盐藻肌动蛋白基因启动子驱动的bar基因表达作为核转化筛选标记(英文)  

The Actin Gene Promoter-driven bar as a Dominant Selectable Marker for Nuclear Transformation of Dunaliella salina

作　　者：姜国忠[1] 吕玉民[1] 牛向丽[1] 薛乐勋[1] 

机构地区：[1]郑州大学医学院细胞生物学研究室,郑州450052

出　　处：《Acta Genetica Sinica》2005年第4期424-433,共10页

基　　金：国家自然科学基因项目(编号: 30270031 );国家"863计划"项目(编号: 2002AA628050 )资助~~

摘　　要：运用基因组步行方法克隆盐藻肌动蛋白基因 5′上游调控序列,发现相对于ATG上游 -573和 -424b的位置上分别有 75bp长的两个重复序列。没有典型的TATA盒,但有两个TATA样结构、一个CCAAT结构和一个与GCTC(G/C)AAGGC一致的序列。以 700bp的盐藻肌动蛋白基因启动子区序列驱动bar基因的表达作为转化盐藻的筛选标记。转化的藻细胞暗光恢复 24h后,在含 0 5μg/mL除草剂的培养基中常规培养生长 1周,然后将细胞平铺于含 0 5μg/mL除草剂的固体培养基上继续筛选培养。约 20d后从固体培养板上挑选出 5个藻落并作了进一步培养和分析。结果显示, 5个转化藻中携带bar嵌合基因的整合位点均位于核基因组内。Southerblotting分析表明,仅有一个转化藻整合单拷贝的bar基因,而另外 4个转化藻株则包含多个拷贝bar基因片段,提示盐藻核基因转化主要是外源基因的随机整合,外源基因在转化盐藻中的整合拷贝数并不影响其除草剂抗性RT PCR方法证明了bar基因在转化藻中的转录。5个转化藻在含除草剂的液体培养基中维持生长了至少 7个月,表明核基因转化的稳定性。A 5′-flanking region of an actin gene from the green unicellular alga Dunaliella salina (D.salina) was cloned using a genome-walking method by PCR and its structural features were characterized.Two repetitive sequences found,over 75 bp in length each,were located at position -573 and -424 bp,respectively,relative to the AUG codon.The actin gene promoter region of D.salina displayed a consensus sequence of GCTC(G/C)AAGGC,a CCAAT motif and two TATA-like motifs that did not have a canonical sequence of a TATA box.The 5′flanking region of the actin gene was exploited to direct expression of the bialaphos resistance gene (bar) from Streptomyces hygroscopicus as a dominant marker in the nuclear transformation of D.salina.Direct selection of bar resistant transformants was achieved by allowing a 24 h period of recovery of cells transformed by biolistic procedure,followed by growth of the cells for one week under standard condition prior to harvesting and plating on the solid medium containing 0.5 μg/mL of phosphinothricin (PPT).Five colonies picked from the plate were analyzed,of which the integration of the bar gene was demonstrated in the nuclear genome.Southern blotting revealed that only one of five transformants contained a single copy of the bar gene whereas others contained multiple copies,suggesting that nuclear transformation of D.salina mainly occurred through illegitimate recombination events,resulting in ectopic integration of the introduced DNA.The integration patterns of the foreign DNA in this experiment appeared not to influence the bar gene expression in the transformants containing single or multiple inserts.The bar gene expression in the five transformants was verified by RT-PCR,confirming transcription of the chimeric DNA.These transformants were maintained on agar plates in the absence of PPT for more than seven months and retained resistance to the herbicide at 1 μg/mL.This work demonstrates that the actin gene promoter-driven expression of the bar gene may be used as a dominant selectable ma

关 键 词：杜氏盐藻 肌动蛋白启动子 重复序列 BAR基因 选择标记 

分 类 号：Q75[生物学—分子生物学]