Cloning And Expression Of Genes Related To Pear Skin Color Mutation In ’Early Red Doyenne Du Comice’ And Primary Study On Function Analysis Of PCMYB2

Color is one of the important factors of fruit appearance quality. The fruits with bright color show higher sales in the market. Usually, the pear germplasm resources can be divided into green, brown and red three types by the color of peel. The red pears has become so popular in generants and consumers because of beautiful appearance, high economic benefit, and high nutritional value. In recent years, it’s a hot spot to cultivate high-quality red pear varieties and pear germplasm in breeding new red pear varieties becomes an important research. In this thesis, the differences of anthocyanin contents, gene sequences, and gene expressions between’Early red Doyenne du Cornice’(Pyrus communis L.) and it’s green variant strain were studied from the physiology and molecular biology levels. The Solexa/Illumina RNA-seq technologies, high-throughput sequencing were used to investigated the different expressed genes between the red and green pears, expected to reveal the differences between the variation of color varieties and the original varieties in molecular level, so as to lay the foundation of the molecular mechanism of red skin formation. The main results are as follows:1. The full-length cDNA of the seven anthocyanin biosynthesis genes (PcPAL, PcCHS, PcCHI, PcDFR, PcF3H, PcANS, PcUFGT) and partical length cDNA of the three transcript factors (PcMYB10, PcbHLH, PcWD40) were cloned in both the cultivar’Early red Doyenne du Comice’and its green variant strain, the accesssion numbers submitted to NCBI as KC460392, KC460393, KC460394, KC460395, KC460396, KC460397, KC460398, KC993864, KC993865, and KC993866. However, there was no sequence difference between the color mutants, which means that the skin color change was not caused by gene mutation. These sequences provide the basic information for the study of the expression of anthocyanin biosynthesis genes.2. The anthocyanin contents of the cultivar ’Early red Doyenne du Cornice’ and its green variant strain were determinationed at different development stages. the expression levels of these seven genes were examined by quantitative real-time PCR (qRT-PCR). Results showed that the anthocyanin contents of ’Early red Doyenne du Cornice’were much higher than the green variant strain and most of the structural genes were upregulated in the red-skin cultivar during fruit development. The expression levels of the transcription factors PcMYB10, PcbHLH, and PcWD40 were also investigated by qRT-PCR, and showed the significant correlation with the’Early red Doyenne du Cornice’ anthocyanin contents. But the expression levels of the transcription factors showed no significant correlation with the green variant strain. It means the three transcript factors coordinated works on the anthocyanin biosynthesis.3. This study investigated the transcriotome profiles of pear using the Solexa/Illumina RNA-seq technologies, high-throughput sequencing, assembly, and annotation of expressed sequences from pear were obtained. Analysis of differential expression in red fruit compared to green fruit revealed 1680 up-regulated genes and 550 down-regulated genes. A total of 4,406 differentially expressed genes between the red and green fruit were found using the Blast2GO program associating with 757 Kyoto Encyclopedia of Genes and Genomes (KEGG) defined pathways. There were two differentially expressed genes in flavonoids metabolic pathway, C3H and ANR. In addition to the known MYB transcriptor family, there were three new family of transcription factors (AP2, WRKY, MADS) showed correlation with anthocyanin biosynthesis. Quantitative real-time PCR was used to confirm the digital transcript abundance measurements results with 12 selected differential expressed genes. The results of qRT-PCR showed the same tendency with digital transcript abundance measurements, the range of correlation coefficients was from 0.822 to 0.993.4. Compared with pear genome database (http://peargenome.njau.edu.cn) in the sequence comparison,10 important MYB factor in Pear were found by the closely related species MYB gene sequences. The expression levels of the 10 MYB factors were confirmed by the Quantitative real-time PCR. The gene Pbr028725.1 was obtained via RT-PCR cloning, containing an open reading frame (ORF) of 609bp and encoding 202 amino acids with protein molecular weight (Mw) 22.8 Kd and isoelectric point (pi) 9.11. The gene Pbr028725.1 was overexpressed in tobacco via Agrobacterium tumefaciens-mcdiaied transformation, and four independent transgenic lines were obtained.