柑桔酸性转化酶基因家族成员的克隆及特性分析

分析植物液泡和细胞壁酸性转化酶基因的保守区序列 ,设计 2对PCR引物 ,以温州蜜柑基因组DNA为模板 ,采用PCR方法扩增出长分别为 74 1bp(A)和 5 2 4bp(B)的 2个DNA片段 ,克隆入pUCm T载体测序 ,序列已在GenBank中登记 (登记号分别为AY0 2 94 81和AF332 881)。在GenBank中进行同源性检索 ,结果表明片段A编码的氨基酸与植物液泡酸性转化酶同源性较高 ,与胡萝卜、马铃薯和番茄同源性分别为 79%、79%和 78%。片段B编码的氨基酸序列与草莓、拟南芥和豌豆细胞壁转化酶同源性分别为 78%、78%和 77%。推测A和B分别定位于液泡和细胞壁。运用Clustalx和Bioedit软件包对已获得的柑桔转化酶基因家族的 7个基因序列进行分析 ,结果表明 7个基因分属转化酶基因家族的 4种类型。推测A和B为 2个新成员 ,分别属于柑桔液泡酸性转化酶基因Ⅱ型(CUAI2 )和细胞壁酸性转化酶基因Ⅱ型 (CUCWI)。Southern杂交结果表明 ,CUCWI和CSCWI 2个探针有时会出现较弱的杂交干扰信号 ,可采用CSCWI与CUCWI同源性较低的另一段序列制作探针进行杂交 ,也可通过缩短杂交时间、提高杂交和洗膜的温度以及降低洗膜SSC的浓度等措施来降低 ;其它成员之间无干扰信号产生

Cloning and Characterization of Members of Acid Invertase Gene Family in Citrus

Two pairs of primers, designed from conserved regions of plant vacuolar and cell wall bound acid invertase genes respectively, were used to amplify 741bp (A) and 524 bp (B) fragments from Citrus unshiu genomic DNA by polymerase chain reaction (PCR). The fragments were cloned into pUCm-T, and then sequenced. The deduced amino acid sequence of fragment A is 79%, 79%, 78% identical to genes from Daucus carota (X75353), Solanum tuberosum (X70368) and Lycopersicon esculentum (Z12025)respectively, and was suggested to encode a soluble acid invertase located in vacuole, while B is 78%, 78%, 77% identical to genes from Fragaria ananassa (AF000521), Arabidopsis thaliana (AP001307) and Pisum sativum (X85327) respectively, and was suggested to encode a insoluble acid invertase in cell wall. Lower homology among A, CUAI1, B and CSCWI suggested that A and B were two new members of acid invertase gene family in Citrus, respectively belong to putative CUAI2 and CUCWI. There was no interaction among these members (CUAI1, CUAI2, CUCWI or CSCWI) except for CUCWI and CSCWI in Southern blot analysis. The interaction between CUCWI and CSCWI could be eliminated by using CSCWI's partial sequence which was lower homologous to CUCWI as probe, and weakened by shortening hybridization time, increasing hybridization and washing temperature, washing membrane in lower concentration of SSC.