The Study On Construction Of CDNA Libray Of Trehalose Production Strains

The methods of extract total RNA in this experiment are Heated Acid Phenol. Trizol reagent Classic Total RNA Isolation Kit E.Z.N.A Yeast RNA Kit. The study had compared the four methods on Saccharomyces cerevisiae total RNA isolation in order to obtain high quanlity total RNA. And we used mRNA which purify from total RNA as template, then synthesize double strands of cDNA melocular when AMV enzyme and some other enzymes exist. Next step we can connect the cDNA and link-up adaptor, after that we connect this production to pUC18 vector because they have the same stickness bottom. Using the recombinat plasmid to transform recept cell.take appropriate volumn the transformed Ecoli.DH5 a and extend on solid LB culture that include IPTG ( 20% ) and X-gal ( 2% ) .Consequently we achieve cDNA library of Saccharomyces cerevisiae AS2. 1416.Afterward use the recombinant plasmid as template, design primer according to tpsl sequence on Genbank, process PCR ampliation, further compare PCR production and tpsl to know how much their gene are same.Experiment consequence show:1) quantifing total RNA according to A260 .average quantity through three times are 1.2ug/ul, 0.48ug/ul, 0.324ug/ul, 1.072ug/ul respective by Trizol reagents Classic Total RNA Isolation Kit, E.Z.N.A Yeast RNA Kit.Among them, Heated Acid Phenol and E.Z.N.A Yeast RNA Kit are better than the other two methods, they can gain 36jjig fc 32.16jig RNA per milliliter. And that,the other two methods are 14.4ug and 9.72ug RNA.And the purify of the total RNA by Heated Acid Phenol method is high.the A260/A280 is 1.917 and A260/A230 is 2.034.It can see three clear bands on electrophoresis.2) It can isolate effectively mRNA from total RNA by SA-PMPs.,the quantity is 0.41(ig,the productive level is 1.14%,the average A260/A280 is 1.977,the purity can satisfy the ask of construct high quality cDNA library.3) After the classfication of double strands of cDNA,the quantity is 20.09 ng,the productive level is 4.9%.4) It is the first time that triumphantly construct the cDNA library of Saccharomyces cerevisiae by pUC18 vector.The reunited level of the library is 96%,the capacity is l.l*109,The range of cDNA is between 0.4*103 to 5.5*103bp. 5) Through the analysis of the published tps1 gene.we have designed PCR primer and used the cDNA clone in the library as template, through PCR ampliation we have triumphantly selected and cloned a DNAsequence which is 1.4kb,after the compare of their isogenous we found that the isogeous of this two sequence is 97%.This research has created a foundation of further research of the theory that trehalose compose.At the same time,it has proved that the cDNA library we have constructed is succeeded.