Construction Of CDNA Library Of Pichia Stipitis And Its ESTs Sequencing

Xylose which accounts for 30% of lignocellusic hydrolysates being fully used is essential for decreasing the cost of ethanol producing. Pichia Stipitis is the most prospect yeast which can ferment xylose to ethanol and has been studied extensively, but at present little is known about its physiology mechanism. EST ( Expressed Sequence Tag ) technology based on cDNA library are efficiency approach to analyze gene function and to discover new genes. In this research we constructed a cDNA library of P5776( Pichia Stipitis CBS 5776 ) and sequecing its 5′-ESTs. These work offer basic resources for gene analyze, for xylose-metabolism study and for gene screen. Yeasts which have different growing-rate were used to extract total RNA, the results indicated it was easy to obtained total RNA when cells were in logarithmic phase( OD₆₀₀ is about 2.1 ). Four methods were used to extract total RNA in this research, they were homogenizing, beading, liquid-Nitrogen grinding, liquid -Nitrogen freezing adding liquid-Nirtogen grinding respectively. And two main reagant were used, one was TRIZOL( a mono-phasic solution of phenol and guanidine isothiocyanate ), the other was a protein-denaturant( mainly composed of β-mercaptoethanol and guanidine isothiocyanate ). The two reagent maintained the integrity of the RNA while disrupting and dissolving P5776 cell components. Addition of chloroform and phenol followed by centrifugation, separated the solution into an anqueous phase and an organic phase, then the RNA remained in anqueous phase were percipitated by isopropyl alcohol and washed by ethanol for ready-to-use. All these reaserch showed grinding yeast cells to powder using liquid-Nitrogen after immediately freezing also by liquid-Nitrogen could greatly improve the efficency of RNA extracting and the quanlity of RNA. By this method, compared with protein-denaturant reagant, about 1.2mg intact total RNA was obtained from 900mg(dry weight) yeast cells in this research which was enough to construct cDNA library. The total RNA yield reached 1.3‰. Total RNA was further purified to obtain mRNA( messager RNA ), mRNA were synthesised to the first strand of cDNA with RT( Revrese Transcriptase ), further changed to double-cDNA strands using "nick-translate "method. The cDNA were fractionated by electrophoresis and the fragments which were longer than 500bp were renovated by gel-purification and ligated to the vector to transform the host by electroporator. Finally the cDNA library of P5776 was constructed. The titer of the primary cDNA library reached 1.0×106pfu/mL, and the clones of amplified library reached 1.25×109. Insert fragments with length exceeding 300bp comprised 71% of recombinants. All index proved that the P5776 cDNA library constructed in this research had high quanlity and could be used for further ESTs sequencing and analyzing. The amplified library of P5776 was screened on white-blue plates, and more than 5,000 white-clones were selected to culture in 96-well plates, then the high quanlity plasmids of white-clones were extracted by traditioal alkaline-lysis method using the Vitagene?96-easy plasmid extract kit. Through PCR( Polymerase Chain Reaction ) primered by T3( the vector has T3 RNA polymerase promoter ) , the target fragments of inserts were get. Then the inserts were taged by four different fluorescence-dye, and fractionated by capillary in ABI Prism? sequencer. More than 5,000 5′-ESTs had been sequenced and through rough selecting, 4,747 ESTs were remained for further research. Among the remained ESTs, more than 90% have longer lenth than 500bp, and there are less than 5% unable-read bases in every sequence. The main work in future is to analyze the ESTs data and to prepare DNA clip. All is the foundation work for bioinformation analyze of xylose-ferment yeast.