CAJ | 학술논문

　　本研究将高分辨熔解分析（high resolution melting assay，HRMA）的方法应用于cry1I类基因的分型。根据已报道的cry1I类基因的保守区设计一对扩增片段为131 bp的通用鉴定引物，利用高分辨熔点仪对产物的熔解曲线进行分析。利用此方法对本实验室保存的标准菌株HD-12进行了cry1I类基因的鉴定，克隆到2个新型的cry1Ib和cry1Id基因，被Bt国际命名委员会分别正式命名为cry1Ib11和cry1Id2。这两种基因在E. coli Rosetta菌株中能正常表达81 kDa的蛋白。Cry1Ib11和Cry1Id2蛋白对小菜蛾Plutella xylostella 二龄幼虫和亚洲玉米螟 Ostrinia furnacalis 初孵幼虫具有显著的杀虫活性，LC50分别为6.13μg·mL?1和6.10μg·mL?1及11.18μg·mL?1和1.43μg·mL?1。但对甜菜夜蛾Spodoptera exigua及鞘翅目的大猿叶甲Colaphellus bowringi均没有明显毒杀作用。高分辨溶解分析法为新型cry基因的快速、准确鉴定提供了有效的工具。
　　본연구장고분변용해분석（high resolution melting assay，HRMA）적방법응용우cry1I류기인적분형。근거이보도적cry1I류기인적보수구설계일대확증편단위131 bp적통용감정인물，이용고분변용점의대산물적용해곡선진행분석。이용차방법대본실험실보존적표준균주HD-12진행료cry1I류기인적감정，극륭도2개신형적cry1Ib화cry1Id기인，피Bt국제명명위원회분별정식명명위cry1Ib11화cry1Id2。저량충기인재E. coli Rosetta균주중능정상표체81 kDa적단백。Cry1Ib11화Cry1Id2단백대소채아Plutella xylostella 이령유충화아주옥미명 Ostrinia furnacalis 초부유충구유현저적살충활성，LC50분별위6.13μg·mL?1화6.10μg·mL?1급11.18μg·mL?1화1.43μg·mL?1。단대첨채야아Spodoptera exigua급초시목적대원협갑Colaphellus bowringi균몰유명현독살작용。고분변용해분석법위신형cry기인적쾌속、준학감정제공료유효적공구。
High resolution melting assay was used to identify cry1I genes in this study. A pair of universal oligonucleotide primers was designed to amplify conserved region fragment with 131 bp of cry1I-type genes, and then PCR products were analyzed by light scanner according to the melting curve of amplified DNA fragments. cry1I genes were identified from Bacillus thuringiensis HD-12 strains on the basis of the differences in melting-peak curve shapes. Two cry1I genes were revealed and cloned respectively, and they were designated as cry1Ib11 and cry1Id2 by International Nomenclature Committee of Bt Endotoxin respectively. cry1Ib11 and cry1Id2 genes could be expressed normally with 81 kDa molecular weight in E. coli Rosetta strain. The two toxins had significantly insecticidal activity against Plutella xylostella second instar larvae with LC50 of 6.13μg·mL?1 and 6.10μg·mL?1 and against Ostrinia furnacalis neonates with LC50 of 11.18μg·mL?1 and 1.43μg·mL?1, respectively. However, it did not show significant activity against neonates of Spodoptera exigua and Colaphellus bowringi. The high resolution melting assay will provide an effective tool for identification of novel cry genes rapidly and accurately.