Cloning And Analysis Of The Aquaporins Gene From Galega Orientalis

Galega orientalis is a perennial leguminous plant. It is characterized by high crop yield, long time utilization, faster reviving growth and high nutrients, especially the crude protein content. G.orientalis could increase lactation content and decrease the threats of bloat risk in dairy cows. G. orientalis can substitute for the Medicago sativa in the form of fresh grass, silage, hay, grass particles and so on.Therefore, G. orientalis is a worthy cultivar of large-scale popularization. However, in the northwest of our country, the population of G. orientalis was limited due to the widespread salinization and aridity.AQP protein belongs to the MIP (major intrinsic proteins), it mediated various physiological pathways. AQP can promote the cell membrane water permeability in order to enforce the water long-distance transportation and transmembrane transport of plant. By regulating its opening and closing, AQP can decrease the water loss, maintain moisture and osmotic balance in cell. This performance can enhance the plant tolerance to water stress. AQP also attend the symplastic transport and transcellular transport of other micromolecule to mediate various physiological processes. These processes include photosynthesis, absorption of nutrients, cellular signal transduction and stress response.Based on the sequence information of a deposited EST fragment, one novel gene encoding aquaporin had been cloned from G.orientalis. This newly identified gene was defined as the GoAQP gene. Analysing the gene sequence bioinformatics and the gene expression, constructing the expression plasmid and analysing the GoAQP subcellilar location has been conducted in order to validate the gene function under water stress. All of the research formed the basis for the cultivation of Gorientalis in the northwest of our country.This study includes the following content and conclusion:(1) Cloning, bioinformatics analysis of cDNA Encoding aquaporins of G.orientalisBased on the sequence information of a deposited EST fragment from cDNA librart of Gorientalis under salt stress by suppression subtractive hybridization, by the aid of RT-PCR and RACE technology, a full-length cDNA of one novel gene encoding aquaporin had been cloned from G.orientalis. This newly identified gene was defined as the GoAQP gene and had been assigned GenBank accession number:HM803185. Bioinformatics analysis showed that the full-length cDNA of this gene is1258bp, including an870bp open reading frame (ORF), which encodes a protein of289amino acids. GoAQP protein possesses the conserved MIP family signal sequence HINPAVT/SFG, the highly conserved sequence GGANXXXXGY and TGITNPARSL/FGAAI/VI/VF/YN which extensively existed in the higher plants. Transmembrane structure analysis by the TMHMM software indicated the GoAQP possesses six typical transmembrane areas. These results implied that it was a transmembrane protein. Phylogenetic analysis showed that GoAQP belong to PIP1subfamily, compared with the AQP family from Arabidopsis thaliana. By the aid of the ProtFun function prediction software, it was found that the protein highly probably possesses transportation function.(2) GoAQP expression characterizationBy Real-Time PCR, the tissue-specific gene expression and gene expression under different abiotic stresses were analyzed. The results revealed that expression of GoAQP was the most abundant in root and the least in stem in Gorientalis. The expression level of GoAQP in leaves were increased with the treatment of NaCl and PEG, and showed a bimodal distribution pattern under NaCl treatment. The expression level of GoAQP in leaves were increased with the treatment of ABA at early stage. with a bimodal distribution pattern, but after8h the expression level declined. These suggested that GoAQP might be involved in the regulation of salt and dtought stress tolerance of G. Orientalis.(3) Construction of plant expression vector of GoAQP and analysis of subcellular localizationThe plant expression vactor pCAMBIA-1302-GoAQP was constructed. This established plant expression vactor formed the primary foundation for the further gene function analysis. Through transient expression of GoAQP-GFP fused gene in onion epidermal cells via direct transformation, subcellullar location of GoAQP were examined by the aid of confocal microscope. The result revealed that GoAQP protein located in the cell membrane.