甜橙液泡转化酶基因(CSVI)的分离及全序列分析  

Isolation and Characterization of Citrus sinensis Vacuolar Invertase Gene (CSVI)

作　　者：安新民[1] 张上隆[1] 徐昌杰[1] 陶俊 秦巧平[1] 

机构地区：[1]浙江大学 [2]扬州大学,扬州225009

出　　处：《林业科学》2004年第5期99-104,共6页Scientia Silvae Sinicae

基　　金：国家自然科学基金重点项目 (3 973 0 3 40 );面上项目 (3 0 170 64 8)资助。

摘　　要：根据不同植物液泡转化酶基因序列的保守区设计合成引物 ,采用滤纸吸附噬菌体PCR法 ,首次从甜橙基因组文库筛选出含甜橙液泡转化酶基因的阳性λ噬菌体 ,经过大量培养并提取其DNA ,限制性内切酶消化后进行DIGsouthern印迹分析 ,将阳性条带回收、加工并克隆测序。序列分析表明 ,该基因全长 4 72 2bp ,编码 5 88个氨基酸 ,包括 6个外显子和 5个内含子。在GenBank进行Blast检索的结果表明 ,该基因编码的氨基酸序列与马铃薯、番茄和酸樱桃液泡转化酶基因编码的氨基酸序列同源性分别为 6 8%、6 8%和 6 2 %。系统进化关系聚类分析结果表明 ,该基因属液泡转化酶基因类型。该基因已经在GenBank登录 (登录号 :AF4 336 4 3)。该基因的获得为从分子水平研究柑桔果实发育过程中蔗糖积累和分解机制以及通过基因工程改良果实品质奠定了基础。A pair of primers, designed from conserved regions of plant vacuolar invertase genes, was used to screen Citrus sinensis vacuolar invertase gene ( CSVI) from C.sinensis genomic library. The positive λ phage was isolated by filter paper absorption phage PCR, and cultured largely. DNA from the positive λ phage was digested with several restriction enzymes. The positive fragments judged by DIG southern blotting analysis, were reclaimed, modified, cloned into pUC19 and pUCm_T, and sequenced respectively. The result indicated that the sequence was 4 722 bp including 6 exons and 5 introns, coding 588 amino acids. It was found to have 68%, 68% and 62% homology respectively to Solanum tuberosum (X70368), Lycoperscion esculentam (Z12025) and Prunus cerasus (AY048579) vacuolar invertases by blast analysis in GenBank. Meanwhile, it was subclassed into plant vacuolar invertase by phylogenetic relation analysis. CSVI had been registered in GenBank (Accession No. AF433643). This work will contribute to a study on the molecular mechanisms of accumulation and catabolism of sucrose during \%Citrus\% fruit development, and improvement of Citrus fruit quality by gene engineering.

关 键 词：甜橙 液泡转化酶 基因 分离 全序列分析 蔗糖 积累机制 分解机制 

分 类 号：S666.4[农业科学—果树学]