Cloning And Analysis Of Four Functional Genes In Pinus Koraiensis

Pinus koraiensis Sieb.et Zucc.mainly exist in Northeast mountain area in China.It has precious value of economy and ecology.cinnamoyl alcohol dehydrogenase?CAD?catalyze the second part of Lignin reduction reaction.That is transform three kinds of cinnamaldehyde,caffeic aldehyde,coniferyl aldehyde,5-hydroxy coniferaldehyde,into caffeol,coniferol,5-hydroxy coniferol.Ent-kaurene synthase?KS?catalyze the reaction of CPP synthetise ent-kaurene in the reaction of gibberellin synthesis pathway.It is a key step in early stage of gibberellin synthesis pathway.GA20-oxidase?GA20?work with GA3,GA2,and transform GA12-aldehyde into all kinds of GA that has biological activity.Chitinase?Chi?in plants mainly work as adversity resistant protein for low temprature and insect pest.By researching these kinds of enzyme in Pinus koraiensis,we can make a contribution to the research of promoting the output and productivity of seed and wood.In this experiment we get CAD,KS,GA20,Chi gene in Pinus koraiensis by cloning,then link these gene to carrier respectively and express in the EscheriChia coli BL21.Laid the foundation of promoting the resistance to disease,output of seed and wood.The results are as follows,1,The Pinus koraiensis CAD gene was cloned and sequenced.The result shows that the length of Pinus koraiensis CAD gene fragment is 1074bp,encoded protein contains 357 amino acids.The molecular weight is 38.9kd,the molecular formula is C₁₇₁₄H₂₇₄₁N₄₅₅O₅₁₇S₂₉,and the isoelectric point is 5.80.The results of multi sequence comparison with other species CAD gene show that Pinus koraiensis CAD gene sequence has high consistency with Pinus taeda and Picea abies.The results of phylogenetic tree analysis showed that the closest relationship with Pinus koraiensis CAD gene was Pinus radiata and Pinus taeda.Express Pinus koraiensis CAD gene in Escherichia coli BL21 cells and the result of SDS-PAGE electrophoresis shows that engineering bacteria express a protein with a length of about 39kD,agree with the theoretical expectation.2,The Pinus koraiensis GA20 gene was cloned and sequenced.The result shows that the length of Pinus koraiensis GA20 gene fragment is 1116bp,encoded protein contains 371 amino acids.The molecular weight is 42.03kd,the molecular formula is C₁₈₉₃H₂₉₁₁N₅₁₁O₅₄₃S₁₆,and the isoelectric point is 6.89.The results of multi sequence comparison with other species GA20 gene show that Pinus koraiensis GA20 gene sequence has high consistency with Pinus tabuliformis and Picea glauca.The results of phylogenetic tree analysis showed that the closest relationship with Pinus koraiensis GA20 gene was Pinus tabuliformis and Pinus densiflora.Express Pinus koraiensis GA20 gene in Escherichia coli BL21 cells and the result of SDS-PAGE electrophoresis shows that engineering bacteria express a protein with a length of about 42kD,agree with the theoretical expectation.3,The Pinus koraiensis KS gene was cloned and sequenced.The result shows that the length of Pinus koraiensis KS gene fragment is 2283bp,encoded protein contains 760 amino acids.The molecular weight is 85.96kd,the molecular formula is C₃₈₁₅H₆₀₀₆N₁₀₅₀O₁₁₃₁S₄₀,and the isoelectric point is 6.10.The results of multi sequence comparison with other species KS gene show that Pinus koraiensis KS gene sequence has high consistency with Picea glauca.The results of phylogenetic tree analysis showed that the closest relationship with Pinus koraiensis KS gene was Pinus radiata and Pinus densiflora.Express Pinus koraiensis KS gene in Escherichia coli BL21cells and the result of SDS-PAGE electrophoresis shows that engineering bacteria express a protein with a length of about 86kD,agree with the theoretical expectation.4,The Pinus koraiensis Chi gene was cloned and sequenced.The result shows that the length of Pinus koraiensis Chi gene fragment is 831bp,encoded protein contains 276 amino acids.The molecular weight is 28.27kd,the molecular formula is C₁₂₂₀H₁₈₇₄N₃₄₀O₃₉₃S₂₁,and the isoelectric point is 8.64.The results of multi sequence comparison with other species Chi gene show that Pinus koraiensis Chi gene sequence has high consistency with Picea glauca and Picea abies.The results of phylogenetic tree analysis showed that the closest relationship with Pinus koraiensis Chi gene was Picea sitchensis and Picea glauca.Express Pinus koraiensis Chi gene in Escherichia coli BL21 cells and the result of SDS-PAGE electrophoresis shows that engineering bacteria express a protein with a length of about 28kD,agree with the theoretical expectation.