茶树P5CS基因克隆及其在渗透和高盐胁迫下的表达分析

Cloning and Expression Analysis of P5CS Gene under Drought and Salinity Stress in Camellia sinensis

△1-吡咯琳-5-羧酸合成酶是植物脯氨酸合成过程中的关键酶。应用同源克隆方法获得茶树P5CS,序列长为1 316 bp,编码323个氨基酸;其编码蛋白分子量为34.7 ku,p I为7.62;N端有1个氨基酸激酶超家族[Amino Acid Kinases(AAK)superfamily]功能区,C端有1个醛脱氢酶超家族[Aldehyde Dehydrogenas(ALDH)superfamily]功能区,预测为亲水性跨膜蛋白;对18个物种的P5CS进行聚类分析,结果生物学分类及进化关系吻合。并与美味猕猴桃高度同源,相似度达89%。应用实时荧光定量PCR分析表明,该基因转录本在水分胁迫24 h内升至对照组水平的2.6倍,而高盐胁迫48 h后才升至9.9倍的最高值;水分胁迫应答速度快,但相对表达量较高盐胁迫低。由此推测该基因被诱导参与了渗透胁迫应答响应,并且对渗透胁迫中的旱害脱水更为敏感。

△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 eDNA fragment of P5CS was cloned from Camellia sinensis ev. 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 CsPSCS 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-sah 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.