Functional Analysis Of R2R3 MYB Transcription Factors About Coloration In Red Pear

In order to figure out the mechanism of the anthocyanin biosynthesis pattern in pear and lay a foundation of further study on the information of the regulatory network of anthocyanin biosynthesis in pear fruits, we cloned anthocyanin biosynthesis related MYB transcription factor genes which were screened by phylogenetic analysis in the former study of our lab.The main results are showed as follows:1. The full-length sequence of three putative flavonoid-related MYB transcription factors was isolated from ’Zaosu’ and the red mutant using homologous cloning. In ‘Zaosu’pear and its red mutant, the promoter of PbMYB10 b has two similar sequences, the promoter of PbMYB9 and PbMYB3 has three similar sequences. The full-length of PbMYB10 b gene containing two introns was 2379 bp. The lengths of the two introns were 104 bp and 1588 bp,respectively. The full-length of PbMYB9 gene containing two introns was 1191 bp. The length of the two introns was 119 bp and 190 bp, respectively. The full-length of PbMYB3 containing single intron was 978 bp. The length of the intron was 207 bp.2. In order to establish and optimize the pear fruit transient expression system, we selected the Agrobacterium strain GV3101 containing the pCambia1301 binary vector to conduct the transient overexpression assay of GUS reproter gene in fruit of Chinese pear cultivars ‘Dangshan’. The key factors affecting the effect of infection, which include the injection volume of bacterial suspension and the Agrobacterium infiltration buffer, were analyzed in this paper. Results show that the optimal injection volume of bacterial suspension was 200?l and the optimal Agrobacterium infiltration buffer was buffer A(Terminal concentration: 5% Glucose, 50 m M MES, 2m M Sodium phosphate, 0.1mM AS).3. The complete cDs of the candidate MYB TFs genes, PbMYB10 b, PbMYB9 and PbMYB3, were fused to the CaMV 35 S promoter in the pCambia1301 binary vector. The transient overexpression assay was carried out in ‘Zaosu’ pear. Results show that PbMYB10 b promoted the expression of PbDFR. It was a specific regulator of the anthocyanin pathways.The expression level of PbANR and PbUFGT1 were up-regulated in the PbMYB9 overpression fruits. PbMYB9 was an important regulator of anthocyanin pathways. PbMYB3 could activate the expression of PbUFGT1, PbFLS.4. The candidate genes were transferred into the left of ‘Zaosu’ pear via agrobacteriummediation.