Cloning And Expression Vector Construction Of GpSRORP、GpPOD、GpMCMF Gene Of Grimmia Pilifera

Grimmia pilifera belongs to the Grimmiaceae Grimmia,it is a sort of typicaldrought-tolerant moss as an excellent anti-drought genetics resource. This study selected threegenes associated with drought from Grimmia pilifera drought stress cDNA library,they arestress response redox protein gene, peroxidase gene, F-H~+-ATPase gene of mitochondrialinner membrane and thylakoid membrane,named as GpSRORP,GpPOD,GpMCMFgene,respectively. we obtained full length cDNA sequence of GpSRORP,GpPOD genethrough3’RACE clone,to get which, analysed these three genes by bioinformatics methodsand constructed expression vectors. The results were:(1) The total RNA of Grimmia pilifera with good integrity, high purity that could beperformed for further experiments.(2) The cDNA reverse transcripted by Grimmia pilifera total RNA was used as templates,three pairs of primers (F1, R1, F2, R2, and F3, R3) were designed for GpSRORP, GpPOD,GpMCMF full length cDNA sequences,the target PCR amplification products wereobtained,the lenth were about1210bp,580bp,470bp respectively, PCR amplification productswere ligated to pMD-18T simple vector, transformed and obtained positive clones. Plasmid ofpositive clones were extracted and detected by PCR and restriction enzyme digestion.Aftersequencing, the similarity between the clone sequence and the original sequence was ashighly as99%, proving that the cloning vector pMD18T-GpSRORP,pMD18T-GpPOD andpMD18T-GpMCMF were constructed successfully. Using bioinformatics software analysis ofthe open reading frame,the physical and chemical properties, hydrophobic and hydrophobicityransmembrane region, secondary structure of the protein, gene family of these three genes.(3) Real-time quantitative PCR was used for identification of expression profiles ofGpSRORP,GpPOD,GpMCMF genes, The results showed that the expression Patterns of thosegenes were different,and preliminary proved the expressions of GpSRORP,GpPOD,GpMCMFgene were all induced by dehydration or rehydration.(4) Using restriction enzyme BamH Iand Nde I for double digests of recombinantplasmids to get the target fragments,the target fragments were ligated with plant expressionvector pRI101-AN by T4ligase to obtain new expression vector pRI101-GpSRORP,pRI101-GpPOD and pRI101-GpMCMF.Expression vectors were checked by double digests,after electrophoresis,clear bands of about1200bp、580bp and470bp were recovered andsequenced,the results showed that the sequences target fragments were99%,similar to the original sequences, proved that target genes were correctly ligtated to expression vectors,Transformed them into competent Agrobacterium tumefaciens EHA105, positive clonescontaining pRI101-GpSRORP, pRI101-GpPOD, pRI101-GpMCMF were checked byPCR,then the GpSRORP, GpPOD, GpMCMF gene expression vectors were successfullyconstructed. The plasmids were transformed into tobacco plantsme diated by Agrobacteriumtumefaciens EHA105, obtained on callus and shoots of tobacco.It provided the foundation forfurther genetic researches of GpSRORP,GpPOD, GpMCMF gene.