Identification Of Interacted Proteins And Functional Studies To RrMYB10 In Rose Rugosa And AtTT2 In Arabidopsis Thaliana

Rugosa rose(Rosa rugosa)is an important economic crop.Flower color,caused by anthocyanins which are water-soluble pigments,is an key quality characteristics in R.rugosa.Anthocyanins belong to the flavonoid family and can provide red,blue and purple colours in a rang of flowers,fruits,foliage,seeds and roots.Anthocyanins are also related in a great rang of function,such as seeds propagation,protection against UV light damage and pathogen attack.It is also benefits to human health including protection against cancer,inflammation,coronary heart diseases and other diseases.Recently,most research focus on the function of anthocyanins to study the flavonoid biosynthetic pathway for reforming plant traits and improving human health.This research concentrates on the function of RrMYB10 in Rugosa rose by ectopical over-expression genetically modified early-flowering tobacco using RrMYB10.Besides this studies,researches also undertaken on the homologous gene AtTT2 of RrMYB10 in order to explore the mechanism of the anthocyanin pathway.The major results were as follows:1.The expression analysis and transcriptome analysis of over-expression in RrMYB10 genetically modified early-flowering tobaccoFor deciphering RrMYB10 affection on related enzymes in the flavonoid biosynthetic pathway,QRT-PCR analysis of CHS CHI、F3H、F3’H、FLS、DFR、ANS、UFGT,which represent the related genes in the flavonoid biosynthetic pathway,was undertaken in transgenic early-flowering tobacco.The result shows that CHS、DFR、ANS with UFGT were up-regulated by RrMYB10,While CHI down-regulated.Transcriptome analysis to the flowers of RrMYB10 transgenic early-flowering tobacco display that a total of 749 and 459 candidate genes were up-regulated and down-regulated respectively.Of which,there were 8 genes involuved in phenylpropane metabolism.2.The subcellular localization of RrMYB10 and the RrMYB10pro::GUS vector was constructedThe RrMYB10::GFP fused protein vector was constructed in this study.Subcellular localization analysis is conducted by the confocal laser scanning microscopy.The results show that the gene locate in the cell nucleus.This study also constructed the RrMYB10pro::GUS vector.3.RrMYB10 Yeast Two-Hybrid screening using a transcription factors library in Arabidopsis thalianaRrMYB1O bait vector was constructed and was transformed into yeast Y187.Screening was performed and the results show that there were 7 proteins identified from the library.Ultimately,5 proteins were verified to interact to RrMYB10 by the X-a-Gal test.4.The phenotype identification of the over-expression screening genes in genetically modified early tobaccoThe 5 interacted genes were coloned to pCAMBIA2300s vector and then transformed into early-flowering tobaccos.Of those transgenic early-flowering tobaccos,two genes result to the white flowers formation,three genes show near or equal to the flower color in the control plants.5.The Yeast Two-Hybrid analysis of the homologous gene AtTT2 of RrMYB10 and 35S::AtTT2 vector construction with BiFC vector construction of the relational genesThe RrMYB10 homologous gene AtTT2 and the 5 interacted proteins were totally transformed into yeast AH 109 in order to further confirm their interaction using yeast two-hybrid system.The analysis revealed AtTT2 also interat to those RrMYB10 interacted genes.The 35S::AtTT2 vector was constructed.In addition,vectors,used specially for BiFC test,of AtTT2,RrMYB10 and the 5 interacted proteins were further constructed to verify their intraction.