马尾松水通道蛋白PmPIP1基因克隆及在干旱胁迫下的表达分析

摘要

克隆马尾松Pinus massoniana水通道蛋白（AQP），并对其生物信息学与干旱胁迫表达模式进行分析。采用逆转录聚合酶链式反应（RT-PCR）以及互补脱氧核糖核酸（cDNA）末端快速扩增（RACE）方法克隆马尾松水通道蛋白基因。采用实时荧光定量聚合酶链式反应（qRT-PCR）分析其在干旱胁迫下的响应模式。结果克隆到一个马尾松水通道蛋白基因，命名为PmPIP1（GenBank登录号为KF582038）。此基因cDNA全长序列为1301 bp，包括867 bp的完整开放阅读框，99 bp 的5′末端非翻译区和335 bp 的3′末端非翻译区。编码288个氨基酸残基，分子量为30.86 kD，等电点（pI）8.48。 PmPIP1与菠菜Spinacia oleracea （2b5fA）水通道蛋白结构相似。 PmPIP1含有6个跨膜区，具有膜内在蛋白（MIP）家族信号序列、高等植物高度保守序列HINPAVTFG和2个天门冬酰胺-脯氨酸-丙氨酸（NPA）保守肽段。 PmPIP1基因属质膜内在蛋白（PIPs）亚族，与挪威云杉Picea abies 水通道蛋白基因亲缘关系最近，同源性达95%。 qRT-PCR表明PmPIP1受干旱胁迫诱导表达。马尾松PmPIP1的克隆丰富了植物AQP基因的资料库，同时推测PmPIP1基因可能参与了马尾松干旱胁迫过程。图8表1参39%The aquaporin (AQP) gene plays an important role in plants adapting to abiotic stresses. To predict the AQP gene function and provide basal data for mechanism of Pinus massoniana’s drought resistance, the se-quence characteristics of the AQP gene from P. massoniana were analyzed and its expression profiling was studied after drought-stress treatment. The AQP gene was cloned using reverse transcription-polymerase chain reaction (RT-PCR) and rapid-amplification of complementary deoxyribonucleic acid (cDNA) ends (RACE). The expression of the AQP gene was then performed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The full-length cDNA of the AQP gene from P. massoniana, designated PmPIP1 with a registered number in GenBank KF582038, was obtained. Results of the sequence analysis showed that the size of PmPIP1 was 1 301 bp, containing an 867 bp open reading frame that encoded 288 amino acid residues with 30.86 kDa molecular weight and an 8.48 isoelectric point, a 99 bp 5′ terminal untranslated regions (UTR), and a 335 bp 3′terminal UTR. The PmPIP1 3D structure had a strong similarity to Spinacia oleracea (2b5fA). PmPIP1 exhibited a typical structure with six transmembrane domains, and had the consensus sequence HIN-PAVTFG of membrane intrinsic protein (MIP) family and two highly conserved peptides Asn-Pro-Ala (NPA). The evolutionary analysis revealed that PmPIP1 shared a 95% identity with Picea abies and belonged to PIPs. The PmPIP1 expression patterns with drought conditions showed that drought did induce PmPIP1. In conclu-sion, cloning of the PmPIP1 gene from P. massoniana enriched the plant aquaporin gene database, and the qRT-PCR analysis indicated that the PmPIP1 gene may be involved in the response related to drought stress.