Study On The Concentration Changes And Related Genes Of Flavonoids In Apple

Flavonoids are the most important secondary metabolites in apple. They play an important role inapple nutritional quality, growth and development and disease resistance, as well as in apple taxonomyresearch. This study utilizes several types of higher flavonoid content in wild-type apples and lowerflavonoid content in cultivated-type apples as experimental mateirals, and takes the different genotypes,different parts and different developmental periods of apple fruits as research objects to study thechange rules of flavonoid content, and the molecular regulatory network and regulatory gene expressionin apple fruit. Additionally, we conducted gene cloning analysis and analysis of the expression oftranscription factor MdMYB12, and have thus laid the foundation for different apple variety breedingwork using molecular biological breeding to cultivate higher flavonoid content in apple fruit. This paperis divided into the following content;(1)W‘e utilized the fruit of ‘Malus sieversii\Malus robusta (Cam) R’’ehd，.，Tsugaru and‘G’uoqing as experimental materials to determine the content difference and change trend ofchlorogenic acid and the total flavonoid content in peel and fruit in the young fruit period and in theincreasing stage by RP-HPLC and spectrophotometry analysis. In doing so, we discovered thatflavonoid content rules have no connection with the chlorogenic acid of apple polyphenols, thatflavonoid content is higher in the peel and that chlorogenic acid content is higher in fruit, that bothflavonoid and chlorogenic acid content are higher in the young fruit period than in the increasing period,and that their content is higher in wild-type apples than in cultivated-type apples. Through theexperiment, which used RP-HPLC to determine the total flavonoids and12kinds of flavonoid’compounds content difference and change trend in four types of apple peels and fruits in the young fruitstage and in the increasing stage, we found out that flavan3-ol and phloridzin are hi‘gher in Malussieversii\and that flavonol content is higher in the cultivated-type apple Jinqing. In the four varietiesof apple peels and fruits, the content of flavonoids decreases with the maturity of the fruit.(2)In order to further prove that flavonoid content changes in relation to fruit development, we‘chose the fruit peel ’’and fruit pulp of the cultivation type apple Tsugaru and the wild type Malus rockiiS’chneid. as experimental materials in five different developmental periods, and found that the fruit sizeof Tsugaru increases with the maturity of fruit enlargement, while th‘at of Malus rockii Schneid.‘exhibits no significant change with maturity. The contents of chlorogenic acid and flavonoids in Malusrockii Schneid: were far higher th‘an those in T，sugaru，and the change of chlorogenic acid content inapple fruit had no direct relation to fruit inlfation, while flavonoid content decreased with fruit inflation.Based on the analysis of content changes of12flavonoids in two varieties of apple, we found that thet’’otal flavonoid content in Tsugaru was much lower than that in ‘Malus rockii Schneid.\but that theflavonoid content in the fruit peel was higher than that in ‘Malus rockii Schneid’.. The change of flavan3-ol content in ‘Malus rockii Schneid: was not obvious with fruit development, and showed no obviousdifference in the fruit pulp and peel, but the content decreased obviously in the early stage of fruit d’’evelopment and had no significant change in later developmental stages in Tsugaru. The phloridzincontent in th’e ’Tsugaru peel decreased slowly with fruit growth, while there was no obvious change infruit pulp. The change in both the ‘Malus rockii Schneid,. fruit peel and pulp was the same; that is,phloridzin content decreased in the early fruit developmental stages and showed no obvious change asfruit matured.(3) With the application of real-time fluorescent quantitative PCR，we analyzed the mRNAexpression differences of five traditional reference genes18SrRNA, ACTB, GAPDH, UBQ and TUB indifferent apple genotypes, different tissues and different developmental periods. Through geNormprogram analysis, we found that the expression stability of the five reference genes is different. UBQ isthe most stable in different genotypes and in gene expression analysis of different developmentalperiods. ACTB expression and UBQ expression are both stable in six different tissues. This study provesthat UBQ gene expression is relatively stable in experimental samples, and the UBQ gene is the firstchoice in apple gene expression analysis.(4) We conducted RT-PCR analysis of11structural genes that regulate the anabolism offl’‘avonoids in ’Tsugaru and Mal’us rockii Schneid.. From this, we found that the expression quantity of10genes (PAL’ CHS，CHI，FHT，FLS，DFR，LAR，ANS，ANR，F3H) in ‘Malus rockii S，chneid.，whichh‘as higher levels of flavonoids than T，‘，sugaru，is more than in Tsugaru，while FGT expressionquantity showed a significant difference between the two kinds of apples. The gene expression quantitychanges with the growth of fruit, and is related to the changes in flavonoid content. The variation trendof CHI gene transcription is the most positively correlated with that of flavonoid content.(5) With the bioinformatics analysis of the publi‘shed apple G’olden Delicious genome, wecloned the mdMYB12transcription factor f‘rom the Malus rockii Schneid,. fruit peel and found that thetranscription is correlated with several kinds of flavonoid substances based on fluorescence quantitativeanalysis of mdMYB12i’‘n ’Tsugaru and Malus rockii Schneid,.