| BANYULS (BAN) is an important structure gene in proanthocyanidin biosynthesis pathyway, which encodes an anthocyanin reductase and transform anthocyanin into proanthocyanidin (PA). Hitherto, the molecular function of AtBAN in Arabidopsis thaliana regulating flavonoid biothesis pathway in seeds has been well studied. Mutation in AtBAN results the lighter color in A. thaliana seed coat. However, functional analysis of BAN in Brassica napus has not been reported yet. Previously, we have screened the differential expressed genes (DEGs) between developing seeds of yellow- and black-seeded B. napus using digital gene expression, and we manily focused on the DEGs participated in flavonoid biosynthesis pathway. Many genes functioned in this pathway were changed in expression level, of which on copy of BnBAN was downregulated significantly in yellow seeds. This paper planed to acknowledge the function of BnBAN regulating seed coat pigments in B. napus, through overexpression and RNA interference of BnBAN. This may help to elucidate the molecular mechanism of seed coat variation in B. napus.Bioinformatic analysis revealed that four copies of BnBAN exist in B. napus, the a and b copies encodes the proteins with the same number of amino acids and molecular weight. Phylogenetic tree analysis of Brassica BAN and AtBAN evealed that BAN in Brassica were highly conserved to AtBAN, and the homology reached to 88.70%. Hence, we may refer to A. thaliana in researching regulation mechanism of BnBAN in B. napus. Expressional analysis of BnaA.BAN.a in different tissues and developmental stages of B. napus using qPCR analysis revealed that was differentially expressed. BnaA.BAN.a was most highly expressed in flower bud, and the expression of BnaA.BAN.a was the lowest in root. Besides, expression of BnaA.BAN.a in seeds was upregulated with the seed development, and reached the maximum at 6 WAR The expression pattern of BnaA.BAN.a in different tissues and developmental stages would help to the researches on regulation mechanism of transcription factors (TFs) in upstream. On the other hand, we carried out sub-cellular localization of BnaA.BAN.a expressed protein in tobacco, and the floresence was observed both in cell membrane and nucleus. This result is a reference in functional analysis of BnaA.BAN.a. However, the sub-cellular analysis of BnaA.BAN.a in tobacco can not fully elucidate its properties, further analysis on protoplasts of B. napus is needed.Since the mutants of ban in B. napus is not available, we plan to downregulate the expression leel of BnaA.BAN.a in B. napus using RNAi, with the aim to know the regulation mechanism of BnaA.BAN.a in pigmentation of B. napus. Meanwhile, we constructed the overexpression vetor to regulate the expression of BnaA.BAN.a in B. napus. Using the Agrobacterium mediated genetic transformation, we transformed the overexpresion vector and RNAi vector into B. napus cv. Yangyou 9. Then we ascertained the positive plants using the PCR identification of screening marker gene hygromycin, as well as expressinal analysis of BnaA.BAN.a using qPCR. Finally, we obtained seven overexpression lines and four RNA interference lines in To generation. qPCR analysis revealed expression of BnaA.BAN.a in five overexpression lines were significantly upregulated with 6 to 12 folds. Expression of BnaA.BAN.a in RNAi lines were all downregulated with 2 to 10 folds. Analysis of polyphenols in seeds of transgenic lines (T1 generation) revealed the content of insolulable PAs were increased with the upregulation of BnaA.BAN.a, whereas the content of soluable polyphenols were decreased the upregulation of BnaA.BAN.a. When the expression of BnaA.BAN.a was downregulated, the changes of insolulable PAs and soluable polyphenols were erratic. Thus, we suspect there might be some other pathways related to PA biosynthesis. On the other hand, we found the content of insoluable PAs in leaves were decreased in overexpresion and RNAi lines, indicating thet regulation mechanism of PA biosynthesis might be different between leaf and seeds. To know the influence of BnaA.BAN.a on other genes participated in flavonoid biosynthesis pathway, as well as lipid biosynthesis and metabolic pathway, we analyzed the expression level of 12 structural genes and TFs. The results revealed that expression level of many genes were changed with the modification of BnaA.BAN.a expression in transgeneti lines. This study is a preminary basis for molecular analysis on pigment composition in B. napus. |
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