The Cloning Of Zm-HINT1 Gene And Its Function Analysis In Abiotic Stress

As one of the three major grains,maize(Zea mays l.)is not only an important food crop,but also an important feed crop and industrial raw material which can plays an extremely important role in China's food security and agricultural economic.During the growth and development period of maize,this has a negative impact on corn productions under the abiotic stress conditions.Abiotic stress such as salinity,high temperature,drought,cold injury and heavy metal pollution,is one of the important factors affecting corn yield and quality.Therefore,the research on the stress-resistance of maize and the exploitation of stress-resistance related genes which is an effective way to improve the ability of maize to adapt to adversity and is also an urgent problem to be solved in modern breeding.Based on the previous proteomic studies,this study focused on one of the differentially expressed proteins,the maize histidine triad nucleotide binding protein Zm-HINT1,and preliminary explored the role of Zm-HINT1 in the stress response.The main contents of this study are as follows:1.Zm-HINT1 was cloned by homologous cloning technique and a 729 bp c DNA sequence was obtained.After amino acid sequence analysis,the results showed that the protein molecular weight is 14.23 KD and the isoelectric point is 5.99 which can encode 129 amino acids.The study found that the amino acid sequence of Zm-HINT1 is highly homologous to mammalian HINT1 and HINTwhich has a conserved domain of HIT family including HIT motif,a-helix and ?-sheet.Simultaneously,Zm-HINT1 is a member of the HINT1 group in the HIT superfamily.Phylogenetic trees analysis showed that Zm-HINT1 have a near relationship with the HINT1 protein of Caenorhabditis elegans which be located on the same evolutionary branch.Further,the PCR analysis showed that Zm-HINT1 among the various organizations,such as roots,stems,leaves and embryos,but not in the pericarp.2.Real-time PCR was used to study the expression patterns of Zm-HINT1 under the abiotic stresses,such as salinity,high temperature,SA,PEG,drought.We found that SA,drought and PEG can induce the expression level of Zm-HINT1,and salinity can decreased the expression level of Zm-HINT1,after high temperature,the expression of Zm-HINT1 was up-regulated first and then down-regulated;while promise that Zm-HINT1 plays an important role in abiotic stress in maize.3.We transformed a fusion expression vector p CAMBIA1304-Zm-HINT1-GFP which is constructed by recombinant DNA technology.We performed subcellular localization of the Zm-HINT1 gene using onion epidermal transient expression experiments.The results showed that Zm-HINT1 is a nuclear localization protein.4.We obtained the transgenic plants by transform the overexpression vector p CAMBIA1304-Zm HINT1-2 into Arabidopsis thaliana by agrobacterium-mediated method,and treated the offspring with different abiotic stress.The result showed that transgenic Arabidopsis the survival rate of transgenic Arabidopsis was higher than the wild-type and empty vector transfected Arabidopsis after high temperature and drought treatment.In addition,the content of proline was used as a physiological index to determine the accumulation under high temperature and drought stress.After high temperature treatment and drought,the content of proline in transgenic lines of Zm-HINT1 was significantly higher than that of empty vector and wild-type Arabidopsis.The above results indicate that Zm-HINT1 may increase plant salt resistance through ABA signal transduction pathway by regulating ABA synthesis.And improve the plant's high temperature and drought resistance by inducing the accumulation of proline which thereby regulating the permeation balance.