SECONDARY METABOLITES FROM Bacillus thuringiensis HS66
SECONDARY METABOLITES FROM Bacillus thuringiensis HS66
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DOI：
10.1007/s10600-022-03687-w
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发表时间：
2022-05-02
影响因子：
0.8
通讯作者：
Zhang, Wen-Fei
中科院分区：
文献类型：
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作者：
Bai, Meng;He, Jia-Li;Zhang, Wen-Fei
苏云金芽孢杆菌是一种普遍存在的革兰氏阳性土壤寄居细菌[1]，属于芽孢杆菌属，与其他物种如蜡状芽孢杆菌、炭疽芽孢杆菌和蕈状芽孢杆菌[2-4]具有许多共同的表型和基因型特性。B最显著的特性。苏云金芽孢杆菌是其产生伴孢杀虫蛋白，所述伴孢杀虫蛋白对鳞翅目（蛾和蝴蝶）、双翅目（蚊子和黑蝇）、鞘翅目（甲虫）和同翅目（蚜虫、粉虱）中的许多昆虫物种有毒。由于其对人类的安全性，B。苏云金杆菌（Bt）是世界上使用最广泛的环境相容性生物农药[5]。然而，B.苏云金杆菌尚未报道。为从B中寻找具有生物活性的化学成分。从苏云金杆菌B的乙酸乙酯萃取物中分离得到12个化合物。苏云金杆菌分离的化合物经鉴定为环（L-Pro-L-Tyr）（1）[6]、环（Pro-Phe）（2）[7]、环（L-Pro-L-Phe）（3）[8]、环（Pro-Val）（4）[9]、环（L-Pro-L-Met）（5）[10]、环（（S）-Pro-（R）-Ile）（6）[11]、环（L-Pro-L-Leu）（7）[12]，N-（2-羟基苯基）乙酰胺（8）[13]、尿嘧啶（9）[13]、2-苯基乙酰胺（10）[14]、邻氨基苯甲酸（11）[15]和S-（4-羟基苯基）乳酸（12）[16]。对棉铃虫初孵幼虫进行了生长抑制试验。化合物1-3、6、7和10对棉铃虫初孵幼虫具有生长抑制活性，IC 50值为50 ~ 200 μg/mL。印楝素作为阳性对照，IC 50值为25 μg/mL。将军在Bruker Av 400 NMR光谱仪上记录1D（1H NMR，13 C NMR，DEPT）和2D（1H-1H COSY，NOESY，HSQC，HMBC）NMR光谱。ESI-MS谱在Agilent 1200系列HPLC上记录，所述HPLC与配备有电喷雾电离源的Bruker Esquire 6000离子阱质谱仪连接。HPLC分离在高效液相色谱仪（Eclipse XDB-C18，10× 250 mm，5 μm）中进行。硅胶和GF 254购自青岛海洋化工厂，Sephadex LH-20购自Pharmacia Co. Ltd.实验材料。从海南吊罗山自然保护区土壤样品中筛选分离出苏云金杆菌HS 66菌株。该菌株（编号20130725 BT）已保藏于海南师范大学热带岛屿生态学教育部重点实验室。B的伴孢晶体。苏云金杆菌HS 66的形态结构进行了扫描电镜观察。为了明确该菌的种类，采用通用引物27 F/1492 R对HS 66的16 S rRNA基因进行扩增，并测序，登录号为MW 227496。菌株HS 66经鉴定为B属。苏云金杆菌的16 S rDNA序列进行Blast搜索分析[17]。利用MEGAX软件进行1000次重复bootstrap构建系统发育树，结果表明，Bacillus sp.菌株HS 66与B的亲缘关系最近。苏云金杆菌
Bacillus thuringiensis is a ubiquitous Gram-positive soil-dwelling bacterium [1] that belongs to the genus Bacillus and share many common phenotypic and genotypic properties with other species such as Bacillus cereus, Bacillus anthracis, and Bacillus mycoides [2–4]. The most distinctive property of B. thuringiensis is its production of parasporal insecticidal proteins, which is toxic to a wide number of insect species among the orders Lepidoptera (moths and butterflies), Diptera (mosquitoes and blackflies), Coleoptera (beetles), and Homoptera (aphids, whiteflies). Due to its safety for humans, B. thuringiensis (Bt) is the most widely used environmentally compatible biopesticide worldwide [5]. However, the chemical constituents of B. thuringiensis have not been reported. In order to search for the bioactive chemical constituents from the B. thuringiensis, 12 compounds were isolated from the EtOAc extract of the B. thuringiensis. The isolated compounds were identified as cyclo (L-Pro-L-Tyr)(1)[6], cyclo (Pro-Phe)(2)[7], cyclo (L-Pro-L-Phe)(3)[8], cyclo (Pro-Val)(4)[9], cyclo (L-Pro-L-Met)(5)[10], cyclo ((S)-Pro-(R)-Ile)(6)[11], cyclo (L-Pro-L-Leu)(7)[12], N-(2-hydroxyphenyl) acetamide (8)[13], uracil (9)[13], 2-phenylacetamide (10)[14], anthranilic acid (11)[15], and S-(4-hydroxyphenyl) lactic acid (12)[16]. The growth inhibition activity against newly hatched larvae of Helicoverpa armigera Hubner was tested. Compounds 1–3, 6, 7, and 10 showed growth inhibition activity against newly hatched larvae of Helicoverpa armigera Hubner with IC50 values ranging from 50 to 200 μg/mL. Azadirachtin was used as positive control with the IC50 value of 25 μg/mL. General. 1D (1H NMR, 13C NMR, DEPT) and 2D (1H–1H COSY, NOESY, HSQC, HMBC) NMR spectra were recorded on a Bruker Av 400 NMR spectrometer. ESI-MS spectra were recorded on an Agilent 1200 series HPLC interfaced to a Bruker Esquire 6000 ion trap mass spectrometer equipped with an electrospray ionization source. HPLC separation was performed in Agilent-HPLC (Eclipse XDB-C18, 10× 250 mm, 5 μm). Silica gel and GF254 were obtained from the Qingdao Marine Chemical Factory, and Sephadex LH-20 from Pharmacia Co. Ltd. Experimental Materials. Bacillus thuringiensis strain HS66 was screened and isolated from the soil samples collected from Diaoluoshan Natural Reserve, Hainan Province, China. The strain (No. 20130725BT) has been deposited in the Ministry of Education Key Laboratory for Ecology of Tropical Islands, Hainan Normal University. The parasporal crystal of B. thuringiensis HS66 was observed by scanning electron microscope (SEM). In order to clarify the kind of species, the 16S rRNA gene sequence of HS66 was amplified using the universal primer sets of 27F/1492R sequenced and deposited in the GenBank database with accession number of MW227496. Strain HS66 was identified as the genus B. thuringiensis by the Blast search analysis using its 16S rDNA sequence [17]. The phylogenetic tree was constructed by the neighbor-joining method with bootstrap 1000 replicates using MEGAX, which also indicated that Bacillus sp. strain HS66 is most closely related to B. thuringiensis.