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Molecular Cloning and Primary Functional Analysis on CpAQP1 and CpAQP2 from Chimonanthus Praecox (L.) Link

Author: ZhangMi
Tutor: LiMingYang
School: Southwestern University
Course: Floriculture
Keywords: Aquaporins molecular-characteristics clone plant expression vector functions
CLC: S685.99
Type: Master's thesis
Year: 2010
Downloads: 53
Quote: 1
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Abstract


Aquaporins (AQPs) are widely distributed in cellular membranes of prokaryotes and eukaryotes, which serve as channels that permit the bidirectional passage of water through cellular membranes. Plant aquaporins is a superfamily, and a large number of aquaporins genes have been found and cloned recently. According to amino acid sequence similarity and subcellular localization can be divided into four categories:plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins and small basic intrinsic proteins. Plant aquaporins play an important role in water regulation and various physiological responses, such as water transport, osmotic regulation, the growth and development, cell growth and biological stress reactions in pants.Based on the previous studies, we had cloned two Chimonanthu Praecox aquaporins gene, named CpAQPl and CpAQP2, and the characteristics of the encoding proteins analyzed with bioinformatic method, then predicted the functions. We have finished the construction of the plant aquaporins vector through Agrobacterium-mediated transformation of Nicotiana tobacum var. xanthi using leaf disc method and the regenerated tobacco plants were obtained, and the functions of two Chimonanthus Praecox aquaporins were analyzed by stress treatment to the transgenic tobacco plants. The main findings are as follows:1.Bioinformatics analysis of CpAQPl and CpAQP2 and the encoding proteinsBased on constructed cDNA library from Chimonanthus praecox flowers and Expressed Sequence Tag (EST) analysis, two genes of Aquaporins named CpAQP1 and CpAQP2P (GenBank accession number are:GU433105 and GU433106) was obtained through full-length sequencing of cDNA library clone. The cDNA of CpAQPl was 1154bp with the largest open reading frame (ORF) of 810 bp, encoding 269 amino acids. The cDNA of CpAQP2 was 1131bp with the largest open reading frame (ORF) of 975 bp, encoding 324 amino acids. Both of them have no intron. Cluster analysis and homology comparison showed that CpAQPl belongs to TIPs subfamily classes, and CpAQP2 belongs to PIPs subfamily classes, containing a highly conserved domain of the MIP family; CpAQP1 had six transmembrane domains and both its N-terminal and C-terminal were in the cell, but CpAQP2 had seven transmembrane domains and N-terminal in the cell and C-terminal out the cell. The result shows that the serine phosphorylation in the C-terminal of two proteins (CpAQP1:Ser-228, CpAQP2:Ser-240) maybe plays an important role in plant stress and subcellular localization using NetPhos2.0 to predict the phosphorylation sites of this protein.2.Construction of plant expression vectorPlant expression vector pCAM-CpAQPl and pCAM-CpAQP2 were successfully constructed by combining the coding sequence of the cDNA with the expression vector pCAMBIA2301g, which is to lay the foundation for further studying the function of the genes via transgenic technology. 3.The genetic transformation of Nicotiana tobacumThe target genes with pCAM-CpAQP1 and pCAM-CpAQP2 were introduced into tobacco genome by leaf disc method through Agrobacterium-mediated transformation.9 transgenic tobacco plants with CpAQPl and 8 transgenic tobacco plants with CpAQP2 were obtained through GUS staining analysis, the Km resistance screening detection and and PCR detection.4. The stress research of transgenic tobacco plantsThe results showed that the resistance capacities of salt stress and drought stress were much greater than the control after the plants were treated with 250mmol/L NaCl solution and 20% PEG6000, which suggested that CpAQPl and CpAQP2 successfully integrated into tobacco, and they played an important role in balance with water of plant body, the damage to permeability, etc. It can be determined that the transgenic tobacco plants have a certain adaptability to adapt to salt stress and drought stress.

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