Construction of Suppression Subtractive Hybridization(SSH) cDNA Library from Two Developmental Stages of Fragaria× ananassa Fruit and Expression Analysis of Related Genes
Abstract：Identification of genes differentially expressed during development, ripening and softening of strawberry fruit is needed to improve strawberry fruit quality and to extend shelf life. In this study a forward and a reverse suppression subtraction hybridization(SSH) cDNA libraries with 516 and 524 positive clones were constructed respectively using cDNA from strawberry(Fragaria×ananassa Duch. cv.Toyonoka) and large green stage fruit as the tester and cDNA from strawberry turning stage fruit as the driver. After removing repeat and redundancy sequences, 707 uniESTs including 369 uniESTs in forward library and 338 uniESTs in reverse library were obtained. The assembling provided a total of 123 contigs and 584 singletons by cluster analyses of the uniESTs. Nucleotide homology searched with Blastn in NCBI non-redundant nucleotide database and 537 uniESTs (75.95% of total uniESTs) were homologous with known genes. Protein homology searched with Blastx in NCBI non-redundant protein database and 509 uinESTs (71.99% of total uniESTs) were homologous with known proteins. The results of gene ontology(GO) annotation showed that 193 uinESTs were involved in biological process with 315 times, molecular function with 332 times and cell component with 409 times respectively. Among these ESTs, putative proteins were related to cell vegetative growth, division, early embryonic development and nutrition transport in large green stage library, and related to metabolites biosynthesis and cell wall-related proteins during fruit softening, pigment synthesis, seed maturation in turning stage library. These results were generally consistent with physiological processes during strawberry fruit development and ripening. In two SSH libraries six genes including ripening-related protein, polygalacturonase(PG)，MYB transcription factor(MYB)，isoflavone reductase-like gene(IRL)，later embryo abundant protein(LEA)and ethylene receptor Etr1(Etr1) genes were analyzed by qRT-PCR in different tissues and stages of fruit development and ripening. The information generated in this study provides new clues to aid the understanding of the development and ripening process in Fragaria×ananassa fruit.
