WO2022095295A1 - 一种芽孢杆菌新菌株hsy204及其杀虫基因和应用 - Google Patents

一种芽孢杆菌新菌株hsy204及其杀虫基因和应用 Download PDF

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WO2022095295A1
WO2022095295A1 PCT/CN2021/075231 CN2021075231W WO2022095295A1 WO 2022095295 A1 WO2022095295 A1 WO 2022095295A1 CN 2021075231 W CN2021075231 W CN 2021075231W WO 2022095295 A1 WO2022095295 A1 WO 2022095295A1
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hsy204
strain
bacillus
insecticidal
orf5878
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French (fr)
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张文飞
吴江玉
魏力
贾璐羽
何佳利
范子钰
蔡雨辰
张旭东
王锐萍
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海南师范大学
海口海森元生物科技有限公司
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the field of environmental microorganisms and the technical field of molecular biology, in particular to a new strain of Bacillus, HSY204, and its insecticidal gene and application.
  • Mosquitoes are a kind of pests we are familiar with. There are many kinds of them. There are more than 3,200 known species in the world. Among them, there are 18 genera, 48 subgenera and 371 species or subspecies of mosquitoes known in my country. Mosquitoes not only seriously affect our lives, but more importantly, they are vectors of many diseases. At present, there are more than 10 species of mosquitoes that suck blood and transmit diseases in my country. Mosquito-borne diseases play an important role in preventive medicine because of their strong transmission power, high morbidity and great harm. The diseases it transmits mainly include malaria, dengue fever, filariasis, Japanese encephalitis and Zika, which seriously endanger people's health.
  • mosquito control In recent years, some old mosquito-borne diseases have been recurring in China, and new mosquito-borne diseases have appeared from time to time. Therefore, the prevention and control of mosquitoes has received extensive attention.
  • the main strategies for mosquito control include environmental control, chemical control and biological control.
  • the environmental control efficiency is low; the chemical control efficiency is high and the effect is quick, but it pollutes the environment, and in a short period of time will lead to the production of resistance, high residues, and even directly lead to human and animal poisoning.
  • Biological control is not only using one kind of organism to control another kind of organism or using the characteristics of the organism itself to control it, its biggest advantage is that it is more friendly to the environment and lower cost.
  • Bacillus is a general term for a class of facultative anaerobic or aerobic, gram-positive bacilli, spore-producing, rich and diverse in physiological characteristics, and extremely widely distributed. It is an important microbial population on plant surface, rhizosphere, soil and air. Bacillus is a species that produces a large number of polypeptide antibacterial substances with different chemical structures. Bacillus cereus is one of the common food-borne pathogens in the environment, facultative aerobic. The growth temperature ranges from 20 to 45 °C. It grows slowly or does not grow below 10 °C. It exists in soil, water, air and animal intestines, etc. It can produce antibacterial substances, inhibit the reproduction of harmful microorganisms, degrade nutrients in the soil, and improve Ecological environment, but there is no report on the application of Bacillus cereus in pesticides.
  • Bacillus cereus HSY204 is a strain isolated from Hainan tropical rain forest by Haisenyuan Biotechnology Co., Ltd.
  • the bioassay results of Bacillus cereus HSY204 showed that the bacteria had high virulence to mosquitoes, and obviously had an impact on the growth of mosquitoes, indicating that the bacteria had good application prospects in mosquito control.
  • the present invention provides a new strain of Bacillus, HSY204, which is classified and named Bacillus cereus HSY204, which was deposited in the China Center for Type Culture Collection on September 21, 2020, and the deposit number is: CCTCC NO.M2020523. The deposit address is: China. Wuhan. Wuhan University.
  • Bacillus cereus (Bacillus cereus) HSY204 includes 3 novel insecticidal genes orf5878, orf5055 and orf5877; wherein, the DNA sequences of orf5878, orf5055 and orf5877 are respectively as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.2, SEQ ID NO. The nucleotide sequence shown in ID NO.3.
  • amino acid sequences of the three insecticidal genes orf5878, orf5055 and orf5877 are respectively the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.
