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溶血磷脂酰基转移酶(LPAT)是植物油脂合成途径的一个关键酶,在植物油脂品质改良和提高种子含油量方面具有重要的应用价值。本研究通过构建花生种子全长cDNA文库,结合大规模EST测序和功能注释,从花生中克隆了溶血磷脂酸酰基转移酶基因,命名为AhLPAT。该基因cDNA全长1629bp,对应的基因组序列5531bp,由11个外显子和10个内含子组成,内含子剪接方式符合GT-AG剪接规则。根据编码区预测AhLPAT编码一条387个氨基酸组成的多肽,预测分子量为43.2kD,等电点为9.42。AhLPAT蛋白含有一个典型的酰基转移酶保守功能结构域以及溶血磷脂酰基转移酶相似的保守区域。该蛋白的氨基酸序列与已报道的其他物种LPAT蛋白序列有较高的一致性。AhLPAT与旱金莲、油菜、海甘蓝、蓖麻和拟南芥的LPAT蛋白氨基酸相似性依次为90%、89%、89%、88%和87%。系统进化分析表明,AhLPAT与拟南芥AtLPAT2亲缘关系较近,且同属于内质网型LPAT蛋白。RT-PCR分析表明,AhLPAT基因在花生根、茎、叶、花、果针和种子中均有表达,在花生开花后50～60d,果针和种子中的表达量最高,且AhLPAT的表达量与花生种子含油量积累速率变化一致,二者显著相关(r=0.63,P<0.05)。推测AhLPAT基因在花生种子油脂合成中起重要作用。
Lysophosphatidyl transferase (LPAT) is a key enzyme in plant lipid synthesis, which has important application value in improving the quality of vegetable oils and improving the oil content of seeds. In this study, we constructed the full-length cDNA library of peanut seeds, combined with large-scale EST sequencing and functional annotation, cloned lysophosphatidic acid acyltransferase gene from peanut, named AhLPAT. The full-length cDNA of this gene is 1629bp in length, corresponding to 5531bp of genomic sequence, consisting of 11 exons and 10 introns. Intron splice conforms to GT-AG splicing rules. According to the coding region, a 387-amino acid polypeptide encoded by AhLPAT was predicted, with a predicted molecular weight of 43.2 kD and an isoelectric point of 9.42. The AhLPAT protein contains a conserved functional domain of a typical acylase and a similar conserved region of lysophosphatidyl transferase. The amino acid sequence of this protein is consistent with other reported LPAT sequences. The amino acid identities of LPAT protein between AhLPAT and Nasturtium officinale, Brassica campestris, Brassica napus, castor bean and Arabidopsis were 90%, 89%, 89%, 88% and 87%, respectively. Phylogenetic analysis showed that AhLPAT is closely related to Arabidopsis AtLPAT2 and belongs to endoplasmic reticulum LPAT protein. The results of RT-PCR showed that AhLPAT gene was expressed in roots, stems, leaves, flowers, fruit needles and seeds of peanut. The expression level of AhLPAT was highest in fruit needles and seeds 50- 60 days after flowering, It was consistent with the changes of peanut seed oil accumulation rate (r = 0.63, P <0.05). It is speculated that the AhLPAT gene plays an important role in peanut oil synthesis.