, 78%, and 77%, respectively. The result showed it was a homologous gene of P5CS in ramie, and named BnP5CS1. Semi-quantitative RT-PCR analysis indicated that the expression of BnP5CS1 had a higher level in stem tip than in root, and the gene induced by the drought stress.P5CS is a key enzyme gene in the synthesis of proline in plants, so the cloning of BnP5CS1 gene might lay a foundation for the adversity resistance molecular breeding of ramie and further function analysis.

Key words: Boehmeria nivea, P5CS, Proline, Drought, Expression analysis

[1]Lehmann S, Funck D, Szabados L, Rentsch D. Proline metabolism and transport in plant development. Amino Acids, 2010, 39: 949–962

[2]Chen J B, Zhang X Y, Jing R L, Blair M W, Mao X G, Wang S M. Cloning and genetic diversity analysis of a new P5CS gene from common bean (Phaseolus vulgaris L.). Theor Appl Genet, 2010, 120: 1393–1404

[3]Bohnert H J, Nelson D E, Jensen R G. Adaptations to environmental stresses. Plant Cell, 1995, 7: 1099–1111

[4]Sleator R D, Hill C. Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence. FEMS Microbiol Rev, 2002, 26: 49–71

[5]Delauney A J, Hu C A, Kavi Kishor P B, Verma D P S. Cloning of ornithin delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis. J Biol Chem, 1993, 268: 18673–18678

[6]Huang Z(黄志), Zou Z-R(邹志荣), Huang H-H(黄焕焕), He C-X(贺超兴), He Z-B(贺志斌), Wang H-S(王怀送), Li J-M(李建明). Cloning analysis and expression of a drought-related gene MeP5CS from melon. Acta Hort Sin (园艺学报), 2010, 37(8): 1279–1286 (in Chinese with English abstract)

[7]Zhang J-S(张积森), Chen Y-Q(陈由强), Li W(李伟), Que Y-X(阙友雄), Ye B-Y(叶冰莹), Chen R-K(陈如凯), Zhang M-Q(张木清). Molecular cloning and expression of the P5CS gene from sugarcane. Chin J Trop Crops (热带作物学报), 2009, 30(9): 1337–1344 (in Chinese with English abstract)

[8]Cao L(曹丽), Sun Z-Y(孙振元), Yi M-F(义放明), Han L(韩蕾), Xin H-B(幸海波). Cloning, expression and subcellular localization of P5CS gene from perennial ryegrass (Lolium perenne L.). Acta Hort Sin (园艺学报), 2010, 37(9): 1477–1484 (in Chinese with English abstract)

[9]Zhang C-B(张春宝), Zhao H-K(赵洪锟), Liu Q-Y(李启云), Liu X-D(刘晓冬), Shen B(沈波), Dong Y-S(董英山). Molecular cloning and express analysis of Δ′-pyrroline-5-carboxylate synthetase (P5CS) gene in wild soybean. Soybean Sci (大豆科学), 2008, 27(6): 915–920 (in Chinese with English abstract)

[10]Chen J-B(陈吉宝), Jing R-L(景蕊莲), Mao X-G(毛新国), Chang X-P(昌小平), Wang S-M(王述民). A response of PvP5CS2 gene to abiotic stresses in common bean. Acta Agron Sin (作物学报), 2008, 34(7): 1121–1127 (in Chinese with English abstract)

[11]Kavi Kishor P B, Hong Z, Miao G H, Hu C A A, Verma D P S. Overexpression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol, 1995, 108: 1387–1394

[12]Chen J-B(陈吉宝), Jing R-L(景蕊莲), Mao X-G(毛新国), Wang S-M(王述民). A response of transgenic tobacco with common bean PvP5CS2 gene to drought stress. J Plant Genet Resour (植物遗传资源学报), 2008, 9(2): 186–189 (in Chinese with English abstract)

[13]Jie Y-C(揭雨成). The Basic Study of the Ramie Drought Physiological (苎麻抗旱生理基础研究). China's Agricultural Science and Technology Press (中国农业科学出版社), 2011. pp 1–163 (in Chinese)

[14]Strizhov N, Abraham E, Okresz L, Blicking S, Zilberstein A, Schell J, Koncz C, Szabados L. Differential expression of two PSCS genes controlling proline accumulation during salt-stress repuires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J, 1997, 12(3): 557–569

[15]Hong Z L, Lakkineni K, Zhang Z H, Verma D P S. Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol, 2000, 122: 1129–1136

