Cloning And Functional Analysis Of Gene POP5in Rice

Stress has been an major factor in restricting the crop growth. Abiotic stress affects the growth and development of crops, and will affect the yields and quality. Statistics showed that crop yield reduction was from50%to80%under abiotic stress. Therefore, study on stress resistance of crops has become an hot topic in biological science. In recent years, with the rapid development of molecular biology, some achievements on the mechanisms for plant to response to stress in molecular level have been obtained, and more stress-inducible genes and stress-tolerance genes have been cloned. Therefore, further study on the genes’ function, not only helps to reveal the stress-tolerance mechanisms of rice in molecular level, but also provides a theoretical basis for rice molecular breeding.The calcium (Ca2+), as a signaling molecule, plays an important role in plant growth and development and stress response. Calmodulin (CaM), as one of the most important classes of Ca2+sensor, involves in many plant physiological responses through interacting with its downstream calmodulin-binding protein.Calmodulin binding protein (CaMBP) existes widely in eukaryotic cells, and the researches on it’s role in plant growth and development and stress response are going on. The expression of many CaMBPs was induced by a variety of stress, so CaMBP was considered to be a very important class of proteins in plant stress research. We founded that OsCaMBP was induced by chilling and heat, suggesting that it might serve as a common regulatory factor involved in the rice adversity reaction. In this study, we did Yeast Two-Hybrid Experiments using OsCaMBP as a bait to screen the proteins interacting with OsCaMBP. Then we selected one prolyl oligopeptidase homologous gene(OsPOP5) for further analysis to explore the role of OsCaMBP played in signalling pathway. The results are as follows:1. According to the approaches of yeast two-hybrid system, the interaction protein OsPOP5is screened from the low tempreture and drought cDNA library of rice taking OsCaMBP as a bait protein. 2. Analyzing OsPOP5by comparing in the database, the results are, the full length of cDNA is2251bp, the gene encodes596amino acid residues, molecular weight is predicted to be67.3KD and isoelectric point is about6.44. We also found that1-310amino acids sequence has a N-terminal domain of dipeptidyl peptidase IV (DPP Ⅳ_N/Peptidase S9B), and395-595amino acids sequence has a Peptidase_S9domain. Besides, the protein was predicted to be located in the extracellular.3. We analyzed OsPOP5expression at low temperature and high salt stress in different tissues using the method of semi-quantitative RT-PCR. OsPOP5was found to express in different degrees in roots, bases and leaves before dealing with adversity stress, while they showed differently expression patterns in different time and tissues under different stresses.4. The constructed expression vector of fusion protein was transformed into expression strain Rosetta. Then the fusion protein of96KD was induced by IPTG.5. We constructed expression vector of sense, antisense cDNA fragment of OsPOP5and established the genetic transform system for rice mediatied by agrobacterium tumefacien to study the physiological function of the gene expression on rice resistant response. Meanwhile, we constructed a green fluorescent protein vector in order to determine subcellular localization of the OsPOP5and to get futher study.