Cloning And Genetic Transformation Of Arabidopsis Thaliana Zinc Finger Protein AtZFN1Gene

Based on the published mRNA sequence of Arabidopsis thaliana, a pair of primers was designed to obtain Arabidopsis thaliana AtZFNl gene. A fragment of1464bp, which included the whole AtZFN1coding domain sequence, encoding396amino acid residues, was obtained by PCR. The amino acid sequence compared by Blast revealed there was high homology with zinc finger protein of other plants and the similarity to Vitis Vinifera zinc finger protein was the highest with73%.The coding region of AtZFNl was inserted into the expression vector pBI121and the recombined vector was named as pBI-ZFN. With vectors pKANNIBAL and pART27, the RNAi expression vector pART-F-R was reconstructed. Then recombined plasmids were transformed into Agrobacterium tumefaciens GV3010. Arabidopsis were transferred by flower-dipping methods, expecting to gain high and low expression level transgenic plants. After selection by50mg/L Kam, the regenerated plants were obtained and PCR analysis of them showed they were all PCR positive lines.Based on the published DNA sequence of Arabidopsis thaliana, a pair of primers was designed to amplify DNA fragment upstream of AtZFNl gene. A fragment of849bp was obtained by PCR, which contained the whole promoter sequence of AtZFNl gene. With plant expression vector pBI121, the promoter of CaMV35S was replaced by AtZFN1promoter, which was transformed into Agrobacterium tumefaciens GV3010. Arabidopsis were transferred by flower-dipping methods, expecting to obtain transgenic Arabidopsis thaliana, harboring a GUS reporter gene driven by AtZFNl promoter.Real-time RT-PCR was performed to reveal transcript level of AtZFN1. The results indicated AtZFN was abundant in flower buds and leaf veins, rather than in root; And the expression of AtZFN1was suppressed by20mM GA3treatment.The open reading framgment of AtZFN1was inserted into pET-30a(+) to construct the expression vectors, and the recombinant plasmids were transformed into E.coli BL21(DE3). The fusion proteins were induced to express by IPTG at37℃. SDS-PAGE analysis revealed that the full-length gene could be expressed and the fusion protein was highly expressed, which made it possible to purify the fusion protein and prepare the corresponding antibody in future study. The coding region of AtZFN1was inserted into pA7-GFP to construct the fusion protein expression vector AtZFN1-GFP. The fusion was then transformed into onion epidermal cells using a gene gun. Subcellular localization of transiently expressed AtZFN1-GFP fusion was detected by a confocal laser scanning microscope. The result revealed that AtZFN1localized in nucleus and plasma membrane.