油菜脂肪酸延长酶基因fae1片段的克隆与SNP分析

 脂肪酸延长酶基因fae1是调控油菜芥酸合成的关键基因。本研究利用GenBank中的fae1基因序列AF490462为模板设计引物，通过多聚酶链式反应（PCR）从白菜、甘蓝和甘蓝型油菜（包括2个人工合成种）的12个不同品种中扩增出长1 007bp的fae1基因片段。PCR产物经与克隆载体pGEM-T vector连接和序列测定，获得12个品种的fae1基因片段的DNA 序列。对来自12个不同品种的fae1基因序列进行比较分析表明：fae1基因具有高度序列保守性，扩增长度均为1 007 bp，核苷酸序列相似度达98%以上，氨基酸序列的保守性更高。在1007 bp的区间内共发现23个SNP(single nucleotide polymorphism)位点，其中有11个单核苷酸变异导致了编码氨基酸的改变。人工甘蓝型油菜和天然甘蓝型油菜的fae1基因片段未发现明显差异。发现了高芥酸品种与低芥酸品种的fae1基因、以及A基因组与C基因组的fae1基因特有SNP位点。

Cloning of fae1 Gene Partial Sequence and SNP Analysis in Brassica Species

fae1 gene is a rate-limiting gene for erucic acid synthesis in Brassica. Partial sequences of the gene were cloned by polymerase chain reaction (PCR) strategy with primers F1 and R1 designed according to the DNA sequence of fae1 reported in Genbank (AF490462). Twelve cultivars with different erucic acid content from B.napus, B.oleracea and B.rapa , including 2 resynthesized B.napus lines were used as genomic DNA source materials. The PCR products were cloned into the vector pGEM-T and sequenced individually. All the 12 cultivars showed similar nucleotide sequences with a same 1007bp in length. A clustal alignment of the 12 sequences showed that fae1 gene is highly conservative among different species tested, and within the 1007bp region showed 23 sites of single nucleotide polymorphism corresponding to 11 amino acid changes at protein level. No significant difference was found between natural and resynthesized lines of B.napus. Specific SNP sites were found to distinguish fae1 genes between high and low erucic acid and between C genome and A genome.