Study On The Mechanism Of Red Development In Malus Sieversii F.neidzwetzkyana (Dieck) Langenf.

Malus sieversii(Ledeb.)Roem.is a remnant of the tertiary species and is also believed to be progenitor of cultivated apple(M.domestica). M. sieversii is rich in genetic diversity. Malus sieversii f.neidzwetzkyana(Dieck)Langenf. is an important plant resource for ornamental and medical function for red coloration of it. Recent years, many researches have been made to clarify the mechanism of red coloration of apple. However, there is few reports on M. sieversii f.neidzwetzkyana. Therefore, M. sieversii f.neidzwetzkyana was used as material to study on the mechanism of red development in M. sieversii f.neidzwetzkyana by HLPC, cloning and transcriptional analysis and AFLP techniques.The main results are shown as follows.1.The anthocyanin composition and content of'xiahongrou'skin and cortex were detected. Cyanidin-3-galactoside was the main anthocyanin composition detected by HPLC. The anthocyanin content of'xiahongrou'skin and cortex displayed a different change trend at different developmental stages. The anthocyanin content of'xiahongrou'skin showed a declined pattern, while the anthocyanin content of'xiahongrou'cortex displayed an opposite tendency.2.The full-length cDNA and gDNA of MsMYB10(GQ500894) was isolated by GenBank-based electronic cloning. It is 3981bp in length. Its ORF was 732bp and encoded a polypeptide containing 492 amino acid residues with a molecular weight of 28.56kDa .The predicted PI of the protein was 8.41.The MsMYB10 has a significant R2R3 domain with conservative tryptophan residues and a transcription activate area rich in acidic amino acids in C end. At protein level, MsMYB10 had 98% identity to MdMYB10 from apples. MdMYB10 had no signal peptide, but had nuclear localization signal. Phylogenetic analysis revealed that MsMYB10 is in the same cluster with apple MdMYB10, which has been proved to regulate anthocyanin synthesis. The genomic DNA sequence of MsMYB10 contained 3 exons and 2 introns. Two introns with length of 256 bp and 2994 bp, respectively, were inserted into the ORF, Three exons with length of 122 bp,132bp and 477 bp, respectively.3. Anthocyanin synthesis is related to the expression level of MsMYB10. Real time quantitative RT-PCR analysis indicated that the expression of MsMYB10 in skin and cortex of'xiahongrou'was very high, and the expression of MsMYB10 showed an increasing pattern in skin and a'high-low-high–low'change trend in cortex during fruit development. While the expression level of MsMYB10 in'bairou'was very low and few the expression of MsMYB10 was in cortex. In addition, MsMYB10 constitutively expressed at different levels in tissues and organs tested. Compared with other tissues, fruit skin produced the highest transcript level. While,leaves produced the lowest transcript level.4.Analysis of the relationship between anthocyanin related genes and apple coloration. The transcripts of anthocyanin related genes MsCHS,MsCHI,MsUFGT,MsDFR1,MsDFR2,MsF3H,MsLDOX were detected by QPCR. The results showed that there is a significant different expression of anthocyanin genes between'xiahongrou'and'bairou'. At the early developmental stage, the expression level of these genes were higher in'xiahongrou'than in'bairou'. Significant difference of the expression of MsCHS,MsCHI,MsLDOX,MsUFGT was observed in the cortex of both varieties. The different expression level of them maybe the molecular basis of differential pigmentation of'xiahongrou'and'bairou'in cortex. The expression of MsDFR1,MsDFR2,MsLDOX was obviously different between'xiahongrou'and'bairou'in skin. The different expression level of them maybe the molecular basis of differential pigmentation of'xiahongrou'and'bairou'skin.5. MsMYB10 was cloned from Malus and expressed in E.coli. The recombinant prokaryotic expression vector pET30a-MsMYB10 was constructed by inserting the cDNA fragment into the prokaryotic expression vector pET-30a,and then transformed into E.coli BL21(DE3). The SDS-PAGE displays that the expressed proteins consistent with the size of 28.8kd expected protein.6. Red-linked AFLP markers were screened. Differentially expressed fragments were amplified by 64 pairs of primer combinations from DNA pools of which only 2 were polymorphic between 2 DNA pools.Whereas,only the 300 bp-sized polymorphic band amplified with the primer conmbination EcoRI-ACC/MseI-CAG showed consistent difference between 2 gene pools.The fragment existed in all 10 extremely red hybrids but not in 10 extremely green,suggesting a linkage to the low-acid trait.