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Study on the Mechanism of Red Development in Malus Sieversii f.neidzwetzkyana (Dieck) Langenf.

Author: WangYanLing
Tutor: ChenXueSen
School: Shandong Agricultural University
Course: Pomology
Keywords: Xinjiang red meat Apple Anthocyanin Transcription factor Structural gene AFLP molecular marker
CLC: S661.1
Type: PhD thesis
Year: 2011
Downloads: 212
Quote: 3
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Abstract


Xinjiang wild apple (Malus sieversii Roem1) case the Tertiary the larvae relic species, is the ancestor of modern cultivated apple (Malus domestica Borkh1) genetic diversity. The Xinjiang Red meat apple (Malus sieversii f.neidzwetzkyana (Dieck) Langenf) Xinjiang wild apple variant of its branches, leaves, flowers, fruit peel and flesh are red, rich in anthocyanin, has a high ornamental value and rich healthcare value. In recent years, the foreign study the molecular mechanisms of Apple coloration been a great breakthrough, but, at home and abroad to see of Xinjiang red meats Apple coloring mechanism never reported. This study as test materials to the Xinjiang Red meat Apple 'summer red meat' determination of the anthocyanin component of skin, pulp, clone red developmental MYB transcription factor; white meat wild apples control, anthocyanin anabolic gene expression patterns were analyzed, and the determination of the anthocyanin content of the corresponding period; 'the summer red meat' x 'Red Fuji' (Spur Fuji) hybrids segregating populations and their parents as test materials , screened AFLP molecular markers tightly linked to red traits aimed at mining and Xinjiang of red meats Apple red form functional genes using genetic engineering, is further proven apple red meat molecular mechanisms, with ornamental fresh and The health features new varieties of apple red meat to lay a theoretical foundation. The main results are as follows: 1. Determination of the 'summer red meat' peel, pulp anthocyanin major component and content. HPLC method detected Xinjiang red meat apple peel, pulp anthocyanin component cyanidin-3 - galacto-glucoside (Cy-3-gal). Use of spectrophotometric determination of the 'summer of red meat' different developmental stages of skin and flesh anthocyanin, anthocyanin content in the pulp and peel trend significantly different, with the development of the fruit peel flower anthocyanin content decreased anthocyanin content in the pulp upward trend. 2 been isolated regulation of anthocyanin synthesis the transcription factor MsMYB10 gene cDNA and gDNA full length. Wherein, cDNA GenBank accession number GQ500894 ORF of region of 732 bp, encoding a polypeptide containing 244 amino acids, predicted PI 8.41, a molecular weight of 28.56 kDa. MsMYB10 having R2R3MYB domain, structural domain conserved tryptophan residue in the C-terminal acidic amino acid-rich transcription activation. With the known MdMYB10 amino acid sequence homology up 98% no signal peptide MsMYB10, has a nuclear localization signal. Phylogenetic analysis showed that, the synthesis MsMYB10 regulation of anthocyanin transcription factor MdMYB10 kinship, in the same clade. The total length of 3981bp of MsMYB10 gDNA, cDNA sequence of ORF region containing two introns and exons, introns, a length of 256 bp intron length of 2994 bp. Two introns the MsMYB10 length cDNA is divided into three exons, length of 122 bp, 132 bp and 477 bp. Closely related to the expression of 3.MsMYB10 Apple anthocyanin synthesis. Using real-time quantitative PCR technology, Xinjiang wild apple 'the summer red meat' and 'white meat' as test materials, determination of fruit development period the regulation of anthocyanin synthesis in apple transcription factor MsMYB10 of expression. The results showed that: the MsMYB10 in 'summer of red meat' expression levels in the peel and flesh is very high, the peel MsMYB10 expression amount showed an increasing trend as ripening, the pulp MsMYB10 expression amount was' high - low - high - Low 'change in trend; expression in control' white meat 'Apple is very low, almost no expression pulp, peel MsMYB10 trend similar to' summer red meat ', as the fruit matures, MsMYB10 expression The sharp rise. 'Summer red meat' and 'white meat' the apple flesh MsMYB10 expression level differences lead to the anthocyanin coloration differences. Measured expression MsMYB10 'summer red meat' of twigs, leaves, peel, pulp. The results found that: the highest amount of gene expression in the peel, followed by pulp, was the lowest in the leaves, consistent with the difference in anthocyanin content in the above organizations. 4 the anthocyanin synthesis structural gene apple coloration differences between analysis. Using real-time quantitative PCR technology to of Xinjiang red meat Apple 'summer red meat' and 'white meat' as test materials, determination of the structural gene MsCHS fruit development period of the anthocyanin biosynthetic pathway MsCHI MsUFGT MsDFR1 MsDFR2 MsF3H, MsLDOX The expression amount. The results show that the structural gene expression in the 'summer Cara' and 'white meat' there are significant differences. Early fruit development, 'the summer red meat' expression levels in the skin and flesh, significantly higher than the controls 'white meat', which both pulp than peel obvious differences in expression levels of genes. The 'summer red meat' flesh, MsCHS, MsCHI MsLDOX MsUFGT the gene expression level of the existence of significant differences. Which MsCHS MsCHI, MsLDOX expression levels in the flesh in the first three measurement periods were higher than the control white meat wild apple, the expression levels were approximately three in 'summer red meat' 'white meat' apple of 3 to 10 times 3 to 37 times and 2 to 7 times. The MsUFGT expression levels in the flesh, the expression levels of the first and the third measurement period was significantly higher than the corresponding period of the white meat wild apples, the first period was measured expression level was about 40 times. Preliminary view that the MsCHS, MsCHI, MsLDOX, MsUFGT with red meat is closely related. The 'summer red meat' peel MsDFR1, MsDFR2, MsLDOX expression level difference between the 'summer of red meat' and 'white meat' obvious. Which the red meats Apple peel MsDFR1, MsDFR2 expression levels after three measurement periods than control white meat apple, both in 'summer red meat' expression levels were approximately 'white meat' Apple 2 to 7 times, 4 ~ 10 times, 'summer Cara' pericarp MsLDOX expression amount of two measurement periods in the middle than the control, approximately 5-20 times controlled. Other analyte gene expression was significantly higher than the control in the second measurement period. The 'summer red meat' peel, in addition to MsLDOX remaining six structural gene expression determination in the second period, the highest expression levels after a downward trend, with the 'the summer red meat' peel anthocyanin accumulation trend. Preliminary view that MsDFR1, MsDFR2, MsLDOX with the Redskins is closely related. 5.MsMYB10 successful expression of genes in prokaryotic to obtain the 28.8kd size of fusion proteins. Connect MsMYB10 coding area to the prokaryotic expression vector PET-30a (), MsMYB10 gene was constructed the original nuclear expression plasmid pET30a-MsMYB10 recombinant plasmid into E. coli BL21 (DE3) competent cells under IPTG induction, to obtain the expected The size 28.8kd fusion protein. 6 screened AFLP markers linked with red meat red apple traits size 300bp about. 64 pairs of AFLP primer combinations of red / green DNA gene pool the red trait screening of molecular markers, which the primer combinations EocRI-ACC / MseI-CAG polymorphism fragments of about 300 bp between the two gene pools F1 progeny further The verification shows that in the between 10 red the DNA pool members and 10 green DNA pool members, the fragment showed continuous differences, the preliminary view that the gene with the red traits.

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