Single Nucleotide Polymorphism Analysis Of Acetyl-CoA Carboxylase Gene In Brassica Napus. L

The acetyl-CoA carboxylase (ACCase), which is the rate-limiting enzyme of fatty acid synthesis, catalyzes the first and ATP dependent reaction of fatty acid biosynthesis in plants, using biotin as prosthetic group and HCO₃^- as carboxyl donor, forming malonyl CoA from acetyl CoA. This enzyme, both at the level of enzyme quantity and at the level of activity, controls the biosynthesis of fatty acids in plants. It is one of the essential enzymes in plants survival.Oilseed rape is one of the five major oil plants in the world and one of the most important oil plants in China as well, occupying an important position in agriculture in China.Single nucleotide polymorphism (SNPs) is the variations of single nucleotide in the genome. It has been viewed as the fourth generation of molecular markers, succeeding restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR), due to its remarkable number, wide distribution, high stability and easy typing.In recent years Brassica napus has attracted many researchers' interests in that it has many good properties especially high oil content. And breeding for high oil content oilseed rape varieties has been the one of the important focuses in oilseed rape investigation. The relationship between the structure of ACCase gene and high oil content of oilseed rape has not been elucidated. This study is to identify the SNP markers and their features among the 12 materials, and analyze the possible SNP markers for high oil content trait. And thus provide a rationale for molecular marker based high oil content breeding in oilseed rape.In this research, 12 inbred homozygous rape lines with different oil content were selected, among which six are in high oil content (42.91～49.75 %) and six in low oil content group (22.5～25.9 %) Their ACCase gene were cloned and sequenced, SNP markers were identified and their features especially in relation to high oil content trait were analyzed. Results are as follows:1. Partial sequences of ACCase gene in Brassica napus L were cloned and sequencedAccording to the gene sequence of homomeric type ACCase of Brassica napus L(AJ13866), nine pairs of overlapping primers with expected PCR product size of about 800—1200bp were designed for nine sections of the gene, which encompasses most of the coding regions.The ACCase genes in the 12 materials were all efficiently and specifically amplified. Through cloning and sequencing, the ACCase gene sequences for all the materials tested were obtained.2. Single nucleotide polymorphism analysis of ACCase gene in Brassica napus LSequences of these PCR fragments were bidirectionally sequenced; and these sequences were subject to CLUSTAL analysis for SNP markers in DNASTAR software package; and then the SNP marker analysis results were subject to polymorphism analyses in DNASP software.CLUSTAL alignment of the 12 sequences of Brassica napus L showed 318 SNPs in 88824bp gene sequence; and the frequency of SNP in ACCase gene is 1/279, 179 of which are in the coding region and 138 are in non-coding region; the number of SNPs in coding region of ACCase is less than in non-coding region; and the value of Ka/Ks is less than 1, suggesting that ACCase gene in Brassica napus L is relatively conservative among the materials tested.3. Relationship between the single nucleotide polymorphism of ACCase gene and oil content in Brassica napus A high-frequency variation region in the high-oil group in sharp contrast to the low oil content group was identified with the sliding window analysis of DNASP software. This region is between 5000 to 6500 bp (corresponding to 9088 ¹0588bp of the complete gene of ACCase), which covers 4 exons (from 24 to 27) and 3 introns (from 24 to 26). This segment codes for the animo acid sequence between position 989 to 1384 (396 amino acid residues in total) of the whole ACCase.This segment of amino acid sequence is between the BCCP domain and CT domain of the ACCase. So far no concrete function has been reported for this segment. It is conjectured that the high frequency variation in this region may somehow increase the activity of ACCase and thus the oil content in rapeseeds. This relationship between the SNPs of ACCase gene and the oil content of rapeseed needs further studies to conclude.