三个陆地棉水孔蛋白基因的克隆与表达分析

从陆地棉SSH-cDNA文库的测序结果中得到一条具有完整ORF的陆地棉水孔蛋白基因序列，将其命名为GhAQP2。以该基因编码的氨基酸序列为探针，在棉花EST数据库经同源搜索得到2个相似性较高的EST，利用RACE技术获得其全长cDNA序列，将其基因命名为GhAQP3和GhAQP4。基因结构分析发现GhAQP2和GhAQP3各有4个外显子，3个内含子；GhAQP4有3个外显子，2个内含子。生物信息分析表明3个基因编码蛋白均含有6个跨膜区，2个NPA结构域，其氨基酸序列具备MIP超家族典型的蛋白保守区序列特征。多序列比对发现3个基因的氨基酸序列与其他物种PIP2类水孔蛋白氨基酸序列具有很高的同源性。qRT-PCR分析表明，GhAQP2在纤维伸长后期优势表达，GhAQP3在下胚轴和子叶中高表达，GhAQP4在纤维伸长前期优势表达，推测3个基因在不同的组织中发挥作用。GhAQP2在20DPA优势表达，为研究该基因在纤维伸长向次生壁加厚期转化过程中的表达调控提供了重要信息。

Cloning and Expression Analysis of Three Aquaporin Genes in Upland Cotton (Gossypium hirsutum L.)

To provide an insight into molecular mechanism of cotton fiber development, we analyzed the sequence characteristics of three aquaporin genes in Gossypium hirsutum L. and their expression profiling in the different tissues. Based on the bioinformatic analysis of the ESTs from the cotton fiber SSH-cDNA library, we obtained a cDNA sequence with an ORF of 927 bp, which has the homolog sequence of aquaporin genes, and it was designated as GhAQP2. With the protein sequence of GhAQP2 as a probe, two homologous ESTs in G. hirsutum database were screened out, and the rapid-amplification of cDNA ends (RACE) was used to amplify the full length cDNAs of GhAQP genes (designated as GhAQP3 and GhAQP4). Bioinformatic analysis showed that GhAQP2 and GhAQP3 were composed of four exons and three introns, GhAQP4 had three exons and two introns in comparison of their sequences of genomic DNA and cDNA by DNAMAN. GhAQP2, GhAQP3, and GhAQP4 exhibited a typical structure with six transmembrane domains which linked by five loops and had the consensus sequence HINPAVTFG of MIP family and two highly conserved peptides Asn-Pro-Ala (NPA). These deduced amino acids showed high identity with the aquaporin of PIP2 subfamily reported from other plant species by multiple alignment analysis. The tissue specific expression analysis by using quantitative RT-PCR assays indicated that GhAQP2 expressed predominantly in 20 DPA fiber cell, while GhAQP3 expressed highly in the hypocotyl and cotyledon. Moreover, GhAQP4 showed highly significant expression level in 10 DPA fiber cell, hypocotyl and cotyledon. These results suggested that the three GhAQP genes might play different roles in different cotton tissues. The result of GhAQP2 predominantly expressed in 20 DPA fiber cell, provides important information for the further research on the gene-expression regulation during developmental stages from fiber cell elongation to secondary cell wall synthesis.