Cry1Ab13杀虫基因的PCR诱变及功能验证  

Verification of PCR mutagenesis and function of Cry1Ab13 insecticidal gene

作　　者：闫鸽 宋阳[1] 张宇婷[2] 张卓[1] 代力强[1] 王丕武[1] YAN Ge;SONG Yang;ZHANG Yuting;ZHANG Zhuo;DAI Liqiang;WANG Piwu(Agricultural College,Jilin Agricultural University,Changchun,J ilin 130118,China;Agricultural College,Yanbian University,Yanji,Jilin 133002,China)

机构地区：[1]吉林农业大学农学院,吉林长春130118 [2]延边大学农学院,吉林延吉133002

出　　处：《西北农林科技大学学报（自然科学版）》2018年第5期141-148,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家转基因生物新品种培育重大专项(2016ZX08004-004)

摘　　要：【目的】通过易错PCR技术克隆一种新的Cry1Ab13基因,以利用Cry类Bt基因提高作物抗虫性,丰富基因资源。【方法】利用易错PCR技术对苏云金芽孢杆菌Cry1Ab13基因进行随机诱变,构建突变体文库,筛选获得突变株Cry1Ab13-1,将该基因构建原核重组表达载体后转化到大肠杆菌中进行异源表达,并利用表达的目的蛋白进行抗虫鉴定。【结果】序列分析表明,突变株Cry1Ab13-1基因序列全长2 036bp,与原始碱基序列的一致性为97.79%,有2个碱基发生突变,导致该基因编码的氨基酸序列中有2个氨基酸改变,分别是第130位由脯氨酸变成丝氨酸,第383位由丝氨酸变成苏氨酸;改变后的氨基酸序列与原始氨基酸序列一致性为96.97%。在线分析表明,该基因与已知过敏源的序列同源性低于35%,不存在致敏性。SDS-PAGE电泳分析表明,表达蛋白分子质量大小为79.5ku,与预期结果相符。抗虫鉴定表明,处理72h时表达的目的蛋白对小菜蛾(Plutella xyllostella)幼虫的致死率达87.88%,明显高于未突变基因对照组的幼虫死亡率;对亚洲玉米螟(Ostrinia nubilalis)幼虫的致死率达81.11%,而且存活的亚洲玉米螟幼虫的生长发育受到明显抑制。【结论】成功克隆了Cry1Ab13-1抗虫基因,且该基因对小菜蛾幼虫和亚洲玉米螟幼虫均具有高效的杀虫活性。【Objective】A novel Cry1 Ab13 gene was cloned successfully by error-prone PCR technology to enrich gene resources for improving crop insect resistance using the Cry-type Bt gene.【Method】The experiment conducted random mutagenesis on bacillus thuringiensis Cry1 Ab13 gene with error-prone PCR technology,built mutant library,and obtained mutant strain Cry1 Ab13-1,which was transformed into Escherichia coli for heterologous protein expression.The protein encoded with Cry1 Ab13-1 gene was used for identification of insect resistance.【Result】The sequence analysis showed that the length of mutant strain Cry1 Ab13-1 gene sequence was 2 036 bp,with the consistency of 97.79%to original base sequence.There were two bases mutates among the amino acid sequence,causing two amino acids changes at the130 th from proline to serine and the 383 th from serine to threonine.The consistency between the changed and original amino acid sequences was 96.97%.The on-line analysis showed that the homology between the gene and the known allergen sequence was less than 35% without sensitization.The SDS-PAGE electro-phoretic showed that the molecular weight of expressed protein was about 79.5 ku,which was coincident with expectation.Insect resistance identification showed that the larval fatality rate of expressed objective protein on Plutella xyllostella reached 87.88%,significantly higher than the non-mutant gene control group.The larval fatality rate of Asian Ostrinia nubilalis reached 81.11%,and the growth and development of survival Asian Ostrinia nubilalis larval were significantly inhibited.【Conclusion】This study successfully cloned Cry1 Ab13-1 insect gene and the gene had efficient insecticidal activity against Asian corn borer larvae and diamondback moth larvae.

关 键 词：易错PCR Cry1Ab13基因 原核表达 玉米螟 小菜蛾 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治] Q784[农业科学—植物保护]