Molecular Cloning And Expression Of Anthocyanins Regulate Gene PyMYB In 'Pingguoli'

As one of the characteristic fruits of Yanbian,Tingguoli,(Pyrus bretschneideri Rehd.'Pingguoli')is also the specialty of Jilin Province and the precious fruit of the north temperate zone.It not only has strong cold resistance,but also the fruit is very big and can be coloured in red.With the good appearance quality and high commodity value,the market demand of red pear becomes bigger and bigger.breeding excellent varieties of red pear can promote the pear trade developing,therefore,it's an important direction of pear trees selective breeding.Because of the rare of red pear resource,breeding the excellent cultivars of red pear is the urgent problem to solve.In recent years,the study of pear fruit coloring mechanism has acquired certain breakthrough,the peel coloration mechanism of Tingguoli' in structure genes has been in-depth study,but the research on the regulatory gene is very little.In this thesis,the fruit peel of Tingguoli' is taken as the material to clone the regulatory gene of MYB10 and MYB114 and studty the anthocyanins nucleoside content in coloring period of bagging and not bagging 'Pingguoli' fruit.At the same time,analyze the expression features of MYB10 and MYB114 with real-time fluorescence quantitative-PCR.The main results were as follows:1.We succesfully obtained the full-length of related regulatory gene in the process of anthocyanins nucleoside synthesis of[Pingguoli' and named it PyMYB10a and PyMYB114 respectively.Sequence analysis showed that cDNA of PyMYB10a was 736bp in length with the OFR of encoding a putative protein of 130 amino acids,the molecular weight of the protein it encodes was 15.17 kDa and the theoretical pI was 4.73.The cDNA of PyMYB114 was 744bp in length with the OFR of encoding a putative protein of 201 amino acids,the molecular weight of the protein it encodes was 23.48 kDa and the theoretical pI was 5.57.2.Similarity and phylogenetic tree analysis results showed that the nucleotide sequence and protein sequence of PyMYB10a and PyMYB114 had high similarity and homology with MYB10 and NYB114 of Rosaceae plants,especially higher with Pyrus plants.3.Study on anthocyanins glycosides content variation in Tingguoli' peel indicated that the anthocyanin glycosides content in the fruit peel of bagging or not existed evident difference in the process of coloring,bagging treatment effectively promoted 'Pingguoli' fruit coloring and anthocyanin nucleoside biosynthesis.4.Real-time fluorescent quantitative PCR analysis showed that the expression level of PyMYB10a and PyMYB114 in the colouring process of bagging fruit rose rapidly after tearing off the bag,the content of anthocyanin nucleoside also increased rapidly.It showed that PyMYB10a and PyMYB114 played an important regulatory role in 'Pingguoli' coloring process.