Cloning And Functional Analyses Of Two Stress-related Zinc Finger Protein Genes Fromammopiptanthus Mongolicus

Ammopiptanthus mongolicus is an unique evergreen broadleaf shrub in the desert area of northwest China and it has strong stress resistance.In this paper,two stress-related zinc finger protein genes,designated AmZFP3 and AmZFP4,were cloned from this species and were functionally characterized through expression profile analysis and transgenic Arabidopsis phenotypic observation under stress conditions.The main results are as follows:(1)The complete coding region cDNA(1101 bp)of AmZFP3 was cloned by RT-PCR method.The predicted protein of AmZFP3 consists of 366 amino acid residues,with a molecular weight of 40.76 KD and an isoelectric point of 9.09.The protein contains a zf-RING-H2 domain and two transmembrane regions,and probably locates in plasma membrane.(2)The expression profiles of AmZFP3 were analysed by semi-quantitative RT-PCR method.The results showed that the transcription level of AmZFP3 in A.mongolicus seedlings was up-regulated to different degrees under cold,drought and salt stresses for 2 h to 48 h,however,the transcription level was almost unchanged under high temperature stress.In young leaves of the A.mongolicus plants growing in the wild,the gene expression level was higher in cold autumn and winter,especially in December,than in warm spring and summer.In addition,the gene was most abundantly expressed in young leaves and young roots,followed by twigs,flowers and immature pods of the A.mongolicus plants growing in the wild in spring.(3)The coding region cDNA fragment of AmZFP3 was successfully inserted into the plant over-expression vector pCAMBIA3300 and was introduced into wild type Arabidopsis.By screening with selective agent PPT(phosphinothricin)and by PCR and RT-PCR detection,seven transgenic lines with high expression levels of AmZFP3 were obtained.(4)The phenotypic characterization of the transgenic lines suggests that over-expression of AmZFP3 could improve drought and salt tolerance of the transgenic lines,nevertheless,it reduced the freezing resistance.Moreover,the sensitivity of the transgenic lines to exogenous ABA was significantly increased.(5)The complete coding region cDNA(471 bp)of AmZFP4 was cloned by RT-PCR method.The predicted protein of AmZFP4 consists of 156 amino acid residues,with a molecular weight of 17.42 KD and an isoelectric point of 8.99.The protein contains two important domains named ZnF-A20 and ZnF-AN1,which probably locates in cytoplasm.Semi-quantitative RT-PCR was performed to analyze AmZFP4 transcript level in A.mongolicus seedlings under different stresses.The results showed that the gene expression was up-regulated in cold stress but was almost unchanged under drought stress.In addition,the expression level was most abundant in flowers,followed by twigs,young roots,young leaves and immature pods of the A.mongolicus plants growing in the wild in spring.