Supplementary Cloning And RNAi Functional Identification Of CHS Gene Families From Brassica Rapa, B. Oleracea And B. Napus

Flavonoids are ubiquitous plant secondary products, which are best known as the typically red, blue, and purple anthocyanin pigments in plant tissues. Flovonols and flavones are pigments of some yellow flower petals. Proanthocyanidins as another group flavonoids are the ingredients of seed coat pigments. Additionally, many flavonoids also are bioactive in human body, such as antioxidation and tumor prevention.Chalcone synthase (CHS) is the first key enzyme of flavonoid pathway, playing an important role in pigment accumulation, defense to UV, and the anti-fungus mechanism. For the model plant Arabidopsis thaliana, which also belongs to the Brassicaceae family, the loss of function mutation of the CHS gene leads to the transformation of the wild-type dark-brown seed to yellow seed, i.e. transparent testa (tt) trait, which was named as tt4. Our previous study has cloned 8,4 and 4 full-length CHS genes from B. napus, B. rapa and B. oleracea, respectively, and B. rapa contains the total sets of CHS genes donated by the two parental species.Brassica is the most important genus of Brassicaceae. It contains many oilseed, vegetable and ornamental crops, which have great economic importance worldwide by providing vegetable oil for human consumption, protein-rich meal for livestock feed and vegetables as well. Oilseed rape (Brassica napus L.) is one of the most important oil plants in China and the world, yellow-seeded B. napus shows many good quality traits, such as lower seed coat pigment content, thinner seed coat, lower meal fiber content, higher seed oil and meal protein content, etc. Creating yellow seed stocks through means like distant hybridizations is time-consuming and low efficient and the expression of the yellow seed phenotype shows drastic sensitivity to environmental factors. It is urgent to elucidate the mechanism and carry out molecular breeding of this trait.In this research, we supplementarily cloned the remaining members of BrCHS, BrCHS and BoCHS gene families. Bioinformatics and functional comparative genomics analysis were systemically conducted. Using RT-PCR, their expression patterns in the various organs of B. napus, B. oleracea and B. rapa were detected, and the expression differences between black-and yellow-seed materials were also detected. The RNAi plant expression vector of Brassica CHS gene family was constructed and used to transform black-seeded B. napus cultivar Zhongshuang 10. Vectors for sense expression of Brassica CHS genes were constructed.(1) Supplementary cloning of the remaining members of B. napus, B. oleracea and B. rapa CHS gene familiesIn this study, full-length cDNAs and gDNAs of 4 CHS genes were cloned from B. rapa, B. oleracea and B. napus, and they were named as BrCHS5, BoCHS5, BnCHS9, BnCHS10. Eight CHS pseudogenes were also cloned and were named BrCHS6pse, BrCHS7pse, BoCHS6pse, BoCHS7pse, BnCHS11pse, BnCHS12pse, BnCHS13pse and BnCHS14pse. The genomic sequences of BrCHS5, BoCHS5, BnCHS9 and BnCHS10 are 1353bp-1656 bp. They all consist of 1 intron and 2 extrons. The full-length cDNAs are 1182-1419 bp, with 5’UTRs of 55 bp and 3’UTRs of 116-182 bp.(2) Protein structural features of newly cloned BrCHS, BoCHS and BnCHS membersThe number of amino acid residues of BrCHS, BoCHS and BnCHS proteins are 393～395 aa, with molecular weights (Mw) of 43.07-43.18 kD and isoelectric points (pls) of 6.09～-9.68; They all are acidic proteins. Hydrophobic amino acids constitute the highest proportion, followed by polar amino acids. They were predicted to have no signal peptide by SignalP and SOSUI. PredictNLS and WoLFPSORT predicted that they were localized in the cytoplasm. NetPhos 2.0 predicted that they all have multiple phosphorylation sites. SOPMA predicted that a-helix is most frequent part in their secondary structures, followed by extended strand.