| Oilseed rape (Brassica napus L.) is one of the four major oil plants in the world and is one of the most important oil plants in China. Its yellow-seeded type shows many quality advantages, such as thinner seed coat, fewer seed coat pigment, lower meal fiber content, higher seed oil content and meal protein content, etc. In recent years yellow-seeded B. napus attracts researchers' interests. The molecular mechanism of the yellow seed trait is still unclear in B. napus. Both B. napus and A. thaliana abelong to Brassicaceae/Cruciferae, and the manner of testa pigment phenotype formation in these two species is much similar, so the research results on A. thalisna TRANSPARENT TESTA (77) loci are helpful to elucidate the molecular mechanism of yellow seed formation in B. napus.Chalcone synthase is the first key enzyme of flavonoid pathway, participating in the formation of many important flavonoid-related traits such as seed coat color, flower color, pigmentations on the sufaces of stems and leaves, etc. In A. thaliana, the CHS gene is identified as the TT4 locus. Study on CHS gene will promote to understand the molecular mechanism of the formation of yellow seed and other flavonoid-related traits, and lay the base for molecularly modifying these traits in B. napus.In this research, full-length cDNAs of 8 members of the B. napus CHS gene family (BnCHS) were isolated, and genomic sequences of 6 BnCHS members were also also obtained. The nucleotide sequences and the deduced proteins of the BnCHS gene family were systematically analyzed. An antisense plant expression vector of the BnCHS gene family was constructed and transformed into a black-seeded B. napus explant cultivar.1. Cloning of full-length cDNAs and genomic sequences of members of the B. napus CHS gene familyBased on multiple alignment of a number of plant CHS genes, primers were designed for rapid amplification of cDNA ends (RACE) of B. napus CHS genes. Twelve 5' ends and 12 3' ends that are most homologous to A. thaliana CHS (AtCHS) were obtained. Based on these sequences, 5' and 3' terminal primers were designed, and the full-length cDNAs of BnCHS1 (1450bp), BnCHS1 (1430bp), BnCHS3 (1496bp), BnCHS4 (1485bp), BnCHS5 (1338bp), BnCHS6 (1314bp), BnCHS7 (1434bp) and BnCHS8 (1481bp) were amplified from the total cDNA template. Using total genomic DNA as template, the genomic sequences of BnCHS1 (1506bp), BnCHS1 (1490bp), BnCHS3 (1743bp), BnCHS4 (1752bp), BnCHS5 (1401bp) and BnCHS6 (1372bp) were amplified. The genomic sequences of BnCHS7 and BnCHS8 were temporally not isolated.The 8 BnCHS genes have typical structural featues of CHS, and show convincing homologies to CHS genes from diverse species including A. thaliana.2. Structure of deduced proteins of the BnCHS gene family membersThe predicted BnCHS1 protein is 395-aa long with a molecular weight (Mw) of 43.02 kDa and an isoelectric point (pI) of 5.92.BnCHS2 is 395-aa in length, with a Mw of 43.05 kDa and a pI of 5.92.BnCHS3 is 394-aa in length, with a Mw of 42.93 kDa and a pI of 6.16.BnCHS4 is 396-aa in length, with a Mw of 43.14 kDa and a pI of 6.03.BnCHS5 is 394-aa in length, with a Mw of 43.01 kDa and a pI of 5.92.BnCHS6 is 394-aa in length, with a Mw of 43.09 kDa and a pI of 6.29.BnCHS7 is 396-aa in length, with a Mw of 43.15 kDa and a pI of 6.61.BnCHS8 is 396-aa in length, with a Mw of 43.24 kDa and a pI of 6.41.In all of the BnCHS 8 members, negatively charged amino acids are a little more than the positive ones. Their frequencies of hydrophobic amino acids are around 36%.All of the 8 BnCHS proteins contain many potential phosphorylation sites and no signal peptide. They were predicted to be target to cytoplasm, but each of them was also predicted with 1-3 transmembrane helices, indicating that they are possibly transmembrane-associated cytoplasmic proteins.All of the 8 BnCHS proteins contain 4 kinds of secondary structures: a-helix, random coil, 6-turn and extended strand. Alpha helix is the dominant type. Random coils are interlaced among the helices. SWISS-MODEL prediction indicated that the tertiary structures of the 8 BnCHS proteins are very similar to each other and they all resemble CHS models in the ExPDB database.All of the 8 BnCHS proteins resemble AtCHS and CHS proteins from other plants in protein structure.3. Homology analysis and evolutionary features of the BnCHS gene familyAll the 8 members of the BnCHS gene family show high homologies to AtCHS, with identities of 73.2%～83.5% on whole genomic scale and of 83.4%～88.5% on ORF level. Variations are located mainly in non-coding regions. 5'UTR-, 3'UTR- and intron-level identities are 53.9%～69.2%, 54.3%～69.9% and 25.5%～69.9% respectively between BnCHS genes and AtCHS.BnCHS1～BnCHS8 show high similarities to AtCHS, with identities of 95.7%, 95.4%, 94.7%, 93.9%, 86.8%, 86.3%, 94.9% and 95.2% and positives of 96.7%, 96.7%, 96.2%, 96.2%, 92.7%, 91.9%, 96.5% and 96.7% respectively. The similarities on protein scale are higher than on nucleotide levels, since many amino acid variations are substitutions by similar amino acids, indicating that CHS proteins are conserved during evolution, and implying that the 8 BnCHS proteins have enzymatic activities similar to AtCHS.Phylogenetic analysis revealed that the 8 BnCHS genes are orthologs of AtCHS, the gene number of BnCHS family conforms to the Brassiceae genome "triplication" assumption, and probably the Brassicaceae ancestor might have evolved 2 CHS genes. The 8 BnCHS genes group into 4 sister pairs. Similarities within each sister pair are much higher than those among sister pairs. This conforms to the amphidiploid feature of B. napus.4. Construction of an antisense expression vector of the BnCHS gene familyAn 806-bp fragment conserved in BnCHS gene family was integrated into the intemdiate expression vector pCambia2301G in the anti orientation to replace the GUS gene driven by the CaMV 35S promoter. Thus a BnCHS antisense plant expression vector was constructed. The vector was named as pCambia2301G-BnCHSA.5. Obtaining of the antisense-BnCHS transgenic pantletsThe transformation of a B. napus cultivar Zhongyou 821 mediated by Agrobacterium tumefaciens carrying the BnCHS antisense expression vector pCambia2301G-BnCHSA was made. 27 plantlets with kanamycin resisitance were obtained, among which one plantlet was identified with stably expression of the GUS reporter gene. The positive ratio was 3.70%. |
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