Cloning, structure analysis and functional verification of MYB10 in Ribes L / 生物工程学报

This study aims to investigate the molecular mechanism of the transcription factor MYB10, which is involved in anthocyanin biosynthesis, in different colors of Ribes L. fruitification. Rapid amplification of cDNA ends (RACE) was used to clone the MYB10 genes from Ribes nigrum L. (RnMYB10), Ribes rubrum L. (RrMYB10), and Ribes album L. (RaMYB10), respectively. Phylogenetic analysis showed that RnMYB10 and RrMYB10 were evolutionarily homologous. Real-time quantitative PCR (RT-qPCR) showed that the expression of MYB10 in the fruits of Ribes nigrum L. was higher than that of Ribes rubrum L. and much higher than that of Ribes album L. The expression of RnMYB10 and RrMYB10 increased at first and then decreased as the fruit diameter increased and the fruit color deepened (the maximum expression level was reached at 75% of the fruit color change), while the expression level of RaMYB10 was very low. Overexpression of RnMYB10 and RrMYB10 in Arabidopsis thaliana resulted in purple petioles and leaves, whereas overexpression of RaMYB10 resulted in no significant color changes. This indicates that MYB10 gene plays an important role in the coloration of Ribes L. fruit.

Cloning and expression of dfr in Ribes L. during fruit maturation / 生物工程学报

Little is known about the molecular mechanism of currant anthocyanin synthesis. We investigated the effect of dfr, a key gene for anthocyanin synthesis in currant, on anthocyanins of different color currant. Black currant (Ribes nigrum L.), red currant (Ribes rubrum L.) and white currant (Ribes albrum L.) were used as test materials to determine the anthocyanin content at different stages of fruit development. Three full-length cDNA sequences of dfr gene were cloned by RACE (Rapid amplification of cDNA ends), and named as Rndfr, Rrdfr and Radfr. Phylogenetic analysis shows that Rndfr, Rrdfr and Radfr had high homology in evolution. The determination of anthocyanin content in different stages of fruit development shows that the content of anthocyanin in black currant and red currant was higher and gradually increased with the ripening of the fruit. While the content of anthocyanin in white currant was extremely low, and almost no anthocyanin was detected. Quantitative RT-PCR analysis shows that the expression level of dfr in black currant was higher than red currant and white currant in each period of fruit development. As the diameter of the fruit increased and the color of the peel deepened, the expression level of dfr in the black currant showed an increasing trend. In the red currant, the expression level gradually increased until the period of 75% fruit color, then the Rrdfr decreased rapidly. In white currant, the overall trend showed a downward trend, and its expression level was the lowest. All the results suggest that dfr gene plays a role in the process of fruit color.

Phylogenetic and expression analysis of SEPALLATA-like gene in Brassica oleracea L. var. acephala / 生物工程学报

The E class MADS-box genes SEPALLATA (SEP)-like play critical roles in angiosperm reproductive growth, especially in floral organ differentiation. To analyze the sequence characteristics and spatio-temporal expression patterns of E-function MADS-box SEP-like genes during kale (Brassica oleracea L. var. acephala) flower development, BroaSEP1/2/3 (GenBank No. KC967957, KC967958, KC967960) homologues, three kale SEP MADS-box gene, were isolated from the kale variety 'Fourteen Line' using Rapid amplification of cDNA ends (RACE). Sequence and phylogenetic analysis indicated that these three SEP genes had a high degree of identity with SEP1, SEP2, SEP3 from Brassica oleracea var. oleracea, Brassica rapa, Raphanus sativus and Brassica napus, respectively. Alignment of the predicted amino acid sequences from these genes, along with previously published subfamily members, demonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins: the MADS domain, intervening (I) domain and keratin-like (K) domain, and the C-terminal domain SEPⅠ and SEP Ⅱ motif. The longest open reading frame deduced from the cDNA sequences of BroaSEP1, BroaSEP2, and BroaSEP3 appeared to be 801 bp, 759 bp, 753 bp in length, respectively, which encoded proteins of 266, 252, and 250 amino acids respectively. Expression analyses using semi-quantitative RT-PCR and quantitative real-time PCR indicate that BroaSEP1/2/3 are specifically expressed in floral buds of kale during flower development process. The expression levels of the three genes are very different at different developmental stages, also in wild type, mutant flower with increased petals, and mutant flower with decreased petals. These different patterns of gene expression maybe cause the flowers to increase or decrease the petal number.