Grimmia Pilifera GH394Gene Cloning And Expression Analysis Ol Bioinformatics And Construction

The plant is one of the pioneers of the natural environment。In the present stage,relatedresearch of drought tolerance mosses and genetic studies and expounds of the function are notcomplete. GH394genes are selected from the Grimmia pilifera p. Beauv drought stresscDNA library of our laboratory which are constructed by the technology of SMARTapplication. By the contrust of Blast matching, enzymes and Glutathione-s-transferase (GST)sequence similarity to89%, preliminary draw the conclusion that GH394gene of Grimmiapilifera P. Beauv genes are related with drought. According to the GST sequence designprimer reported in the NCBI, research by constructing GH394gene cloning and expression ofthe carrier, gene function test eukaryotic laid a good experimental basis on the subsequentgene expression. This study of the method and the results are as follows:1.Use the improved SDS law to extract materials total RNA. Get clear18S,28S RNAstrip, use (NanoDrop2000) test, OD260/280at1.8-2.0rooms, comply with the requirements.2.Through the3’RACE for the whole length of the gene sequence, design and GST thathas been reported according to the NCBI sequence specific primer, the use of gene sequencesDNAMAN6.0GH394enzyme cut analysis, confirmed the gene sites can be inserted BamHⅠ and SacⅠ. The material for the total RNA transcription chain-free cDNA, andsingle-strand cDNA for template PCR primers. Get clear strip, position and expected657bpin size, recycling purpose strip.3.Using bioinformatics software: ORF-dimensional structure, physicochemicalproperties of proteins, amino acids, secondary structure, the gene family analysis.4.The purpose of the recovery and pMD18pieces-T Vector link, product into e. coli(Escherichia coli) DH5α, inspection bacteria, sequencing, extract the plasmid enzyme cut.Recycling purpose extract.5.The carrier of the pRI101-AN enzyme is cut, large fraction of the recovery. Use T4connection enzyme will purpose extract and carrier larger pieces connected, product into e.coli to repair, again into the root cancer agrobacterium-mediated (Agrobacterium tumefaciens)GV3101, inspection bacteria, through the plasmid PCR evaluations.Through the sequencing result and electrophoresis, enzyme and image shows that hassuccessfully constructed pMD-GH394cloning vector and pRI101-AN-GH394expressionvector and will be restructured plasmid load into the purpose of strains.