| Blackcurrant (Ribes nigrum L), a small shrubs, belongs to the Saxifragaceae genus Ribes. It is one of the advantaged fruit trees of cold region in Heilongjiang Province because of its the larger cultivated area and rich variety of resources. Its fruit is rich in anthocyanins and other phenolic compounds. The presence of these phenolic substances not only impact the color of black currant products, but also has a variety of physiological effects on the prevention of cardiovascular disease and cancer, etc. So black currant has attracted the widespread attention of scholars at home and abroad. In this study,26 varieties of resources from the black currant germplsm garden of the Northeast Agricultural University were systematic evaluated from the perspective of the fruits of economic traits. HPLC-MS/MS was used to analysis the type and content of anthocyanins and non-anthocyanin phenolic of 26 varieties of ripe black currant fruit, revealing the difference of species, composition and content between anthocyanins and non-anthocyanin phenolic in ripe fruit of different varieties of resources. RnANS1, RnF3’5’H1, RnF3’Hl and RnDFRl, mechanism genes associating with anthocyanin synthesis, were cloned from the black currant fruit by RT-PCR and RACE technology and complete corresponding the bioinformatics analysis. Real-time PCR was used to analyze the expression of the four genes RnANS1, RnF3’5’H1, RnF3’H1 and RnDFRl in fruit veraison of the black currant. The main results are as follows:According to the isometric grouping method, adding up frequency distribution of economic traits of blackcurrant fruits, using 1 to 5 grading system they proposed grading standards of era yield, berry weight, anthocyanin content, ear length, organic content, soluble sugar content as six traits. Using principal component analysis methods to determine that the main indicators impacting the economic evaluation traits of black currant fruit are ear yield, anthocyanin content, soluble sugar content and organic acid content, the main factor of fruit is sugar content of fruit. Application of factor analysis method, defining the condition. The first common factors are ear yield and berry weight, the second common factors are the fruit soluble sugar and anthocyanin. From the perspective of economic traits of fruit, people make comprehensive evaluation to black currant varieties. And they think the kind of (?)Sofia,Kim Dae-grain,Grew Bucky,Ussuri and other varieties fruit have better comprehensive economic traits.Thirty three anthocyanins were detected in the ripe fruit peel of 26 black currant cultivars, which contains two kinds of double glycoside, fourteen kinds of monoglycoside, six kinds of pyranoid anthocyanins, nine kinds of glycosides, one kind of three glycoside and six kinds of unidentified anthocyanins. Most of black currant varieties could be detected about 20 kinds of anthocyanins, and (?) ’is the most abundant breed in anthocyanin species; anthocyanin composition is very complex in ripe fruit peel of different varieties black currant, and eleven anthocyanins is co-owner in them. Delphinidin, anthocyanidins, glucoside and rutinoside types of anthocyanins have larger proportion. Anthocyanins content of most varieties of fruits is between 30mg/g and 40mg/g. opcкий, HecTop Kозин, Black smitn, BeAopycKa (?) and Y-Ce(?) (?) have more rich anthocyanin than other species. Content of second anthocyanidin glucoside was significantly higher than the proanthocyanidin monoglycoside, delphinidin and anthocyanidins content are predominant in Total anthocyanin. Principal component analysis showed that anthocyanins, methyl delphinidin, malvidin and delphinidin type of anthocyanins play a decisive role in the composition of anthocyanins of black currant peel. Twenty-six black currant varieties could be divided into two categories, Fortodi; YccypHH; ojebyn; Risager; Brodforp; daisha; Earapa are the first category and the other 19 varieties are the second category.Ten kinds of non-anthocyanin phenolic compounds were detected in the peel of 26 kinds of black currant fruit, including nine kinds of flavonols non-anthocyanin polyphenols.4 to 5 kinds of non-anthocyanins phenolic compounds can be detected in most varieties of fruit; non-anthocyanin phenol of myricetin types could be detected in most species peel. Non-anthocyanin content is more abundant in three species pericarp which are (12) Modified CTAB method was used to extract total RNA and reverse transcribed intocDNA from the ripe fruit of Brodforp. Full-length cDNA sequences of four genes that are anthocyanin synthase(RnANS1), flavonoid-3’,5’-hydroxylase(RnF3’5’H1), flavonoids-3’-hydroxylase(RnF3’H1) and dihydroflavonol-4-reductase (RnDFR1) were cloned from Blackcurrant fruit by RT-PCR and RACE. The GenBank accession numbers were KC493686, KC493688, KC493689 and KC493687 respectively. Bioinformatics analysis methods suggest that the four genes encode polypeptides containing 357,509,524 and 366 amino acid residues, and the polypeptides’molecular weights are respectively 40.51 kDa,57.12kDa,58.40kDa and 40.57kDa. The similarity of the genes with related genes of other species are more than 70%, the molecules phylogenetic tree was constructed to analysis the genetic relationship of the four genes with the corresponding gene in other species. The signal peptide structure and hydrophobic of the each gene were analyzed and the secondary structure of the protein was initially identified. The expression characteristic of associated structural gene in fruit veraison of blackcurrant is as follows: ① With the fruit color deepened, RnANS1 was up-regulated expression and the transcription reach the highest level during mature period;② In the process of fruit turn color, RnF3’5’H1 was up-regulated expression and the expression of RnF3’5’H1 is basically in the highest level when fruit coloring up to 50% and 80%. The transcriptional level decreased When the fruit ripening;③RnF3’H1 is down-regulated after the fruit turned color and its expression trend was up-regulated when the fruit coloring reaches a certain level. The level of transcription is the highest when the fruit coloring degree reached to 80% and it is lower when the fruit is mature;④With the deepening of fruit coloring degree, the expression of RnDFR1 was up-regulated trend. the level of transcription is highest when the degree of turned color is up to 80%. The transcription levels is lower when fruit ripening. Transcription levels of RnANS1 and RnF3’5’H1 showed a positive correlation with the content of anthocyanins and soluble solids. There is no correlation with fruit pH value. The expression of RnF3’5’H1 plays a key role in the anthocyanin synthesis of black currant fruit. There is no correlation between the transcription level of RnF3’H1 and RnDFR1’s and anthocyanin content, soluble solids content and pH. Flavonoid-3’-hydroxylase encoded by RnF3’H1 and Dihydroflavonol-4-reductase encoded by RnDFR1 are not the key enzyme in the process of anthocyanin synthesis of black currant fruit. |
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