The Isolation, Identification And Biochemical Analysis Of DHHC-type Zinc Finger Protein Gene At5g04270in Arabidopsis

Formation of plant architecture is a complicated biological phenomenon. It is influenced by a variety of factors such as genotype, hormone, environment and nutrition. The initiation and outgrowth of axillary organs play very critical roles in plant architecture. In this study, the T-DNA activation taging mutant library was contructed on crylcry2background, and got an activation-tagging mutant scc10-D （suppressor of cry1cry2） grown in long-day （16-h light/8-h dark） condition. Then the gene who associated cryptochrome and plant architecture of scc10-D was isolated and identified through cloning, recombination, heredity resistance screening bioinformatics and all that analysis methods.The main results were obtained as followed:（1） A gain-of-function mutant library was built on cry1cry2background by T-DNA activation tagging technology and nearly2000independent transformant lines were got by Basta screening.4lines that had obvious phenotype change were found from transformant lines. The mutant scc10-D who showed enhanced shoot branching was selected as our research material. The flanking sequence of T-DNA insertion site in scc10-D mutant was defined through IPCR analysis. The results showed that the insertion locus of T-DNA was the nucleotide90708of the fifth chromosome. The mRNA expression of six genes adjacent to the T-DNA insertion locus was analyzed by reverse transcriptase polymerase chain reaction （RT-PCR） and real time PCR. The result showed that the transcript level of a DHHC-type zinc finger protein gene, At5g04270, was found to increase markedly in the scc10-D. So, we presumed the phenotype of scc10-D mutant caused by the overexpression of At5g04270gene.（2） The At5g04270gene was cloned into over-expressed vetor pEGAD, and the pEGAD-At5g04270was transformed into agrobacterium tumefaciens strain GV3101and integrated into genome of Arabidopsis Col-4by GV3101infection. The At5g04270gene over-expressed lines35S::GFP-At5g04270were successfully constructed and the homozygote was obtained by Basta screening. It was found that the At5g04270over-expression lines35S::GFP-At5g04270had the features of enhanced shoot branching, while the T-DNA mutant of At5g04270gene, SALK₀06515, showed decreased shoot branching when compared to the wild type （Col-4）. At the same time we found that the At5g04270mRNA expression of35S::GFP-At5g04270was incremental by RT-PCR. These results suggested that the phenotype of enhanced shoot branching of the mumant scc10-D At5g04270resulted from the over-expression of At5g04270gene. At5g04270T-DNA munant SALK₀06515homozygote was identified and screened by three-prime technique, and the At5g04270mRNA expression was analyzed by RT-PCR. The result showed that At5g04270mRNA had not been expressed in SALK₀06515homozygote. At5g04270mRNA expression in root, stem, rosette leaf, cauline leaf and flower of Arabidopsis Col-4were analyzed by RT-PCR. The result showed that At5g04270mRNA could be examined in all organs, but the mRNA expression was more in flower and rosette leaf than that in root, stem and cauline leaf.（3） The genome sequence, amino acid sequence of At5g04270were analyzed and contrasted with that of correlated other gene thought TAIR and NCBI website. At the same time, the amino acid modification site, signal peptide sequence, transmembrane domain, hydrophilicity and hydrophobicity of the At5g04270and DHHC PCR site-directed mutagenesis At5g04270C105A were analysed and contrasted. The results showed that the At5g04270was a membrane protein owing to it had three major transmembrane structures and a secondary transmembrane structure, and the At5g04270had the activation of protein acyltransferase by reason of it contained DHHC-CRD domain. The cysteine （C） was the fatty group polar amino acid, but the alanine （A） was the fatty group nonpolar amino acid. Due to change of amino acid polar after DHHC mutated to DHHA, the binding ability of zinc ion may be changed and the activation of proteinl acyltransferase may be losed.（4） The DHHC domain was changed to DHHA by PCR in At5g04270, then the At5g04270and site-directed mutagenesis At5g04270C105A was cloned into pYES260vetor, and the recombinant pYES260-At5g04270C105A and the pYES260-At5g04270were transfomated into yeast ark1Δ. The result showed that At5g04270but not At5g04270C105A restored the morphology of akr1A yeast cells grown at30℃in complete selective galactose liquid culture. The result suggested that At5g04270may be in the nature of protein palmityl acyltransferase activity and the DHHC domain may be the function domain of the transferase. The prokaryotic expression vetors pCold-At5g04270was constructed by cloning, restriction endonuclease digestion and T4DNA ligase reaction and pDEST17-At5g04270was constructed by gateway technique for further research the function of At5g04270gene.