Molecular Cloning And Functional Study Of ACCase Genes From Camellia Oleifera

Tea-oil tree(Camellia oleifera Abel), one of most important ligneous edible trees, is originated in China. To improve the seed oil rate is increasingly becoming the significant objective of C. oleifera breeding. To boost the seed oil rate is a long-term objective and technical difficulty. Moreover, it is difficult to reach the aim though traditional breeding. In order to realize the great goal, and to accelerated the C. oleifera improvement as well, one possible method is to look up a key enzyme involved in lipid biosynthesis (fatty acid biosynthesis), and to carry out the molecular biology research of C. oleifera seed's fatty acid biosynthesis so as to discover the fundamentals of lipid biosynthesis. The heteromeric form acetyl coenzyme A (CoA) carboxylase (ACCase) in plant organs of seeds catalyzes the formation of malonyl CoA from acetyl CoA, and is a rate-limiting step and a key enzyme in de novo fatty acid biosynthesis. The four components that constitute heteromeric ACCase are biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), the α-and β-subunits of carboxyltransferase (α-and β-CT), encoded by accC, accB, accA and accD genes, respectively. In this research, the four cDNAs and genomic DNAs sequences of C oleifera ACCase were cloned. Then, their expression patterns were analyzed in'Huashuo' and other five C. oleifera species, as well as in different tissues and developing stages of 'Huashuo' seeds. Finnally, studies of overexpression and RNA interference of them were carried out in Arabidopsis thaliana, respectively. Major research results are as follows:1. Isolation and cloning of the four cDNAs of C. oleifera ACCase subunits. Using the single-stranded cDNA generated from total extracted RNA of'Huashuo'seeds as template, the cDNAs of four ACCase subunits were cloned by the degenerate PCR, rapid amplification of cDNA ends (RACE) and touch-down PCR, and designated as Co-accB, Co-accC, Co-accD and Co-accA, respectively.(1) The Co-accB (encoded BCCP) was1153bp with an816-bp open reading frame (ORF), encoded272amino acid residues; it had the precursor and mature protein, and its plastid-processing site for C. oleifera BCCP occurs between residues Ser64-Ala65. It contained the biotinylation motif CIIEAMKLMNEIE harboring the biotinyl-Lys residue.(2) The Co-accC (encoded BC) was1638bp with a1602-bp ORF, encoded533amino acid residues. The subcellular localization of the Co-accC protein was in plastid, and the plastid-processing site for C. oleifera BC was postulated to occur between residues Arg46and Val47. It comprised four conserved motifs BC-1, ATP-binding site, BC-2and Biotin carbxylation site.(3) The partial Co-accA (encoded a-CT) was1293bp, and its amino acid residues155-190are acetyl-CoA binding domains.(4) When cloning the accD encoded β-CTsubunit of C. oleifera ACCase, a2574- bp fragment including the partial rbcL, rbcL-accD intergenic spacer and the full-length accD gene was obtained. The ORF of accD was1530bp, encoding510amino acids. It contained a zinc-binding domain and five conserved regions. The putative binding sites for Acetyl-CoA and carboxybiotin were located at amino acid residues332-346and351-368, respectively, and followed by the putative catalytic site of carboxyl-transferase at amino acid residues379-392. The nucleotide sequences of rbcL, rbcL-accD and accD shared high identity with those of rbcL, rbcL-accD and accD from other20Theacae genera species, indicating that there existed the close genetic relationship among them. The amino acid sequence of four genes for C. oleifra ACCase subunits shared some identities with thoese of the corresponding genes for ACCase subunits from G. hirsutum, A. hypogaea and G. max of oil plants, as well as from E. coli, suggesting that they belonged to the heteromeric ACCase genes; moreover, they possess the binding sites of the two-half reactions and structure domains of interaction among ACCase subunits. Their precursors of three nuclear-encoded genes aceA, accB and accC are processed and binded (3-CT to form the heteromeric ACCase with activity after the plastid processing.2. Isolation and cloning of the genomic DNA sequences of BC and β-CT subunits of the C. oleifera ACCase. According to the corresponding cDNA sequence of accC, the primers were designed to clone the genomic DNA sequences using the total DNA of 'Huashuo' leaves by the touch-down PCR. The1638-bp genomic DNA for accC was obtained, including the1602-bp ORF encoden533aa. Furthermore, the2222-bp genomic DNA sequence including rbcL, rbcL-accD intergenic and accD was isolated, and it contained the1530-bp ORF of accD. Compared with their cDNAs, the genomic DNA sequences of accC and accD were verified to be intronless. The up-stream region of the2222-bp sequence was about529bp in the length. It contained no