《植物生理学报》 2015, 51(11): 1933-1941

海马齿Δ1-吡咯啉-5-羧酸合成酶(SpP5CS)基因的克隆和表达分析

申龙斌¹, 杨成龙^2,*, 郭建春³, 段瑞军³

¹中国热带农业科学院热带作物品种资源研究所, 农业部华南作物基因资源与种质创制重点开放实验室, 海南儋州571737; ²贵州省亚热带作物研究所, 贵州兴义562400; ³中国热带农业科学院生物技术研究所, 农业部热带作物生物学与遗传资源利用重点实验室, 海口571101

通信作者：杨成龙；E-mail: yangchenglong208@163.com；Tel: 0589-3911295

摘　要：

以海马齿为材料, 根据已克隆的P5CS基因的保守序列设计简并引物, 利用RT-PCR和RACE技术从海马齿中获得了一个海马齿Δ1-吡咯啉-5-羧酸合成酶基因。该基因cDNA序列全长2 452 bp, 其中包括开放读码框为2 169 bp、5′-UTR 52 bp和3′-UTR 231 bp, 推断其编码722个氨基酸, 分子量79.4 kDa, 根据序列比对将其蛋白命名为SpP5CS。对该蛋白质序列进行生物信息学分析表明: SpP5CS与冰叶日中花、海蓬子和葡萄等双子叶植物相似性较高, 而与小麦、狗尾草和水稻等单子叶植物相似性较低。荧光定量PCR分析表明, 该基因在海马齿的叶中表达量最高, 在茎中其次; 海马齿SpP5CS基因在各部分中的表达量随着盐胁迫的时间延长在叶中变化最为明显: 在400和600 mmol•L^-1 NaCl胁迫9 h表达量最高, 在800 mmol•L^-1 NaCl胁迫12 h最高, 其表达均表现先升高后降低的趋势。P5CS基因是植物体内合成脯氨酸的关键酶基因, SpP5CS基因的克隆将为作物抗逆分子育种和进一步的功能分析打下基础。

关键词：海马齿; SpP5CS; 盐胁迫; 表达分析

收稿：2015-07-02   修定：2015-11-02

资助：海南耕地改良关键技术研究与示范专项(HNGDhs2015)和中国热带农业科学院中央级公益性科研院所基本科研业务费专项(ITBB130302和ITBB130505)。

Cloning and Expression Analysis of Δ1-Pyrroline-5-Carboxylate Synthetase (SpP5CS) from Sesuvium portulacastrum

SHEN Long-Bin¹, YANG Cheng-Long^2,*, GUO Jian-Chun³, DUAN Rui-Jun³

¹Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, China; ²Guizhou Institute of Subtropical Crops, Xingyi, Guizhou 562400, China; ³Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China

Corresponding author: YANG Cheng-Long; E-mail: yangchenglong208@163.com; Tel: 0589-3911295

Abstract:

In this study, taking Sesuvium portulacastrum as an experimental material, SpP5CS gene has been cloned by designed primers according to conserved sequence of cloned P5CS genes through the means of RTPCR and RACE technology. There are 2 452 bp in this gene cDNA which include 2 169 bp of open reading frame, 52 bp of 5′-URT and 213 bp of 3-UTR. 722 amino acids were coded according to estimation with 79.4 kDa formed this proton which named SpP5C5 by alignment. This kind of SpP5CS sequence has higher similarity to dicotyledon such as Mesembryanthemum crystallinum, Salicornia and grape, etc., but lower similarity to monocotyledon such as wheat, rice and foxtail through bioinformatics analysis. As a result, it has highest gene expression in the leaves of S. portulacastrum, and low expression level in its stems through RT-PCR analysis. With the prolonging the stress of salt, the SpP5CS gene expression level in the leaves of S. portulacastrum has been significant impacted: with treatment of 400 and 600 mmol•L^-1 NaCl, the highest expression level reached at 9 h, with treatment of 800 mmol•L^-1 NaCl, the highest expression level reached at 12 h, and with the same trend that: firstly increase and then decreasing. P5CS gene is a key enzyme gene in the synthesis of proline in plants, so the cloning of SpP5CS gene might lay a foundation for the adversity resistance molecular breeding of crop and further function analysis.

Key words: Sesuvium portulacastrum; SpP5CS; salt stress; expression profiles