[16]Székely G, Abrahám E, Cséplo A, Rigó G, Zsigmond L, Csiszár J, Ayaydin F, Strizhov N, Jásik J, Schmelzer E, Koncz C, Szabados L. Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J, 2008, 53: 11–28

[17]Hur J, Jung K H, Lee C H, An G. Stress-inducible OsP5CS2 gene is essential for salt and cold tolerance in rice. Plant Sci, 2004, 167: 417–426

[18]Ginzberg I, Stein H, Kapulnik Y, Szabados L, Strizhov N, Schell J, Koncz C, Zilberstein A. Isolation and characterization of two different cDNAs of Δ1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress. Plant Mol Biol, 1998, 38: 755–764

[19]Fujita T, Maggio A, Garcia-Rios M, Bressan R A, Csonka L N. Comparative analysis of the regulation of expression and structures of two evolutionarily divergent genes for Δ1-pyrroline-5-carboxylate synthetase from tomato. Plant Physiol, 1998, 118: 661–674

[20]Kishor P B K, Hong Z, Miao G H, Hu C A A, Verma D P S. Overexpression of [delta]1-pyrroline-5-carboxylate synthetase increase proline production and confers osmotolerance in tansgenic plants. Plant Physiol, 1995, 108: 1387–1394

[21]Sayari A H, Bouzid R G, Bidan A, Jaoua L, Savouré A, Jaoua S. Over-expression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers salt tolerance in transgenic potato plants. Plant Sci, 2005, 169: 746–752
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 李祎君, 吕厚荃. 气候变化背景下农业气象灾害对东北地区春玉米产量影响[J]. 作物学报, 2022, 48(6): 1537-1545.
[3] 李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO2浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118.
[4] 王霞, 尹晓雨, 于晓明, 刘晓丹. 干旱锻炼对B73自交后代当代干旱胁迫记忆基因表达及其启动子区DNA甲基化的影响[J]. 作物学报, 2022, 48(5): 1191-1198.
[5] 晋敏姗, 曲瑞芳, 李红英, 韩彦卿, 马芳芳, 韩渊怀, 邢国芳. 谷子糖转运蛋白基因SiSTPs的鉴定及其参与谷子抗逆胁迫响应的研究[J]. 作物学报, 2022, 48(4): 825-839.
[6] 靳容, 蒋薇, 刘明, 赵鹏, 张强强, 李铁鑫, 王丹凤, 范文静, 张爱君, 唐忠厚. 甘薯Dof基因家族挖掘及表达分析[J]. 作物学报, 2022, 48(3): 608-623.
[7] 丁红, 徐扬, 张冠初, 秦斐斐, 戴良香, 张智猛. 不同生育期干旱与氮肥施用对花生氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 695-703.
[8] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[9] 谢琴琴, 左同鸿, 胡燈科, 刘倩莹, 张以忠, 张贺翠, 曾文艺, 袁崇墨, 朱利泉. 甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析[J]. 作物学报, 2022, 48(1): 108-120.
[10] 曹亮, 杜昕, 于高波, 金喜军, 张明聪, 任春元, 王孟雪, 张玉先. 外源褪黑素对干旱胁迫下绥农26大豆鼓粒期叶片碳氮代谢调控的途径分析[J]. 作物学报, 2021, 47(9): 1779-1790.
[11] 张明聪, 何松榆, 秦彬, 王孟雪, 金喜军, 任春元, 吴耀坤, 张玉先. 外源褪黑素对干旱胁迫下春大豆品种绥农26形态、光合生理及产量的影响[J]. 作物学报, 2021, 47(9): 1791-1805.
[12] 岳丹丹, 韩贝, Abid Ullah, 张献龙, 杨细燕. 干旱条件下棉花根际真菌多样性分析[J]. 作物学报, 2021, 47(9): 1806-1815.
[13] 曾紫君, 曾钰, 闫磊, 程锦, 姜存仓. 低硼及高硼胁迫对棉花幼苗生长与脯氨酸代谢的影响[J]. 作物学报, 2021, 47(8): 1616-1623.
[14] 高璐, 许文亮. 脯氨酸羟化酶GhP4H2在棉花纤维发育中的功能研究[J]. 作物学报, 2021, 47(7): 1239-1247.
[15] 李洁, 付惠, 姚晓华, 吴昆仑. 不同耐旱性青稞叶片差异蛋白分析[J]. 作物学报, 2021, 47(7): 1248-1258.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2