苏云金芽孢杆菌cry基因芯片检测方法的研究  

Study of Microarray Detection Method of cry Genes from Bacillus thuringiensis

作　　者：刘旭光[1,2] 宋福平[1] 文思远[3] 王升启[3] 黄大昉[4] 张杰[1] 

机构地区：[1]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室 [2]连云港师范高等专科学校初教系,连云港222206 [3]军事医学科学院放射医学研究所/全军生物芯片重点实验室 [4]中国农业科学院生物技术研究所

出　　处：《中国农业科学》2004年第7期987-992,共6页Scientia Agricultura Sinica

基　　金："973"计划资助项目(2003CB114201;2001CB109005)

摘　　要：建立了一种不经PCR扩增而直接应用寡核苷酸芯片从苏云金芽孢杆菌总DNA中检测cry基因的方法。对探针长度、浓度、总DNA含量、标记方法以及灵敏度等条件进行了研究。结果表明,用高浓度Klenow酶随机标记高浓度的总DNA与寡核苷酸探针(40～50mer、40μmol·L-1)杂交可用于检测Bt菌株中含有的cry基因;不同长度探针之间对杂交信号的影响存在显著性差异(P<0.01),而不同浓度的探针之间没有显著性差异。此方法的其它条件为杂交温度42℃、时间3h;检测的样品总DNA的灵敏度约为2.5μg;标记产物杂交用量(0.625～20μg)的对数值与信号值的对数值之间存在明显的线性关系(r2=0.985～0.989)。In order to directly detect cry genes from total DNA of Bacillus thuringiensis, a new method, which canidentify cry genes based on oligonucleotide microarray hybridization, but without amplification, has been established. Theapproach involves fluorescent labeling for high concentration of total DNA using high concentration Klenow fragment,and hybridization to the oligonucleotide probes(40-50 mer, 40molL-1) specific for each cry gene. Other key parameterssuch as length, and concentration of probe, content of total DNA and labeling method were also studied. The resultsshowed the sensitivity difference among the length of probe (P<0.01) was notable, but no significant sensitivity differenceamong the concentration of probe. The condition of hybridization was 42℃ and 3 hours. The detection limit with randomlylabeled total DNA under these conditions was estimated to be approximately 2.5g; A linear quantitative relationship (r2 =0.985 to 0.989) was observed between signal intensity and labeling sample concentration over a range of 0.625-20g.

关 键 词：苏云金芽孢杆菌 CRY基因 基因芯片 检测方法 杀虫晶体蛋白 生物防治 

分 类 号：S476[农业科学—农业昆虫与害虫防治]