Cloning And Expression Analysis Of Drought Induced Gene Kdnovel1007 From Kalancho(e|") Daigremontiana

Drought Stress severely affects the normal growth and development of plants, resulting in an immeasurable impact on agricultural production. Therefore, the research on drought resistance of plants is essential. Under drought stress, plants tend to take certain strategies to respond to drought stress positively, so that they can grow normally.With the plantlet of leaves from Kalanchoe daigremontiana chosen as research material, this study selects the specific expression EST (singletl007) sequence of subtractive cDNA library of Kalanchoe daigremontiana for the research. There is no homologous sequences of the specific expression EST sequence in GenBank. Through cloning the full-length sequence of kdnovel1007 gene by the means of RACE and performing bioinformatic analysis, we can get the information of open reading frame and the protein sequence encoded. In order to better understand the function of the protein encoded by kdnovel1007 gene, we performed bioinformatic analysis of the protein sequence. The physicochemical properties, functional domains, a transmembrane region, subcellular localization, signal peptide prediction and spatial structure information of the protein was obtained. To explore whether the gene is drought-stress response gene, real-time fluorescence quantitative PCR was carried out on the expression of the gene under natural drought conditions.The full-length sequence of kdnovel1007 gene was obtained by RACE and analyzed by the means of bioinformatic. The full-length of kdnovel1007 gene is 1022bp, the open reading frame is 468bp, which encodes 155 amino acids. Through the analysis of the encoding protein bioinformatics, it is achieved that the molecular formula of the predicted protein was C733H1194 N216O234S8 which contains 2385 atoms in total. The molecular mass of the protein is about 1.7kDa and the theoretical isoelectric point (pl) is 8.35. The instability index is computed to be 63.12. This classifies the protein as unstable. The hydrophobicity/hydrophilicity prediction shows that amino acids of hydrophobic are mainly located in transmembrane region, while the amino acids of hydrophilic are located in non-transmembrane region. After the subcellular localization was carried out, it was found in the chloroplast of plant cells. Conservative domain prediction was carried out, and it found that in the 21-89 bit of the protein sequence there was the conserved domain which was related to GrpE super family. Through the transmembrane domain prediction, we found that the protein is a transmembrane protein and in the C-terminal of the protein sequence there was a transmembrane region. After the prediction of the secondary structure of the protein it was found that the protein has random coil structure, α-spiral and extended chain structure. Through the tertiary structure prediction it was found the protein also has β-folded structure and doesn’t have β-corner structure. Signal peptide prediction was carried out, and it was found that there is no signal peptide, therefore the protein isn’t secreted protein.Real-time PCR was carried out on the expression of kdnovell007 gene under natural drought conditions. The expression of this gene is increased with the decrease of the relative water content of the soil in a certain extent. When the relative water content of the soil was 40%, the expression of the kdnovel1007 gene was 5.73 times that of the control group, while 5.93 times that of the control group when the relative water content of the soil was 30%. The expression of the kdnovel1007 gene reached the highest level when the relative water content of the soil was 20%, and it was 13.43 times that of the control group. Compared with last drought level the gene expression level decreased rapidly when the relative water content of the soil was 10%, and its expression was still 2.51 times that of the control group. The drought stress induces up-regulation expression of kdnovel1007 gene, which indicated that this gene is a drought stress response gene. This gene is playing an important role in plant resistance to drought stress.