Construction Of A Suppression Subtractive Hybridization Library For Red Skin Mutant Of 'Zaosu' Pear And Expressed Sequence Tag Analysis

Red pear breeding is one of the major domestic and international pear breeding projects. Separation and identification about red skin development-related genes is the basic molecular breeding.In this experiment, red mutant of'Zaosu'and its mother plant were used as the materials, we construct a subtracted cDNA library by the suppression subtractive hybridization (SSH)method.we got differentially expressed clonings and sequenced. These differentially expressed genes were identified and functional analysis. Main conclusions of this paper are as follows:1.In this study, we used red mutant of'Zaosu'and its mother plant as the materials , green pear'Zaosu'as driver, and the red mutant as tester to construct a subtracted cDNA library by the suppression subtractive hybridization (SSH)method. 1039 cDNA library clones were selected, 970 positive clones were chosen from the library,the inserted segments were 250-1000 bp in length.2. After clearing pollution, redundant, low quality(﹤100bp)sequence, according to blast screening and functional annotation in GenBank database, 314 unique genes were obtained. Among the obtained fragments, 203 (64.7%)ESTs were found to have homologous genes with known functions,49(15.6%) ESTs with unknown functions, and 62(19.7%) ESTs with no homologous genes. ESTs were found with no significant similarity which may represent new genes or high variant non-coding regions of cDNAs. 212 ESTs of them had been submitted to GenBank,and the accession number is HO774921-HO774955,HO840420-HO840592 and HS573389-HS573392.3. Results showed that the differentially expressed genes are involved in anthocyanin biosynthesis, signal transduction, energy and metabolism, transcription factors, disease resistance and bud mutation. What's more, those best matched protein were from many plant species, such as pear(Pyrus),apples(Malus x domestica),arabidopsis thaliana (Arabidopsis thaliana),tobacco(Nicotiana tabacum),maize (Maize), ricinus communis (Ricinus communis)and grape(Vitis vinifera ).4.We have found 3 genes related to bud mutation,which are BEL1-like homeodomain transcription factor, retrotransposon protein and chromatin remodeling complex subunit.5. We have found expressed genes which are involved in anthocyanin biosynthesis mainly have MYBR domain class transcription factor ,bHLH ranscription factor, Cytocrome P450 like protein, flavanone 3-hydroxylase, anthocyanin-O-methyltransferas, zinc finger and ARF domain class transcription factor.6. The disease and defence genes obtained as flows: ubiquitin-protein ligase,dehydrin, heat shock protein,leucine rich repeat protein,cysteine proteinase, polyphenol oxidase, defensin protein and elicitors et al.