Studies On The Cloning And Evolution Relationships Of Homologous GH120and GH395Genes In Girmmiaceae

Drought is the main environmental factors that affect plant growth and development, ithas become a bottleneck which restricting the development of agriculture in our country.Using of the biotechnology to enhance drought resistance of crops has become an effectiveway to solve the drought problems.Due to the special and extreme resilience,mosses hasbecome a model plant for drought mechanism research and relative gene cloning. Majorityspecies of the Grimmiaceae can survive in harsh environmental conditions.Because they havea strong resistance to resist adversity.Molecular biology researching on Grimmiaceae couldmake a great significance in improving crops and economic crops quality.（1） Cloning the ubiquitin extension protein from Grimmia pilifera for the firsttime.Total RNA was extracted from plant, then reverse transcription to cDNA.According tothe EST sequence of ubiquitin extension protein from drought cDNA library of Grimmiapilifera constructed by our laboratory to design primer. Using3’RACE technology（RapidAmplification of cDNA End） to amplification,GenBank Accession No. JQ659260.Thefull-length cDNA of ubiquitin extension protein gene is673bp,containing a471bp openreading frame（ORF）, encodes a residue of156amino acids with a predicted molecular massof17.8kD. The theory isoelectric point is9.81, it mainly distributed in the cell nucleus andmitochondria matrix. By analysising the homology of encoded amino acid sequence showthat, the deduced amino acid sequence showed more than96%similarity with thecorresponding ubiquitin extension protein fragment of the Physcomitrella patens、Piceasitchensis、Vitis vinifera、Ricinus communis、Populus trichocarpa.（2）In this experiment,the total RNA was extracted from the Rhacomitrunmjaponicum,then reverse transcription to cDNA.And a pair of primers was designed accordingto the conserved regions of the Cu/Zn SOD mRNA from Grimmia pilifera in GenBank.A565bp fragment was got by PCR amplication，GenBank Accession No. JQ659261.The ORFof Cu/Zn SOD gene with465bp and could express a protein of154amino acids.Themolecular weight was15.5kD,and its theoretical isoelectric point was5.77.It has18Negativecharge residues（Asp and Glu）,and12positive charge residues（Arg+Lys）,unstablecoefficient is13.36.The sequence of amino acids encoded Cu/Zn SOD gene contains fiveconserved regions of the protein,all are the Cu-Zn Superoxide Dismutase superfamily.The sequence with Cu/Zn SOD of other plants was blasted on NCBI website,and it was found thatthey had high homology.By the Blastn comparison analysis shows that the fragment showed99%and82%similarities of the related gene with Grimmia pilifera and Physcomitrellapatens.（3）We designed the specific primers according to the conserved region ofGH395,GH120gene.GH395homologous genes were isolated from11species inGrimmia,Rhacomitrunm and Schistidium of Grimmiaceae by RT-PCR amplification，toGH120gene was amplified in18kinds.These homologous genes were highly conserved，thesimilarities at GH395nucleotide level are over80%and such as GH120are over90%.Producing the Phylogenetic trees are based on these homologous sequences,we canspeculate that Grimmia are very closely to Schistidium in the evolutionary process.As a classSchistidium liliputanum is always together with Grimmia elatior,Grimmiaobtusifolia,Grimmia ovalis and Grimmia decipiens,it indicate that their genetic distance arevery closer.