Cloning,Expression Analysis And Transformation Of Wheat Aquaporin Gene

Wheat is one of the three staple foods,and its output and quality are closely related to world food security.However,wheat is facing various stresses during its growth.Aquaporins are a class of membrane-intrinsic proteins that can efficiently and specifically transport water molecules and play an important role in regulating the transmembrane transport of water.In addition,aquaporins play an important role in the process of plants responding to various stresses such as drought,salt,and low temperature.However,due to the large wheat genome and the heterologous hexaploid,the research on wheat aquaporins is not thorough.In this study,copies of wheat aquaporin genes TaTIP1-1c and TaTIP3-1b on 4BL and 1DL were obtained from wheat variety KN199 by PCR amplification.Taking these two aquaporin genes as research objects,the following progress has been made:1. A preliminary analysis of TaTIP1-1c was carried out through a bioinformatics analysis tool.The analysis result shows that the coding region of TaTIP1-1c gene is 753bp in length and encodes 250 amino acids in total.TaTIP1-1c protein has a molecular weight of25755.92 Da,6 transmembrane domains,no signal peptide,and is a non-secreted,weakly acidic,stable,and hydrophobic protein.The secondary structure is composed of 26.8%alpha helix,25.6%extended straight chain and 47.6%irregular coil.The tertiary structure model shows that the protein can form a homotetramer,and phylogenetic analysis shows that the relationship between wheat and Aegilops is closer.Analysis of cis-acting elements in the promoter region revealed that the gene has multiple regulatory elements,most of which are related to plant hormones（abscisic acid,methyl jasmonate,auxin）and light.2. A preliminary analysis of the TaTIP3-1b gene was carried out using bioinformatics analysis tools.The analysis results show that the coding region of TaTIP3-1b gene is 792 bp in length,encoding a total of 263 amino acids.TaTIP3-1b protein has a molecular weight of27550.09Da,6 transmembrane domains,no signal peptide,and is a non-secreted,weakly alkaline,stable,and hydrophobic protein.The secondary structure is composed of 34.22%alpha helix,21.29%extended straight chain,3.8%β-turn and 40.68%irregular curl.The tertiary structure shows that the protein also exists as a homotetramer.Analysis of cis-acting elements in the promoter region revealed that the gene has multiple regulatory elements,most of which are related to abscisic acid and light.3. TaTIP1-1c gene expression analysis by real-time fluorescence quantitative PCR technology.The results showed that the TaTIP1-1c gene was expressed in different tissues of wheat variety KN199,with the highest expression in the stem and the least expression in the root.Analysis of TaTIP1-1c gene expression in wheat leaves under different stresses showed that the gene was down-regulated under drought stress,up-regulated under salt stress,and expressed under abscisic acid（ABA）and hydrogen peroxide（H₂O₂）treatments Up.The expression analysis of TaTIP1-1c gene in wheat seedling and booting stage under drought stress showed that the expression level of this gene was significantly up-regulated in the roots of seedling stage and down-regulated in the leaves of seedling stage;The expression levels were all increased.It is speculated that this gene is involved in the process of wheat responding to adversity stress.4. Genes of TaTIP1-1c and TaTIP3-1b genes located on 4BL and 1DL were obtained from wheat variety KN199.The overexpression vectors of TaTIP1-1c and TaTIP3-1b genes were constructed.Using the gene gun method,the overexpression vectors of TaTIP1-1c and TaTIP3-1b genes were transformed into wheat immature embryos,and transgenic positive plants were obtained by tissue culture.