Cloning Of The Related Drought-resistant Gene From Pinus Massoniana And Its Expression Analysis

Drought,as one of the important factors affecting the normal growth of plants in all kinds of biological constraints,directly affects the normal metabolism of Pinus massoniana growth and development.When cells are dehydrated due to drought-stress,membrane and enzyme systems are destroyed and the cell functions are lost,leading to plant metabolism disorders.Subsequently,the protein synthesis system breaks down and hormone balance is damaged.It is therefore desirable to study the P.massoniana drought resistance mechanism in order to improve P.massoniana drought resistance capacity and cultivate drought resistance breeds.To investigate the molecular mechanism of pine response to drought stress,P.massoniana(No.83 family),an elite genotype with a significantly higher tolerance to drought,was used as the research material in this study.The main research contents and results are as follows:We cloned the related drought resistant gene fragments of APX?AQP?CBL?DHN?ERF?GPX?NAC?PEPC?POD with homologous cloning technique,which confirmed that these genes were present in P.massoniana.The full-length cDNA,named as PmNAC?PmERF?PmCBL3?PmDHN?PmGPX6?PmPIP1and PmPOD,respectively,were obtained using RT-PCR and RACE technique.Bioinformatics analysis revealed that PmNAC contained NAM domain and showed a high sequence identity with Picea sitchensis;PmERF contained AP2-ERF domain and showed a high sequence identity with Pinus tabuliformis;PmCBL3 contained EF-hand domain and showed a high sequence identity with Picea sitchensis;PmDHN contained Dehydrin domain and showed a high sequence identity with Pinus mugo;PmGPX6 contained GSH_Peroxidase domain and showed a high sequence identity with Pinus tabuliformis;PmPIP1 contained MIP domain and showed a high sequence identity with Picea abies;PmPOD contained Peroxidase domain and showed a high sequence identity with Picea sitchensis.Its expression were analyzed by Real-time PCR at different organizations and different periods in P.massoniana under drought treatment.The results demonstrated that PmCBL3?PmDHN?PmGPX6? PmPIP1 and PmPOD were induced by drought tolerance.Based on the analysis of genes expression using control 18 S,an initial up-regulation,followed by a down-regulation,was observed in roots,stems and leaves.This expression pattern showed that these genes likely participate in the drought process.At the same time,we cloned a PmACTIN gene fragments in P.massoniana.The plant expression vectors of pBI121-Pm PIP1?pSH737-PmCBL3?pBI121-PmGPX6?pBI121-PmPOD?pBI121-PmDHN were built with restriction enzyme site,which lay a foundation for further genetic transformation of Arabidopsis thaliana by gene transformation technique.The plant expression vectors of pBI121-Pm PIP1?pSH737-PmCBL3?pBI121-PmGPX6?pBI121-PmPOD?pBI121-PmDHN were transformed into A.thaliana using gene transformation technique,and the PmGPX6,PmPOD and PmDHN genes that overexpressed in A.thaliana were obtained.The gene function analysis results showed as follows:The results showed no differences in the phenotypes and root lengths among the WT and OP plants in the MS medium under normal conditions containing 0% PEG-6000.Under drought stress in MS medium containing 3.0% PEG-6000,the differences in phenotypes between the WT an OP plants were minimal,while the root lengths of the OP seedlings were longer than those of the WT lines.GFP analysis of the roots in transgenic A.thaliana showed that green fluorescence was excited by blue light in the dark,which also indicated that PmGPX6 can be transcribed in transgenic A.thaliana.The results showed no differences in the phenotypes and root lengths among the WT and OP plants in the MS medium under normal conditions containing 0% PEG-6000.Under drought stress in MS medium containing 4.0% PEG-6000,the differences in phenotypes between the WT an OP plants were minimal,while the root lengths of the OP seedlings were longer than those of the WT lines.GFP analysis of the roots in transgenic A.thaliana showed that green fluorescence was excited by blue light in the dark,which also indicated that PmPOD can be transcribed in transgenic A.thaliana.The rate of water loss in leaves of the WT and of the transgenic plants maintained an increasing trend as a function of treatment time.The rate of water loss of the WT plants were higher than those of transgenic plants,indicating that water loss occurred faster in the WT plants than those detached from the transgenic plants(P<0.05).In addition,an increase in proline content was observed in the OP and WT plants under drought stress,and the proline content of the transgenic plants was increased faster.The MDA content in the WT plants was higher and increased more rapidly than in the OP plants under drought stress.The contents of proline and MDA were statistically different(P<0.05).The results showed no differences in the phenotypes and root lengths among the WT and OP plants in the MS medium under normal conditions containing 0% PEG-6000.Under drought stress in MS medium containing 3.5% PEG-6000,the differences in phenotypes between the WT an OP plants were minimal,while the root lengths of the OP seedlings were longer than those of the WT lines.The results of RT-PCR analysis showed that PmDHN can be transcribed.Compared to the normal treatment,the transcriptional levels of six PmDHN lines were enhanced under drought condition.The results of the Southern blot analysis showed that no hybridization signal was detected in the wild-type A.thaliana,and two copies of PmDHN were present in transgenic A.thaliana of D1 and D3 lines.At the same time,GFP analysis of the roots in D3 line showed that green fluorescence was excited by blue light in the dark,which also indicated that PmDHN can be transcribed in transgenic A.thaliana.The rate of water loss in leaves of the WT and of the transgenic plants maintained an increasing trend as a function of treatment time.The rate of water loss of the WT plants were higher than those of transgenic plants,indicating that water loss occurred faster in the WT plants than those detached from the transgenic plants(P<0.05).In addition,an increase in proline content was observed in the OP and WT plants under drought stress,and the proline content of the transgenic plants was increased by 7.79% and 15.79% respectively.The MDA content in the WT plants was higher and increased more rapidly than in the OP plants under drought stress,and the MDA content of the WT plants was increased by 4.08% and 11.52% respectively.The contents of proline and MDA were statistically different(P<0.05).The genes were identified as PmAQP?PmCBL?PmDHN?PmERF?Pm GPX6?PmNAC?PmPEPC?Pm POD?PmACTIN genes and had been assigned GenBank.The results showed the genes of PmGPX6?PmPOD?PmDHN had participated in the drought process.