基于基因组与蛋白组分析挖掘高效杀虫菌株Bt S2480-1毒蛋白基因

Exploration of insecticidal genes in Bt S2480-1:based on the analysis of genome and proteome

摘要:

[目的]鉴定苏云金芽孢杆菌野生型菌株Bt S2480-1的杀虫活性并挖掘该菌株含有的杀虫基因资源。[方法]采用Illumina Hiseq2000测序平台对Bt S2480-1菌株进行全基因组测序,通过生物信息学方法获得该菌株的基因组信息、基因预测以及毒蛋白基因预测识别;随后,利用LTQ-Obitrap nano-LC-MS/MS系统对该菌株的总蛋白进行了质谱分析;最后以致倦库蚊幼虫和甜菜夜蛾幼虫为靶标昆虫对该菌株进行了生物活性测定。[结果]Bt S2480-1基因组大小为6.2 Mb,GC含量为35.11%,拼接得到1个拟核和3个大质粒的可视化草图,预测编码基因6 297个,其中含有12个预测毒蛋白基因。Bt S2480-1菌株总蛋白在LTQ-Orbitrap MS/MS质谱分析中共有 1500个蛋白质获得鉴定,鉴定获得11个毒蛋白。Bt S2480-1菌株总蛋白对致倦库蚊幼虫表现出非常高的杀蚊活性,而对甜菜夜蛾幼虫杀虫活性相对偏弱,其LC₅₀分别为27.636 μg/mL (95% FL:12.559‒61.707μg/mL)和496.833 μg/mg (95% FL:320.134-776.964μg/mg)。[结论]Bt S2480-1菌株基因组分析显示共含有12个预测毒蛋白基因,LTQ-Orbitrap MS/MS鉴定获得11个毒蛋白,Bt S2480-1菌株总蛋白对致倦库蚊和甜菜夜蛾幼虫都具有杀虫活性。

关键词:苏云金芽孢杆菌;Bt S2480-1;毒蛋白;LTQ-Obitrap;杀虫活性

Abstract:

[Objective]To identify the insecticidal activity of wild-type Bacillus thuringiensis strain Bt S2480-1 and explore the insecticidal gene resources.[Methods]The whole genome of Bt S2480-1 was sequenced by Illumina Hiseq2000.The genome information,genes,and insecticidal genes of the strain were analyzed with bioinformatics methods.The total proteins of Bt S2480-1 were analyzed by LTQ-Obitrap nano-LC-MS/MS and the biological activity of Bt S2480-1 against Culex quinquefasciatus and Spodoptera exigua larvae was determined.[Results]The genome of Bt S2480-1 was 6.2 Mb,with GC content of 35.11%,and one nucleoid and three large plasmids were yielded through assembly,containing 6 297 coding genes with 12 encoding insecticidal proteins.Meanwhile,a total of 1 500 proteins of Bt S2480-1 were identified by LTQ-Orbitrap MS/MS,among which 11 were insecticidal proteins.The total proteins of Bt S2480-1 showed strong activity against C.quinquefasciatus larvae but relatively weak activity against S. exigua larvae,with LC₅₀ of 27.636 μg/mL (95% FL:12.559-61.707 μg/mL) and 496.833 μg/mg (95% FL:320.134-776.964 μg/mg),respectively.[Conclusion] Bt S2480-1 contains 12 genes encoding insecticidal proteins and displays insecticidal activity against C. quinquefasciatus and S.exigua larvae.

Key words:Bacillus thuringiensis;Bt S2480-1;insecticidal protein;LTQ-Orbitrap;insecticidal activity