Molecule Cloning And Functional Analysis On CpPR-1 From Chimonanthus Praecox(L.) Link

A series of defense responses may take place when the plant is treated with some compounds,salt and pathogens,such as fungi,bacterium and virus,or any other stresses,usually accompany with the generation of some new proteins,named pathogenesis-related proteins(PRP). According to the latest researches,PRs can be classified into 17 classes,such as PRl,PR2,PR3,and PR4. PR-1 have high sequence conservation in the structure.Based on constructed cDNA library from Chimonanthus praecox Expressed Sequence and Tag (EST) analysis, a gene named PR-1 was obtained through full length sequencing of cDNA library clone.The characteristics of the encoding proteins analyzed with bioinformatic method,then predict the functions.We have finished the construction of the plant aquaporins vector through Agrobacterium-mediated transformation.Using leaf disc method and the regenerated tobacco plants were obtained,and the functions of PR-1 were analyzed by stress treatment to the transgenic tobacco plant.The main findings are as follows:1. Cloning of CpPR-1 from Chimonanthus praecox flowerIn this paper, a PR-1 gene was obtained by sequencing the randomly selected clones, on the basis of cDNA library construction of Chimonanthus praecox (L.) Link. The bioinformation analysis showed that the cDNA sequence had a length of 1199 bp, containing ORF of 486 bp which encoded 162 amino acids.It have no intron.2.Construction of plant expression vectorPlant expression vector pCAM-CpPR-1 Were successfully constructed by combining the coding sequence of the cDNA with the expression vector pCAM2301g,which is to lay the function for further studying the function of the genes via transgenic technology.3.The genetic transformation of Nicotiana tobacumThe target gene with pCAM-CpPR-1 were introduced into tobacco genome by leaf dics method through Agrobacterium-mediated transformation.11 transgenic tobacco plants were obtained through GUS staining analysis,the Km resistance screening detection and PCR detection.4.The stress research of transgenic tobacco plantsThe results showed that the resistance capacities of heavy metal stress were much greater than the control after the plants were treated with 50mmol/L CuSO4solution.It can be determined that the transgenic tobacco plant have a certain adaptability to adapt to heavy metal stress but have no significant resistance to tobacco mosaic virus.