光杆菌(NLK-1) Txp40毒蛋白基因克隆表达及预测

Cloning, expression and prediction of insecticidal toxin Txp40 from Photorhabdus luminescens NLK-1

摘要:

【背景】光杆菌存在于嗜菌异小杆线虫肠道内，并与其互惠共生，其能够产生多种高效、广谱的杀虫蛋白及毒素，是近年来继苏云金芽胞杆菌(Bt)之后挖掘新型杀虫蛋白及杀虫基因的热点研究对象。【目的】克隆Photorhabdus luminescens (NLK-1) Txp40毒蛋白基因，分析其与已知其他同属共生菌相似毒蛋白在基因序列、蛋白组成、理化性质及构象的区别，构建原核表达载体并转化大肠杆菌进行诱导表达，初步测定其杀虫活性。【方法】采用侵染的大蜡螟幼虫血腔直接分离初生型共生细菌，根据已报道的序列经比对分析设计引物，扩增目的基因，连接克隆质粒pMD19-T后测序，利用Expasy在线ProtParam tool预测其基本理化特性参数，NPS@-Network Protein Sequence Analysis在线工具进行二级结构预测。通过克隆、酶切、连接目的基因在pET28a原核表达载体上，转化大肠杆菌BL21中，利用蓝白斑筛选阳性克隆，测序验证后进行IPTG诱导表达；菌体超声破碎离心，以毒蛋白含量较高的上清溶液对大蜡螟幼虫进行饲喂和血腔注射毒性测定。【结果】Photorhabdus luminescens (NLK-1) Txp40 毒蛋白基因全长为1 008 bp，与已知相关基因的序列相似性为94%，与已知40 kD相关蛋白的氨基酸相似性达到99%，分子量37.9 kD，pI 8.37，二级结构预测表明其主要由α螺旋35.71%，无规卷曲54.46%，延伸链9.52%组成，跨膜区域与已知蛋白基本相似，克隆构建了原核表达载体pET28a-(NLK-1) Txp40，SDS-PAGE分析其在38 kD处有特异条带，蛋白分子量与预测值基本一致，且表达相对单一，表达量较高。Photorhabdus luminescens (NLK-1) Txp40蛋白对大蜡螟幼虫具有较高的血腔毒性，大蜡螟幼虫注射5 μL蛋白粗提液剂量下48 h内致死率达100%，未发现胃毒活性。【结论】获得Photorhabdus luminescens (NLK-1) Txp40毒蛋白基因，比对、分析了与已知基因在序列组成、蛋白基本理化性质和二级结构的异同，构建了原核表达载体并成功诱导表达，验证了Photorhabdus luminescens (NLK-1) Txp40毒蛋白具有较高的大蜡螟幼虫血腔毒性，为进一步发掘Photorhabdus luminescens (NLK-1)中的杀虫功能基因和蛋白奠定基础。

Abstract:

[Background] Photorhabdus sp. is preserving in the intestine of Heterorhabditis sp., and it can produce variety efficient and broad spectrum insecticidal proteins and toxins. In recent years, it’s the novel research object on new insecticidal protein and gene after Bacillus thuringiensis. [Objective] To clone Txp40 toxin gene of Photorhabdus luminescens (NLK-1) and analyze the differences on gene sequence, protein composition, physicochemical properties and conformation similarity to other symbiotic bacteria. Txp40 toxin gene was cloned in prokaryotic expression vector of Escherichia coli and induced its to express, then utilized Galleria mellonella larvae to preliminary detect its insecticidal activity. [Methods] The primary symbiotic bacteria were isolated from the infected larvae by Heterorhabditis bacteriophora, and the primers were designed according to the reported sequences. The target gene was amplified and sequenced by the cloning plasmid pMD19-T. The Expasy Online ProtParam Tool to predict its basic physical and chemical properties parameters, NPS@-Network Protein Sequence Analysis online tools for secondary structure prediction. Txp40 toxin gene was detected by cloning, digestion and ligation on pET28a and transformed into Escherichia coli BL21 then induced by IPTG, and identified by blue-white blotting. Utilizing Photorhabdus luminescens (NLK-1) Txp40 protein crude extract to test the insecticidal activity to Galleria mellonella larvae by fed and hemocole injection. [Results] Txp40 toxin gene open reading frame is 1 008 bp (335 amino acid), and the similarity to the known gene was 94%. The similarity with the known 40 kD related protein was 99%, molecular weight was 37.9 kD, pI 8.37, and it was mainly composed of Alpha helix 35.71%, Random coil 54.46%, Extended strand 9.52% by secondary structure predicting. The expression of pET28a-Txp40 was detected by SDS-PAGE, which showed that there were specific band at 38 kD, and the molecular weight of protein was similar with prediction, and the expression was relatively single and the level was higher. The insecticidal activity tests showed that Photorhabdus luminescens (NLK-1) Txp40 protein had high virulence to Galleria mellonella larvae, and the lethality reached 100% at 48 h under 5 μL Txp40 protein crude extract per larvae, but oral toxicity was not detected. [Conclusion] Photorhabdus luminescens (NLK-1) Txp40 toxin gene was successfully obtained, and the physicochemical properties of the protein were analyzed by comparison to the known gene, and secondary structure were predicted and analyzed to other known protein. At the same time, the prokaryotic expression vector was constructed, the preliminary insecticidal test showed Photorhabdus luminescens (NLK-1) Txp40 protein had high hemocole toxicity to Galleria mellonella larvae. The research laid the foundation for the further study and discovery of insecticidal genes and toxic protein from Photorhabdus luminescens (NLK-1).