Functional Characterization Of Gene CpEXP2

Expansin gene family is a kind of a huge gene family.It is mainly divided intoα-expansin andβ-expansin two categories,both of which have a lot of members.It posesses the existence of a broad variety of plants.A large number of expansin genes have been cloned recently.The expansin encoding protein belongs to the cell wall zymoprotein,expressed in the plant with a high degree of specificity.Its expression and regulation was controlled by the plant's own development process,as well as exogenous hormone regulation and control of environmental factors.Study shows that,Expansin gene play an important role in plant seed germination,root growth,stem,leaf growth and development,fruit ripening and softening,abscission of organ,etc.It has almost participated in the entire process of plant growth and development.Based on the previous studies in this laboratory,in this study,we have cloned another Chimonanthus Praecox expansin gene,named CpEXP2.We have finished the construction of the plant expression vector through Agrobacterium-mediated transformation of Arabidopsis thaliana,obtaining T1-generation transgenic plants;We also have constructed prokaryotic expression vector,expressing CpEXP2 in E.coli. The main findings are as follows:1.Cloning of CpEXP2We have found the eDNA sequence with the size of the 1115bp,by rough randomly selection from cDNA library sequencing.Sequence analysis showed that the gene contains an ORF frame of 771bp, encoding 257 amino acids.BLASTX comparison showed that theα-expansin homology higher prima facie by the expansin of the cDNA encoding proteins Expansin,named CpEXP2.2.Construction of Plant Expression VectorCpEXP2 gene is in the eDNA library cloning vector pTriplEx2,activate bacterial liquid respectively including pTriplEx2 and pMD-18TM and extract plasmid,then digest plasmid by restriction endonuclease Sill.Recycle target gene and then link to cloning vector pMD-18TM.The recombinant plasmid pMD-18TM-CpEXP2 is.successfully constructed and confirmed by restriction endonuclease digestion and PCR.Digest pMD-18TM-CpEXP2 by double restriction endonuclease XbaI,SmaI,then recycle the target gene and link to expression vector pC2301.The identification of PCR and enzyme cutting show that the construction of the recombinant pC2301-CpEXP2 plasmid could be confirmed. 3.The genetic transformation of Arabidopsis thalianaWe transplanted the Chimonanthus Praecox expansin gene CpEXP2 into Arabidopsis by Agrobacterium tumefaciens.Through the Km+ resistance screening detection and PCR amplification.It proves that the initial CpEXP2 gene has been integrated into the Arabidopsis genome primarally and transgenic plants is growing normally.4.Construction of prokaryotic expression vectorDesign the primer with the BamHI and HindⅢsites,then cloned the CpEXP2 gene through PCR.Digest the CpEXP2 and prokaryotic expression vector PET-28a by double restriction endonuclease BamHI and HindⅢ,then recycle the target gene and link to expression vector PET-28a.Digestion and PCR verification showed that the prokaryotic exprssion vector with the purpose of gene——PET-28a-CpEXP2 has been successfully constructed.5.Prokaryotic expressin vector PET-28a-CpEXP2 expressed in E.coli BL21Under the propriate induce condition,we tranplanted the prokaryotic expressin vector into E.coli to induced CpEXP2 gene'expressing in E.coli BL21,and then proved the expression of CpEXP2 through the SDS-PAGE detection on the recombinant protein.