PEP prometer sites, while had NEP prometer sites,-10PEHVPSBD, CAAT BOX1, YACT and napA E-box, etc. Genomic DNAs of BC (encoded by nuclear) and β-CT (encoded by plasid) in C. oleifera are probably considered as'small genomics', and their introns might be gradually loss. There are some differences between accD genomic DNA and cDNA, the reason might be corelated with gene family, RNA editing as well. The acquired genomic DNA sequences of accC and accD could provide the materials and theoretical basis for lipid biosynthesis and seed-oil ratio research.3. The studies of the expression pattern of the four genes for C. oleifera ACCase subunits. Total RNAs were extracted from C. oleifera matured seeds of'Huashuo'and other five C. oleifera species, and different tissues and developing stages of seeds of 'Huashuo', and used to generate the corresponding single-stranded cDNAs. Then, the reference gene GAPDH were selected from references genes of GAPDH, Actinl and UBC30, et al. According to optimized multiplex RT-PCR conditions, the expression patterns of the four ACCase genes were analyzed, and the reliability of this data was confirmed by real-time PCR analysis.(1) The expression level of accB was the highest in the pistil of 'Huashuo', next is in the8-month-old young fruits. It had a similar expression patterns in tissues of flower bud, stamen, ovary and leaf. There were no distinct differences in six varieties of C. oleifera matured seeds. There were the high expression levels in 'Hengdong17'and in'Hengdong65'.(2) The expression level of accC was higher in the8-month-old young fruits and in flower buds of'Huashuo'; there were relatively lower levels in tissues of stamen, pistil, ovary and leaf. There were distinct expression differences in the six C. oleifera matured seeds. There were high expression levels in'Hengdong17'and 'Huaxin', followed by in 'Hengdong31','Huajin','Huashuo' and 'Hengdong65'.(3) The expression amount of accA was higher in'Hengdong17'and in 'Huaxin'. Moreover, its expression amount was more in young fruits and matured seeds, while less in leaves and stems.(4) There were distinct expression differences in six C. oleifera matured seeds. There was the highest expression amount in'Huaxin'. Its transcription levels are parti call y corelated with seed-oil ratio of some C. oleifera species. The expression levels of accD were higher in leaves and developing seeds. Taken together, the four genes of heteromeric ACCase are coordinately expressed, and there exsited trends that transcription levels of the four genes are gradually increased, which are in agreement with requirement of the lipid biosynthesis in the development from flowering stage to mature in the growth and development of seeds, and with that of Ke's research. Their expressions also present two transcription peaks. The former are not abvious, while the later are relative distinct. These suggest that there are close relationships between the transcription levels of the four ACCase genes lipid biosynthesis, and thus C. oleifera ACCase may be the rate-limiting step and a key enzyme. Our results support the conclusion that the expressions of the four ACCase genes are significant to the activity and seed oil ratio, and that ACCase can be used as a marker in the breeding program. Furthermore, our research also provides a visual detection method of the four ACCase genes of C. oleifera from transcription levels.4. Overexpression of accC from C. oleifera and RNA interference (RNAi) expression of accAlaccBlaccClaccD. The overexpression pCambia1304-accC vector was constructed, and introduced into Agrobacterium tumefaciens by chemical poration, and transformed the wild Arabidopsis thaliana. Regenerated T1and T2Arabidopsis transformation lines were obtained, respectively. The interference vectors of pJawohl8-RNAi-Co-accA/Co-accB/Co-accC/Co-accD were constructed via Gateway technology, and introduced into A. tumefaciens, and transformed the wild A. thaliana. Regenerated T1Arabidopsis transformation lines of Co-accB/Co-accC/Co-accD RNAi were obtained, respectively. The rearches will provide the material and theoretical basis basis for overexpression of C. oleifera ACCase genes.In summary, the sequences of four cDNAs genes and two genomic DNAs of C. oleifera ACCase subunits were obtained. Their structure features, expression patterns and their roles in fat acid biosynthesis and lipid biosynthesis were analyzed. It provided a visual detecting method for C. oleifera variety assessment as well as regulatory measures through high yield cultivating techniques. The research will lay the fundamental theoretical basis for improving seed-oil ratio and oil production of per unit area yield and total output, and for applying of some cultivation measures for C. oleifera high yield, and thus possess the significant potential values of exploitation and application.