Functional Analysis Of AtHD2D Gene On Abiotic Stress In Arabidopsis

The HD2 s family is plant-specific HDACs.Previous studies have shown that HDACs is involved in plant development and related to stress.In order to study the response mechanism of AtHD2 D gene over-expression plants to abiotic stress in Arabidopsis,this study used WT Arabidopsis(ecotype,Columbia)as the experimental material,we amplified the full-length CDS sequences of four genes in the HD2 s family and the AtHD2D’s promoter sequences by RT-PCR.The expression vector was constructed and Arabidopsis transformation followed the floral dip method.Transgenic positive plants were obtained by antibiotic screening and leaf PCR.q RT-PCR technique and GUS histochemical staining were used to analyze the expression patterns of AtHD2 D gene in the spatial and temporal;Abiotic stresses(Na Cl,4 ℃and drought)were treated on transgenic lines to analyze the response of AtHD2 D gene and promoter to stress and their role in adversity.The results are as follows:1.Four genes(AtHD2 A,AtHD2 B,AtHD2 C and AtHD2D)in the HD2 s family were amplified from Arabidopsis with sequence lengths of 738 bp,885 bp,921 bp and 612 bp,encoding 245,294,306 and 203 amino acids,respectively.DNA sequence analysis showed that the overall homology of AtHD2 s family genes is 35%,among which AtHD2 A and AtHD2 B have the closest homology(59%),followed by AtHD2 C by constructing phylogenetic tree,while AtHD2 D has the furthest homology with the other three genes,which is unique in evolution.2.The HDACs activity of AtHD2 D over-expression,WT and hd2 d mutant SALK-095273 C was measured in Arabidopsis.The HDACs activity of AtHD2 D over-expression plant was the highest,HDACs activity of hd2 d mutant plants was the lowest.3.The four genes in the HD2 s family(AtHD2 A,AtHD2 B,AtHD2 C and AtHD2D),were all expressed in the roots,stems,leaves,flowers and young fruits in Arabidopsis by q RT-PCR,and the expression levels of HD2 s gene were the highest in the roots,while the gene expression levels were relatively low in the stems,flowers and young fruits.4.Stress tolerance of AtHD2 D over-expression Arabidopsis was tested.The results showed that under the stresses of Mannitol and Na Cl,the seed germination rate and root length of AtHD2 D over-expression plants were higher than those of WT plants;DAB and NBT staining as well as the determination of leaf water retention indicated that the degree of cell membrane damage in WT Arabidopsis was higher than that in over-expression Arabidopsis;Under Na Cl and drought stresses,the four physiological indexes(SOD,POD,chlorophyll and proline)were determined,and it was found that the POD,SOD activity,chlorophyll and proline contents of AtHD2 D over-expression plants were higher than those of the WT.The expression levels of stress responsive genes were determined in AtHD2 D over-expression and WT Arabidopsis stressed for 20 days,and q RT-PCR results showed that the expression levels of drought stress genes in WT was significantly lower than that of over-expression plants,suggesting that AtHD2 D gene could up-regulate the expression of drought related genes through some mechanism,thus improving the resistance and adaptability of over-expression plants to adversity.The short-term stresses(4 ℃,Na Cl and Mannitol)of WT Arabidopsis showed that the expression of AtHD2 D gene in leaves increased significantly.5.Bioinformatics analysis indicated that there is co-sequenced gene upstream of AtHD2 D gene,thus determining the sequence of AtHD2 D gene’s promoter with size of 559 bp.The DNA was extracted from Arabidopsis by CTAB method,and the promoter of AtHD2 D gene was cloned.Transferred into the p CAMBIA1305 binary vector,the expression of downstream GUS gene was driven by the promoter of AtHD2 D gene.By histochemical staining,the promoter of AtHD2 D gene could drive GUS gene expression in roots,stems,leaves,flowers and young fruits in Arabidopsis,showing promoter activity and activating the expression of exogenous genes in different tissues.