Cloning And Antisense Transformation Of The TT8 Gene Family In Brassica Napus L.

Oilseed rape (Brassica napus L.) is one of the four major oil crops in the world and is one of the most important oil plants in China too. In recent year the yellow-seeded B. napus has attracted researchers' interest in that it has many good properties compared to black seeds, such as thinner seed coat, fewer seed coat pigment, lower meal fiber content, higher seed oil content and meal protein content, etc. It is one of the most important objectives to breed yellow-seeded rapeseed cultivars with stably inherited yellow seed trait and good agronomic traits. The molecular mechanism of the yellow seed trait is still unclear in B. napus. However, the achievements on the Transparent Testa (TT) trait in Arabidopsis thaliana are available for an insight into the formation of the yellow seed color in B. napus. Up to now, 21 TT loci and a BAN locus have been identified, and many of the TT genes have been cloned in A thaliana.The A. thaliana TT8 (AtTT8) gene encodes a regulative factor in the flavanone synthesis way. The AtTT8 belongs to the basic helix-loop-helix domain (bHLH) transcription factors which play an important role in the biologic development. In A thaliana, Zea mays, Petunia hybrida, Antirrhinum majus and so on, the TT8 (or analogue) gene mutation alters seed color or flower color. In A. thaliana, the TT8, TTG1 and TT2 proteins all regulate the BAN and DFR genes' transcription. Study on B. napus TT8 gene is helpful to understand the molecular mechanism of the yellow seed color formation and may lay a foundation for creating stable yellow-seeded lines by genetic engineering.In this research, full-length cDNAs of 3 members of B. napus TT8 gene family (BnTT8) were isolated, together with genomic sequence of one member. The nucleotide sequences and the corresponding polypeptides of the BnTT8 gene family were systematically analyzed. The mumber of the BnTT8 gene family members was identified through Sourthern-blot. A BnTT8 gene family antisense plant expression vector was constructed and some regenerated plantlets were obtained from black-seeded B. napus explant cultivar transformed with this vector.1. Cloning and analysis of the 3 members of BnTT8 gene family in B. napusBased on the conserved regions of the A. thaliana TT8 gene, the PCR pimers were designed. 3 full-length of cDNA sequences of the B. napus TT8 gene famliy were cloned using total RNA from reproductive organs of black-seeded B.napus line 5B by RACE (rapid amplification of cDNA ends) technique. They were named as BnTT8-1, BnTT8-2 and BnTT8-3, respectively. Furthermore, the genomic sequence of BnTT8-1 was also cloned.BnTT8-1 has a genomic sequence of 2 949bp with 6 introns. Its mRNA (excluding the polyA tail) is 1 771 bp, with an open reading frame (ORF, including the stop codon) of 1 566bp. Its 5' UTR and 3' UTR are 147bp and 58bp respectively. BnTT8-2 mRNA is 1 783bp, with an ORF of 1 566bp, a 5' UTR of 137bp, and a 3' UTR of 80bp. BnTT8-3 mRNA is 1 676bp, with an ORF of 1 308bp, a 5' UTR of 137bp and a 3' UTR of 231bp. There is a typical polyadenylation signal AATTAAA in 3' UTR of them, and there are microsattilites rich in AG-repeats in their 5' UTRs. The nucleotide composition is varied in different regions of them. The G+C content of the non-coding region is obviously lower than that of the coding region. In BnTT8-1, the total intron length is much shorter than A. thaliana.The deduced BnTT8-l protien is 521 aa, Mw=59.669 kDa, pI=5.61; BnTT8-2 is also 521 aa, Mw=59.955 kDa, pI=5.65; and BnTT8-3 is 435 aa, Mw=50.071 kDa, pI=5.47. Glutamic acid is the most abundant residue in them, and acidic residues are more than basic ones. BnTT8-3 mRNA shows mutation at position corresponding stop codon of typical TT8 genes, and a 107-bp deletion exists just upstream this site, causing the loss a 86-aa C-terminal region in its deduced protein.BnTT8-1, BnTT8-2 and BnTT8-3 have very high homology at amino acid level, BnTT8-1 and BnTT8-2 share 96.7% identities and 98.1% positives, BnTT8-1 and BnTT8-3 share 97% identities and 98.6% positives, while BnTT8-2 and BnTT8-3 share 98.4% identities and 99.3% positives. The nucleotide sequence and the protein sequence of BnTT8 genes shared high homologies with those of the known plant's TT8 genes and the bHLH genes, especially with the highest homologies to those of the AtTT8 gene. The BnTT8-1, BnTT8-2 and BnTT8-3 share 83.4%, 83.9% and 83.9% of similaritis and 76.5%, 76.9% and 77.5% identities with AtTT8, respectively.BnTT8-1, BnTT8-2 and BnTT8-3 all have a bHLH conserved domain from L₃₆₆ to H₄₁₇. At this region and other conserved residues, they are extremely similar to AtTT8. BnTT8-1, BnTT8-2 and BnTT8-3 had 33, 33 and 32 potential phosphorylation sites respectively, suggesting that phosphorylation might be related to the activity of the BnTT8 proteins. The secondary structures of the BnTT8 proteins are primarily composed of alpha helix and random coil, except for certain proportions of extended strand and beta turn.Like AtTT8, the 3 BnTT8 members is probably involved in regulation of flavonoid structural genes. But since BnTT8-3 lacks the 86-bp C-terminal region, whether it is bioactive and how active it is deserve further study.2. Number of the members of BnTT8 gene familyIn Southern blot results, each restriction enzyme yielded 3 or less hybridization bands. In this study, only 3 members were cloned. Thus it is postulated that there are only 3 members of BnTT8 gene famliy in B. napus.3. Construction of a BnTT8 antisense plant expression vectorA 817-bp fragment conserved in BnTT8 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 BnTT8 antisense plant expression vector was constructed. The vector was named as pFBnTT8A..4. Obtaining of the antisense-BnTT8 transgenic pantletsThe transformation of a B. napus cultivar Xiangyou 15 mediated by Agrobacterium tumefaciens carrying the BnTT8 antisense expression vector pFBnTT8A was made. 24 plantlets with kanamycin resistance were obtained, among which 13 plantlets were identified as transgenic plants by detecting the expression of the GUS reporter gene. The transformation effciency was 2.23%.