Cloning And Analysis On PmSnRK2s Genes Form Pinus Massoniana

The Masson pine?Pinus massoniana?,is distributed widely and a unique native tree species in China.It has important economic and ecological values,and excellent resistance ability that is particularly resistant to infertility.However,the the mechanism research on the drought resistance of Masson pine is rarely reported.This thesis mainly studied three genes of PmSnRK2.3,PmSnRK2.6a and PmSnRK2.6b,which play an important role in drought resistance.The results are as follows:?1?The complete cDNA sequences of PmSnRK2.3,PmSnRK2.6a and PmSnRK2.6b were obtained by homologous cloning and rapid amplification?RACE?.The lengths were 2356bp,1985bp and 1674bp,respectively.362,356 and 362 amino acids are encoded.The physicochemical properties of the above three genes were analyzed by online software ProtParam.The results showed that the molecular formula of PmSnRK2.3 protein was C₁₈₁₂H₂₈₂₅N₄₉₅O₅₅₀S₁₆,the molecular weight was 40857.31Da,the molecular formula of PmSnRK2.6a was C₁₇₇₂H₂₇₇₃N₄₈₁O₅₄₅S₁₆,the molecular weight was 40048.37Da,and the molecular formula of PmSnRK2.6b was C₁₇₉₄H₂₇₈₈N₄₈₈O₅₅₉S₂₁,the molecular weight was 40810.07Da.And except the PmSnRK2.6b is unstable hydrophilic protein,the rest genes are stable hydrophilic protein.?2?The real-time fluorescence quantitative expression analysis of adult parts of Pinus massoniana showed that the lowest expression organ of PmSnRK2.3 was the new stem,the highest expression organ was the flower,and the expression level of the gene in the flower was1.34 times as much as the old leaf;the lowest expression organ of PmSnRK2.6a was the old stem,and the highest expression organ was flower,and the expression level of this gene in flower was4.5 times as much as the old leaf;the lowest expression organ of PmSnRK2.6b was the old stem,the highest expression organ was the flower,and the expression level of the gene in the flower was 2.9 times as much as the old leaf.The results showed that the PmSnRK2.3,PmSnRK2.6a and PmSnRK2.6b all showed different degrees of expression in various organs of adult Pinus massoniana,and the expression in flowers was the highest,while the stems were the tissues with lower expression.30 days old Pinus massoniana seedlings were hydroponically cultured in 10%PEG6000 and ABA solution,and seedlings at 0h,0.5h,1h,2h,4h,8h,16h and 24h were used as materials for real-time quantitative fluorescence analysis and expression.It was concluded that peaks appeared at 2h and 24h under PEG6000 treatment.The highest expression level was about2.3 times as much as 0h and the lowest was 1.1 times as much as 0h.Under ABA treatment,peaks appeared at 1 h and 24 h,and the highest expression level was 2.8 times of 0 h,and the lowest was about 1 h of 0 h.?3?The GUS fusion expression vector of PmSnRK2.3 was constructed by homologous recombination,and the transgenic plants were obtained after infecting the leafs of tobacco by Agrobacterium tumefaciens.The differences between wild-type and transgenic tobaccos were analyzed by genomic PCR,GUS staining phenotypic analysis,drought simulation,and physiological indicators determination.After drought treatment,both the phenotypes of the plant growth potential,leaf and root,and the physiological indicators were analyzed.The results all showed that the drought resistance of the transgenic tobacco was stronger than that of the control group.The results of this dissertation can help to reveal the drought-resistance mechanism of Pinus massoniana and help the excavation and utilization of resistance genes.