苹果液泡酸性转化酶基因片段的克隆及序列分析

在分析植物可溶性转化酶基因的保守区序列基础上，设计了一对PCR引物，以苹果(辽伏)基因组DNA为模板，采用PCR方法扩增出长约743bp的DNA片段，克隆入pUCm—T载体，测序结果表明，所获得的片段推测为苹果酸性转化酶基因的片段，该基因片段长度为743bp，是开放阅读框的一部分，无内含子。其序列已在GenBank中登记(登记号为AF433644)。在Gen-Bank中进行同源性检索，结果表明该成员编码的氨基酸与植物可溶性酸性转化酶的氨基酸同源性较高，与温州蜜柑(GenBank AF014925)、胡萝卜(X67163)、甜菜(GenBank X81796)和绿豆(GenBank D10265)液泡酸性转化酶基因编码蛋白质的氨基酸分别具有80％、80％、78％和77％的同源性。而与定位于细胞壁的酸性转化酶(不溶性)同源性较低，由此可推测出研究获得的苹果转化酶基因可能定于液泡。该基因片段的获得对于深入研究苹果蔗糖代谢以及果实发育和品质形成具有重要的意义。

COLONING AND ANALYSIS OF THE VACUOLAR ACID INVERTASE GENE FRAGMENT FROM MALUS PUMILA

A pair of primers was designed according to the conserved sequences of plant so luble acid invertase genes, and a 743bp fragment was amplified by polymerase cha in reaction (PCR) using the genomic DNA of Malus pumila (Liao Fu) as a templa te. The fragment was cloned into pUCm T, and then sequenced. The results suggest t h at this fragment is a part of open reading frame(ORF), and there is no intron in this fragment. The sequence has been registered on GenBank (AF433644), and show s much more homology to soluble than to insoluble invertases of plants by Blast in GenBank. The deduced amino acid sequence of the invertase gene fragment is 80 %? 80%? 78%? 77% identical to that of the proteins from Citrus unshiu (AF0149 25) , Daucus carota (X67163), Beta vulgaris (X81796)and Mung bean (D10265) , respective ly.The gene is putatively recognised as a soluble acid invertase(vacuolar acid i nvertase) gene. Our results are important for study on transportation and metabo lism of sucrose, development and quality of apple fruit.