(3) Evolutionary relationships among B. napus, B. rapa and B. oleracea based on CHS genesPair-wise alignment of genomic sequences results revealed that BrCHS, BoCHS, BnCHS, BnCHS1, BnCHS2, BnCHS3, BnCHS4, BnCHS5, BnCH6, BnCHS7, BnCHS8, BnCHS9, BnCHS10, BnCHS11pse, BnCHS12pse, BnCH13pse and BnCHS14pse share 99.1%,99.1%,98.2%,100%,98.9%,100%,99.7%, 99.8%,98.7%,99.2%,99.9%,99.9%,99.8%and 99.9%identities with BrCHS1, BoCHS1, BrCHS2, BoCHS2, BrCHS3, BoCHS3, BrCHS4, BoCHS4 BrCHS5, BoCHS5, BrCHS6pse, BoCHS6pse, BrCHS7pse and BrCHS7pse, respectively.This indicates that the 1～14 CHS gene members of B. napus were orginated from BrCHSl,BoCHS1, BrCHS2, BoCHS2, BrCHS3, BoCHS3, BrCHS4, BoCHS4, BrCHS5, BoCHS5, BrCHS6pse, BoCHS6pse, BrCHS7pse and BoCHS7pse, respectively. B. napus is the fusant of B. rapa and B. oleracea. Sequence alignments and phylogenetic relationships of BrCHS, BoCHS, BnCHS and AtCHS proteins also certified the above conclusion.This study also indicated that there were two CHS genes in the ancestor of Brassicaceae, and they both were "triplicated" in the the ancestor of Brassiceae. Though most of the triplicated members were kept in the following evolution, some members have experienced pseudogenization.(4) Organ-specificity of CHS genes from B. rapa, B. oleracea and B. napusOrgan-specificity of CHS genes of B. rapa, B. oleracea and B. napus in typical black-seeded lines were detected by RT-PCR. In transcription level, BrCHS, BoCHS and BnCHS had similar patterns, which expressed in all detectecd organs and have the highest expression in bud, flower and the early-stage developing seeds.As the seeds mature, the expression reduced.(5) Relationships between CHS expression and yellow seed trait of B. rapa, B. oleracea and B. napus Differences of expression levels of CHS genes in major reproductive organs of B. rapa, B. oleracea and B. napus in black-and yellow-seeded lines were detected by semi-quantitative RT-PCR. The results showed that in parental species the overall and member-specific expressions in main reproductive organs of yellow seed materials were down-regulated as compared with black-seeded stocks. B. napus, however, no down-redulation was detected. This implied that the yellow seed traits in parental species are related to CHS down-regulation, but no relation in B. napus. In Brassica, relationships between CHS and yellow seed traits are different among species. Simultaneous down-regulation of all members in parental species also implies that the real reason lies in the upstream regulation signal of CHS.(6) Relationship between seed coat pigmentation and Brassica CHS gene family revealed by RNAiAn 831-bp RNAi fragment BCHSI targeted to Brassica CHS gene family was cloned using the BnCHSl gene from B. napus as the template. Its antisense and sense fragments were inserted to the promoter-spacer and spacer-terminator cloning sites of an improved plant RNAi basic vector pFGC5941M using NcoI+AatII and BamHI+Xbal double digestions respectively. The resulted 11814-bp RNAi vector was pBCHSI, which was verified by multiple PCR checking and successfully transformed into Agrobacterium tumefaciens strain LBA4404 to form engineering strain. RNAi expression vectors were transformed into Brassica napus cultivar Zhongshuang 10 by Agrobacterium-mediated transformation. we have gained 81 regenerate plants. Basta detection and PCR detection screened out 14 positive transgenic plants of highly resistant to Basta.Investigations indicated that the transgenic showed 4 obvious changes as compared with the CK. First, the decrease of seed coat pigmentation, and the yellow-brown transgenic seeds were much lighter in color than the black seeds of the CK.Second, their seeds turn smaller, lower plumpness and with a decrease in 1000-seed weight. Third, transgenic plants had lower setting rate and shorter pod length. Fourth, they have fewer number of effective collateral compared with the CK. These indicated that Brassica CHS gene family participates not only in seed coat pigmentation but also in the formation of other traits such as branch formation, pollinationa and fertilization, seed development, etc.The results of this study indicated that in Brassica plants, CHS genes simultaneously regulate the trait formation of seed coat seed coat color trait, branch formation, pollination and fertilization, seed development.(7) Vector construction for sense expression of Brassica CHS genesCHS genes from B. rapa, B. oleracea and B. napus were integrated into intermediate vector pCambia2301M1B to form 8 plant expression vectors which were named as pBnCHSIS, pBnCHS2S, pBnCHS3S, pBnCHS4S, pBnCHS5S, pBnCHS6S, pBnCHS7S, pBnCHS8S, respectively. And their Agrobacterium engineering strains were gained.