Cloning And Expression Analysis Of Flavonoid-related Transcription Factors In Crabapple

As a kind of landscape and ornamental plant, crabapple has a long history of planting in China. The number of new crabapple varieties is increasing in recent years because of the unremitting efforts of breeding researchers. They brought lots of changes in leaf color, flower color, fruit color, florescence, tree form and so on. Because the plentiful ornamental of crabapple, it is becoming one of the main ornamental plant in the modern landscape.Flavonoid is one of the material taking participating in forming flower color and leaf color in the plants. This experiment did the research in the leaf coloring mechanism and regulation pathway of the ornamental crabapple. Three kinds of transport factors(TFs), MYB, bHLH and WD40, which related of flavonoid synthesis pathway in Malus ’Royalty’ are cloned, and the expressing of the TFs was analysised. On the other hand, 3 different growing period leaves of 3 different cultivars of crabapple were taken as samples, detecting the flavoniods content and analysising the related structural genes and TFs gene expressing level. Here is the result:(1) The red tender leaf of Malus ’Royalty’ was chosen as experiment material, cloning the three kinds of TFs: MYB, bHLH and WD40. As the result, we got four genes, MRYMYB10(Gene Bank: KF772789), MRYMYB6(Gene Bank: KJ026364), MRYLC(Gene Bank: KP222538) and MRYWD40(Gene Bank: KJ542656). The bioinformatic analysis show that the amino acid sequence of them are similar to the correspondence genes in apple: MdMYB10(GeneBank: AB592747),MdMYB6(GeneBank: DQ074461), MdbHLH33(GeneBank: DQ266451) and MdTTG1( GeneBank: GU173814). And the similarities are 100%、96.79%、98.92% and 99.77%. The results of secondary structure analysis and tertiary structure analysis of 4 protins show that MRYMYB 10 protein and MRYMYB 6 protein both contain typical R2R3 structure and located in the cell nucleus; Meanwhile, MRYMYB 10 owns high homology between different species, while the MRYMYB 6 has a low homology with other plants except Malus species; The MRYLC protein has the basic helix-loop-helix(bHLH) structure and most possible located in the cell nucleus, and it has a high homology in the same family and low homology in the different families; The MRYWD40 protein has the classical repeated WD conservative structure and conservatism is high between different genera and families, and the subcellular localization shows that the MRYWD 40 protein may be existed in the cytoskeleton and cell nucleus.(2) Choosing the leaves of Malus ‘Royalty’, Malus ‘Radiant’ and Malus ‘Snowdrift’ as experiment materials, and leaves of 3 different growing periods(tender leaf, changing leaf and mature leaf) of 3 crabapples were taken as experiment samples. The analysis of pigment content in samples shows that: 1. The red coloring level of crabapple leaves has the positive correlation relationship to the anthocyanin content in the leaves; 2. There is a competing relationship of substrate consuming and product accumulation in different branches of flavonoid synthesis pathway in crabapple; 3. Some of the phenolic compounds in the leaves of Malus ‘Royalty’, which are nutritious, are more than in other tow crabapples leaves in the same leaf period, and the phenolic compounds content in the leaves has the positive correlation relationship to the red coloring level of leaves.(3) The expressing level of the structural genes and TFs in the flavonoid synthesis pathway in leaf samples of 3 crabapples in 3 periods was analyzed. The results show that: 1.Anthocyanin accumulation is controlled by ANS and FGT gene, but the anthocyanin accumulation is not consistent to the ANS gene expression level, while consistent to the FGT gene expression level; 2.ANS and FGT gene expression levels are correlated to the MRYMYB10 and MRYMYB6. MRYMYB10 has positive influence to the anthocyanin accumulation while the relationship between MRYMYB6 and MRYMYB10 is competition, and MRYMYB6 shows the negative influence to the anthocyanin accumulation; 3. MRYLC and MRYMYB10 protain may form a complex and regulate the FGT, having a positive influence to the anthocyanin accumulation, while MRYMYB6 has inhibiting effects on the expression of MRYLC; 4. The expression levels of MRYWD40 and MRYLC are similar. they are maybe both controlled by the same factor, or maybe they form a complex and influence each other; 5. The complex formed by MRYWD 40 and MRYLC may directly take participate in the regulation of anthocyanin combination. The complex may also form a MBW complex with MYB protein and regulate the combination of anthocyanin; 6. The MRYWD40 protein may not only have the function of regulating anthotyanin combination,but also can regulate other pathways during plant growth and development.