茶樹P5CS基因克隆及其在滲透和高鹽脅迫下的表達分析

王麗珊　林清凡　陳蘭平　池福鈴　郭春芳　張積森

摘 要 △1-吡咯琳-5-羧酸合成酶是植物脯氨酸合成過程中的關鍵酶。應用同源克隆方法獲得茶樹P5CS，序列長為1 316 bp，編碼323個氨基酸；其編碼蛋白分子量為34.7 ku，pI為7.62；N端有1個氨基酸激酶超家族[Amino Acid Kinases（AAK） superfamily]功能區(qū)，C端有1個醛脫氫酶超家族[Aldehyde Dehydrogenas（ALDH）superfamily]功能區(qū)，預測為親水性跨膜蛋白；對18個物種的P5CS進行聚類分析，結果生物學分類及進化關系吻合。并與美味獼猴桃高度同源，相似度達89%。應用實時熒光定量PCR分析表明，該基因轉錄本在水分脅迫24 h內(nèi)升至對照組水平的2.6倍，而高鹽脅迫48 h后才升至9.9倍的最高值；水分脅迫應答速度快，但相對表達量較高鹽脅迫低。由此推測該基因被誘導參與了滲透脅迫應答響應，并且對滲透脅迫中的旱害脫水更為敏感。

關鍵詞 茶樹；P5CS；實時熒光定量PCR；水分脅迫；高鹽脅迫

中圖分類號 S571.1 文獻標識碼 A

Cloning and Expression Analysis of P5CS Gene

under Drought and Salinity Stress

in Camellia sinensis

WANG Lishan1，2，3， LIN Qingfan1，2，3， CHEN Lanping1，2，3， CHI Fuling1，2，3

GUO Chunfang4 *， ZHANG Jisen1，2，3

1 College of Life Sciences， Fujian Normal University， Fuzhou， Fujian 350108， China

2 Key Lab for Sugarcane Genetic Improvement， Ministry of Agriculture， Fuzhou， Fujian 350108， China

3 Genomics and Biotechnology Research Center， Fujian Agriculture and Forestry University，F(xiàn)uzhou， Fujian 350002， China

4 Fujian Institute of Education， Fuzhou， Fujian 350025， China

Abstract △1-pyrroline-5-carboxylate synthetase（P5CS）is considered to be the key enzyme for proline biosynthesis in plant. In the study，a 1 316 bp-length cDNA fragment of P5CS was cloned from Camellia sinensis cv. Tieguanyin through homology-based cloning strategy. The cDNA encodes a polypeptide with 323 amino acids containing an amino acid kinases domain in N terminal and an aldehyde dehydrogenase motif in C-terminus. Based on the conserved domain search analysis in NCBI，the deducted polypeptide was predicted to be a hydrophilic transmembrane protein. The BLAST results showed that the fragment shared 89% similarity with Actinidia deliciosa（ADU92286）. Phylogenetic analysis of P5CS from different species showed an evolutional consistency between the gene and higher plant. Moreover，the expression patterns of CsP5CS under the drought and salt stress were detected by real-time quantitative PCR. The CsP5CS gene expression level of the plant with 24 h PEG stress and 48 h high-salt stress was 2.6 and 9.9 times，respectively，as the control. The significant up-regulation expression under both PEG and salt stress treatments suggested that CsP5CS involved in the response to osmotic stress and might function to dehydration resistance by relegating the accumulation of proline. Meanwhile，CsP5CS is more sensitive to drought stress than salinity stress in the response to osmotic stress.

Key words Camellia sinensis； P5CS； Real-time quantitative PCR； Drought stress； Salinity stress

doi 10.3969/j.issn.1000-2561.2015.01.012

土壤水分是茶樹生理與生態(tài)需水的主要來源，旱害給茶樹生長、茶葉產(chǎn)量和品質(zhì)帶來較大影響。脯氨酸是植物面對水分和高鹽脅迫積累的最為重要的滲透調(diào)節(jié)物質(zhì)，以增強自身對滲透脅迫的抵抗能力[1-3]。一般認為脯氨酸的生物合成途徑有2種，即谷氨酸途徑和鳥氨酸途徑[4-5]。其中谷氨酸途徑中的關鍵酶是△1-吡咯琳-5-羧酸合成酶（△1-pyrroline-5-carboxylate synthetase，P5CS：EC2.7.2.11/1.2.2.41）[6-13]。近年有關茶樹抗旱性與葉片組織中脯氨酸含量變化的動態(tài)關系其研究結果不盡相同。王守生[14]、吳伯千[15]、Handique等[16]認為脯氨酸的積累與茶樹滲透調(diào)節(jié)呈正相關；伍炳華[17]、李華均[18]、李金昌[19]認為脯氨酸的積累與茶樹滲透調(diào)節(jié)無明確關系；……