Function Analysis Of BcNAC2-Embryogenesis Related Gene In Turnip

Flowers development is one of the most attractive events during the plant developmental process, and bud formation means the beginning of reproductive growth (Lu Hai-Yu et al.,2009). In 1991, floral organ development of ABC model has been proposed by Coen and Meyerowitz, which was a breakthrough of plant development biology and which could explain the identity of the four whorls of the typical eudicot flower. In Brassica crops, male sterility phenomenon occurs generally, and available heterosis to increase their yield and quality by application of male sterility. The female sterility phenomenon, however, has hardly been reported in Brassica. In this study, a novel preferentially expressed cDNA sequence, has been isolated and characterized from turnip(Brassica campestris L. ssp. rapifera (Matzg) Sinsk, syn. B. rapa L.). The full-length cDNA and genome DNA was subsequently amplified by homology-based cloning method, named BcNAC2. Moreover, the gene sequence was analyzed and protein functions were predicted. By using NCBI database, we downloaded 30 NAC sequences of plants, subsequently, the homology analysis was performed and the phylogenetic tree was constructed. In addition, the expression pattern of BcNAC2 was analyzed by RT-PCR (Reverse transcriptase polymerase chain reaction) during the different stages of plant development. The expression of BcNAC2 after fertilization process was described by in situ hybridization method. In this paper, firstly, antisense. RNA expression vector was constructed; secondly, Brassica campestris L. ssp. chinensis var. parachinensis (Bailey) Tsen et Lee was transformed by agrobacterium-mediated method to obtain their loss-of-function mutant. Lastly, their function in pistil development process was analyzed. The major study results as follows:(1) Based-on the cDNA-AFLP differential fragments, the cDNA sequence of BcNAC2 has been isolated by homology-based cloning method. Subsequently, genomeic DNA sequence was also obtained. Sequence' analysis result indicated that the full-length of BcNAC2 was 2080 bp and the largest open reading frame (ORF) was composed of 1683 bp with a deduced 560 amino acids, which composed of six extrons and five introns of 67 bp,102 bp,269 bp,81 bp and 83 bp, and the "GT-AG" splicing rule of BcNAC2 at the exon-intron boundaries. Sequence analysis results reveal that BcNAC2 belongs to NAC gene because one NAC domain has been predicted in the 9th～159th amino acids of deduced BcNAC2 protein. Moreover, it is a NAM protein by SMART datebase. The secondary structure of BcNAC2 protein contained mainly 19.64%α-helix,15.00%β-sheet and 65.36% coil and a saddle-shaped was the third structure. Moreover, compared with function domains of NAC2 protein in Arabidopsis thaliana, the motifs number in function domains of BcNAC2 was significantly different. In the function domains of BcNAC2, there were two N-glycosylation site (79～82,103～106 amino acids), four protein kinase C phosphorylation site (83～85,87～89,95～97,110～112 amino acids), five Casein kinase II phosphorylation site (18～21,42～45,71-74,95～98,137～140 amino acids), two N-myristoylation site (80～85,109～114 amino acids). But in NAC2, we found two N-glycosylation site (57～60,79～82 amino acids), one cAMP- and cGMP-dependent protein kinase (53～56 amino acid), three protein kinase C phosphorylation site (42～44,61～63,72～74 amino acids), three Casein kinaseⅡphosphorylation site (30～33,37～40,72～75 amino acids), two Tyrosine kinase phosphorylation site (11～17, 11⒍18 amino·acids), three N-myristoylation site (4～9,58～63,85～90 amino acids). However, whether the different sequences can lead to genetic function of difference, may need a follow-up study.(2) By NCBI database, we downloaded 30 NAC protein sequences from the different plants. Then the homology analysis was performed and the phylogenetic tree was constructed. The amino acids sequence alignment confirmed that BcNAC2, AtNAC2, TERN in tobacco and GmNAC6 in soybean can be clustered as a single category, NAM subfamily. Cluster results indicated that AtNAC2 was the closest relatives to BcNAC2, which were the same family plant. The homology analysis of proteins sequences in NAM subfamily described that it comprised a variety of plant proteins that are identifiable by the presence of a highly conserved N-terminal NAC domain, accompanied by diverse C-terminal domains and do not contain any known protein domains. Therefore, NAC transcription factors may control the transcriptional activity through the diversiform C-terminal sequence, accord with NAC transcription factor structure characteristics.(3) Gene expression analysis carried out with RT-PCR analysis and in situ hybridization. The expression pattern of BcNAC2 was analyzed by RT-PCR during the different stages of plant development, we found that BcNAC2 gene presented the character of constitutive expression partially, and highest expression at 8 days after pollination. BcNAC2 not only in prophase vegetative organs, and higher expression in the later seeds and embryo formation process, suggesting BcNAC2 play an important role in throughout the growth process in turnip. In situ hybridization results proved that higher expression in the embryo at 8 days after fertilization. Therefore, BcNAC2 probably involved in the development of seed or embryo of turnip.(4) The plant antisense expression vector of BcNAC2 was constructed containing pBI35s-BcAC2 regulated by the constitutive promoter CaMV35S. Then the expression vector was introduced into flowering Chinese cabbage by agrobacterium-mediated method. At last,6 kanamycin resistant shoots were obtained successfully so far.