Cloning And Functional Analysis Of CpPLP Gene From Chimonanthus Praecox (L.) Link

As a new family of lipolytic enzyme protein, patatin-like proteins have several activity of acyl hydrolase with esters (Lipid acyl, hydrolase; LAH), esterase and acyltransferase (wax synthase). Recent researches have shown that the activity of wax esters and acyl hydrolase enzyme can not only as a means of defensing against insect pests in plants, but also helping plants to cope with drought stress.On the basis of the constructed cDNA library from Chimonanthus praecox and its ESTs analysis, a patatin-like protein gene was obtained by sequencing and cloning randomly, named CpPLP. In this paper, we analyzed the sequence characterristics and transcriptional expression level of CpPLP in Chimonanthus praecox. Then constructing the plant expression vector and transforming into tobacco, we analyzed the related functions of CpPLP gene. The main results are as follows:1, The molecular characteristics and Bioinformatics Analysis of CpPLP geneThe cDNA full-length of CpPLP gene is1489bp, and the ORF frame is1131bp, it encoded376amino acids. Analysis of the conserved domain in NCBI, the protein of CpPLP gene encoding is high similar to patatin-like protein family. According to analysis of its structure, the relative molecular mass of this protein is41122.5, the isoelectric point is4.84, and the secondary structure is mainly composed of α-helix (Alpha helix,41.88%), extension chain (Extended strand,22.65%), random coil (Random coil,29.06%), β-angle (Beta turn,6.41%); hydrophobicity analysis showed that CpPLP gene encoding protein had good hydrophilicit.2, Expression analysis of CpPLP geneThe analysis of CpPLP by using real-time fluorescence quantitative PCR showed that, CpPLP gene in wintersweet flower organs especially in stamens has a highest expression. And there is no expression of CpPLP gene in root, stem, leaf and pistil. The results showed that the expression of CpPLP gene has very strong tissue specificity. The transcripts of CpPLP gene is most in stamen, speculated that the gene is related to morphogenesis in stamens, to protect the stamens from outside insect pest or mechanical damage; and the fluorescent quantitative analysis of flowers during different florescence showed that, CpPLP gene is mainly expressed in the later period and open flap, reaching the highest expression level in the blooming period, and no expression in the germination stage and bud stage. This result further confirmed that CpPLP gene has protective effect in stamens during development stage; under drought stress, the fluorescent quantitative analysis showed, the expression of CpPLP was significantly increased, suggesting that the CpPLP gene can help plants to survive in the hydrophobic conditions.3, The constmction of CpPLP gene expression vector and transformation of tobaccosPlant expression vector pCAMBIA2301g/CpPLP was constructed, then CpPLP was transformed into Agrobacterium tumefaciens by using electro-poration method. The vector transformed into tobaccos by using the leaf disc transformation procedure. Then the transgenic tobaccos were obtained by Km+resistance screening, DNA and RNA detection, GUS staining, real-time fluorescent quantitative PCR. Finally, five lines transgenic tobaccos were obtained, and there were15plants each line. By measuring soluble protein content and LAH activity, it can be seen that transgenic tobacco had a marked higher soluble protein content and LAH activity than the control.4, Resistance Analysis of transgenic tobaccoTo test the stress tolerance of transgenic tobaccos, we used the half leaf blight spot method to transform tobacco mosaic virus (TMV) into the tobaccos. It showed that the disease symptom of wild type was more serious than transgenic tobacco plants. By inoculating with Pseudomonas solanacearum from root, it showed that the symptom was lighter compared with control. We used the foliage spray method to inoculate tobacco with tobacco powdery mildew fungus disease, it showed that the symptoms of transgenic tobacco plants were lighter than wild type, and there were not blockbuster of circular spots. The results showed that the expression of CpPLP gene is improved the diseases tolerance of tobacco to some extent.By using a PEG treatment of tobacco, we measured the content of proline and MDA. The results showed that the drought tolerance in transgenic tobaccos were better than wild type. It shows that the expression of CpPLP gene can help plant to survive in hydrophobic condition.