柑桔转化酶基因家族新成员的克隆和序列分析

Cloning and sequencing of a novel mem ber of citrus invertase gene family

分析植物可溶性转化酶基因的保守区序列,设计一对PCR引物,以甜橙基因组DNA为模板,采用PCR方法扩增出长约1000bp的DNA片段,克隆入pUCm-T载体,测序结果表明获得柑桔酸性转化酶基因家族的一个新的成员,基因片段长1096bp,包括4个外显子和3个内含子。其序列已在GenBank中登记(登记号为AF433643)。在GenBank中进行同源性检索的结果表明,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,与宽皮柑桔(GenBank登记号,AF312229)可溶性酸性转化酶基因编码的氨基酸具有77%的同源性,而与定位于细胞壁的(不溶性)酸性转化酶同源性较低,最高仅33%(Fragariaananassa,GenBankAF000521)。聚类分析结果表明该成员与我们已报道的2个柑桔酸性转化酶基因不同,推测我们获得的基因是转化酶基因家族的又一新成员(CS-VI1)。它与CU-AI1和CSCWI-1的核苷酸同源性分别为45.28%和44.85%,氨基酸同源性分别为27.58%和10.35%,3个成员间核苷酸和氨基酸序列较低的同源性,不会在South-ern、Northern和Western杂交中产生交叉干扰反应。

A pair of primers,designed from conserved regions of plant soluble acid i nvertase genes,was used to amplify 1096bp fragment by polymerase chain reaction(PCR).The fragment was cloned into pUCm-T,and then sequenced.It includes fou r exon and three introns.The deduced amino acid sequence is 77%identical to the gen e from Citrus reticulata(GenBank ,AF312229),and only 33%identical to the gene from Fragaria ananassa(GenBank AF000521),was suggested to encode a soluble acid invertase.It is suggested that a noved member of citrus acid invertase gene family was o btained.There are no cross reaction between it with two members(CUAI-1,CSCW-I)of citrus acid invertase gene family in Southern,Northern and Western bl ot analysis because of lower identity t o each others.