Cloning And Function Identification Of Enzyme Of Deadenylation CpCAF1 From Wintersweet

Wintersweet, Chimonanthus praecox(L.) Link, is a famous traditional species originating from China, where it is appreciated as cut flower with high economic and ornamental value. CAF1 is an enzyme kind of deadenylation, playing an important role in the m RNA degradation. Deadenylation is the first step in most eukaryotic cells m RNA degradation. The progress of m RNA degradation can clear the m RNA which outdated and harmful to mutate, also ensuring that cells of normal life activities, changing gene expression and the biological character. as the gene expression after transcription regulation, it is one of the key steps of biological transcriptome reconstruction. For now, CAF1 gene has been research in the Arabidopsis thaliana, Capsicum annuum and Oryza, the results show that CAF1 gene plays an important role in plant growth and biological and abiotic stress response in the process.1. The structure of CpCAF1 gene and features of its protein By random sequencing, the c DNA sequence of CpCAF1 was separated on basis of EST analysis of constructed c DNA library. DNAstar software was used to analyze the sequence features. The c DNA sequence contains a 807 bp open reading frame. The online prediction of Prot Param software indicated that the deduced CpCAF1 has a prediction molecular formula of C1371H2087N349O410S11, contains 269 amino acid residues, with the molecular weight of 30.37 KD, an isoelectric point(p I) of 4.70. The protein stability factor was 25.90, showing stable protein character. Signal P software showed that there is no Signal peptide and subcellular localization analysis showed that the protein may existed in nucleus possibly. Multiple sequence alignment by DNAstar 5.0 software revealed that CpCAF1 has high homology up to 98% with other protein, such as Arabidopsis thaliana、Prunus mume、Vitis viifera、Theobroma cacao、Elaeis guineensis、Nelumbo nucifera、Ricinus communis., sharing some conserved structures. CpCAF1 protein of nucleic acid enzyme family belongs to the DEDD subtribe, The active area consist of 3 asparagus nucleotide(D), 1 glutamic acid(E) and the adjacent histidine(h).2. Expression analysis of CpCAF1 gene Expression features of CpCAF1 in various issues, florescences and under different treatments were analyzed by Quantitative Real-time PCR. The result proved that CpCAF1 had tissue specificity as the transcript was found in the root, stem, leaf and flower tissue, especially higher in the decline of leaf and the lowest in the root. During different florescences, the expression of CpCAF1 was in every period, the highest expression in senescence, the lowest expression in the bud period. The treatment with SA induced the expression of CpCAF1, otherwise, the treatment with ABA,GA3 Inhibition of the expression of CpCAF1. The results above indicated that CpCAF1 involved in signal transduction.3. Analysis of CpCAF1-transgenic Arabidopsis thaliana The constructed expression vector p CAMBIA2301G-CpCAF1 was transferred into the agobacterium EHA105, followed transforming Arabidopsis thaliana by inflorescence infection method. 7 T4 generation transgenic Arabidopsis thaliana were eventually gained after the process of Kan-resistance, GUS staining and DNA identification. Quantitative Real-time PCR was used to check the expression of CpCAF1 in the transgenic Arabidopsis thaliana. Finally, CpCAF1-5 of the highest expression and CpCAF1-3 the lowest of expression were selected to observe the phenotype and process some treatments. The observation of the transgenic and wild type Arabidopsis thaliana showed that the bolting, pumping of the lateral branch, the first inflorescence, the first flower and the first seed pod of the T4 generation of transgenic Arabidopsis thaliana came earlier. In conclusion, root growth, lateral branch growth, Indus leaves and pod growth status are positively correlated with the amount of gene expression. The transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana were put in 4 ℃ low temperature processing respectively 0 h, 24 h, 48 h, recording the change of proline, MDA and SOD. The experimental results show that the cold tolerance of transgenic Arabidopsis thaliana better than wild-type Arabidopsis thaliana. By using Non-invasive Micro-test Technique, the experiment measured the flow velocity of potassium, when transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana under the condition of low potassium. Results show that under the condition of potassium deficiency, three seedlings of potassium are induced by the different levels of internal flow.