Establishment Of CDNA-AFLP System And Cloning Of UFGT Gene For Strawberry Fruit

Strawberry(Fragaria ananassa Duch)is a major berry fruit which is plantedwidely around the world, and well received by producers and consumers for its earlymaturity, brilliant color, high nutrition and fragrant flavor.'Fa Lan Di' is one maincultivar grown under open-air or in the greenhouse in the South.In this experiment, the relationships among the content changes of anthocyanin,polyphenols, flavonoids and sugar acids during the ripening of 'Fa Lan Di' werestudied. And differential expressed cDNAs were separated from different ripeningstrawberry fruits, using the technique of cDNA-AFLP. The full-length cDNA of UDPglucose-flavonoid3-o-glucosyl transferase(UFGT) which is the last key enzyme in theanthocyanin pigments biosynthetic pathway was cloned by RACE. The main role ofUFGT is to make anthocyanin stabile by means of combining unstable anthocyaninwith glycosyl. Therefore, the cloning of UFGT played an important role on thecontrol of anthocyanin biosynthesis and exploitation of anthocyanin in strawberryfruit.1. The content changes of anthocyanin, polyphenols, flavonoids and sugar acidswere compared in different ripening fruits of 'Fa Lan Di':15days after flowering,25days after flowering,30days after flowering,35days after flowering. The resultsshowed that acidity decreased and sugar increased, and anthocyanins accumulatedgradually along with the maturing of fruits. The contents of polyphenols andflavonoids kept a high level on green ripe stage, then declined and rised up to redmature stage.2. The reaction system of cDNA-AFLP was optimized, and differentialexpressed cDNAs connected to the color of fruit were separated. The result showedthat the final concentration in selective ampliation reaction system was30ng ofcDNA,0.1mmol/L of dNTP,0.5μmol/L of primers,2μl of buffer,12.6μl of ddH2Oand1U of Ex-taq polymerase in the total volume of20μl, the amplification programwas as follows: Advanced-denaturing at94℃2minutes, denaturing at94℃30 seconds, annealing at65℃30seconds(each cycle was reduced by0.7℃), extendingat72℃80seconds(12cycles), denaturing at94℃30seconds, annealing at56℃30seconds(25cycles), extending at72℃7minutes after the last cycle was completed.3. False positive of the separated different expressed TDFs were verificatedusing the technique of reverse Northern hybridization, and33positive differentialexpressed cDNAs were obtained. Sequence homology analysis showed that thecDNAs involved in RNA transcription and translation, protein synthesis andtranslation and post-translational modification, protein aging, the post-modification ofterpenoids formation and so on. In these sequences, N-acetyltransferase(Y1E0M3),short-chain alcohol dehydrogenase(Y3E5M4), NAD+synthase(Y3E5M4) andglycosyltransferase enzyme(Y3E0M1) were closely related to the coloration ofstrawberry fruit.4. A complete sequence of1122bp named UDP glucose-flavonoid3-o-glucosyltransferase(UFGT), containing an ORF851bp, encoding276amino acids.759bp5'-end sequence and331bp3'-end sequence, with high homology with various plants'UFGT.