  • the cloning method of the three insecticidal genes orf5878, orf5055 and orf5877 is as follows: using the genome of Bacillus cereus HSY204 as a template, using PCR amplification method to obtain the complete orf5878, orf5055 and orf5877 respectively. Insecticidal gene fragments.
  • primer sequences and restriction endonucleases amplified by the PCR include:
  • restriction endonucleases are Nde I and Xho I;
  • primer F taagaaggagatatacatatgaattcaaatattcaaaattcta
  • R gtggtggtggtggtgctcgagccggtaaggtaccaattcaat
  • restriction endonucleases are Nde I and Xho I;
  • Orf5877 primer F taagaaggagatatacatatggttactacaaaatgatacctag
  • R gtggtggtggtggtgctcgagaatttgatccgattcgtataact
  • restriction endonucleases are Nde I and Xho I;
  • the application of a new Bacillus strain HSY204 is the application of Bacillus cereus HSY204 in killing Aedes mosquitoes.
  • the beneficial effects of the present invention are: the present invention conducts experiments such as screening, identification, the insecticidal activity of the insecticidal protein in the vegetative phase, and the cloning and expression of its genes through Bacillus cereus HSY204,
  • the biological activity assay showed that Bacillus cereus HSY204 strain and its insecticidal protein were highly toxic to Aedes aegypti larvae and had a significant effect on the growth of mosquitoes, but had no effect on the growth of diamondback moth and cotton bollworm , which can be applied to prevent and control mosquitoes and effectively reduce the risk of large-scale transmission of mosquito-borne diseases.
  • Fig. 1 is the light microscope picture of Bacillus cereus (Bacillus cereus) HSY204 under different field magnifications of the present invention
  • Fig. 2 is the scanning electron microscope picture under different field magnifications when Bacillus cereus HSY204 of the present invention is cultivated to spore stage;
  • Figure 3 is the SDS-PAGE analysis of Bacillus cereus HSY204 of the present invention, in the figure, 1: HSY204; 2: Bti (Bacillus thuringiensis israelensis)
  • Fig. 4 is the agarose electrophoresis analysis diagram of the PCR amplification product of Bacillus cereus HSY204 insecticidal gene of the present invention, in the figure, A is the 5877 gene; B is the 5878 gene; C is the 5055 gene;
  • Fig. 5 is the SDS-PAGE analysis chart of Bacillus cereus HSY204 insecticidal gene 5055, 5877, 5878 protein of the present invention, in the figure, A is 5877 protein; B is 5878 protein; C is 5055 protein.
  • Enzymes, kits and other reagents used in the following examples were purchased from Shanghai Bioengineering Co., Ltd.
  • the primers used in the following examples were synthesized by Shanghai Bioengineering Co., Ltd.
  • Soil sample collection The sampling site is selected in the tropical rain forest of Hainan province. In order to increase the probability of acquiring new strains, the sampling site is selected where the vegetation is intact, the ecological environment is free from human intervention, and the soil humus is rich;
  • strain HSY204 using the sodium acetate-temperature screening method, weigh about 5g of soil into 20ml of BPA medium, shake fully, culture at 30°C for 4-5h, take a water bath at 75°C for 15min, take 1ml of supernatant to dilute To 10 -3 , 10 -4 and 10 -5 , each draw 200 ⁇ L of NB plates, invert at 30° C. for 3 days, and pick out single colonies with different shapes and plate them.
  • Bacillus cereus HSY204 After the Bacillus is cultivated for 3-5 days to form spores, further use carbolic acid fuchsin staining and optical microscopy to isolate whether the isolates contain parasporal crystal protein. Bacillus, named Bacillus cereus HSY204, and then further observe the shape of the spore with an electron microscope, as shown in Figure 1 and Figure 2, the specific shape of bacteria and crystals can be obtained. In order to clearly screen the insecticidal activity of wild strains, Aedes aegypti was used as the target insect for bioassay, and a strain HSY204 was screened to show good insecticidal effect on Aedes mosquitoes and named Bacillus cereus HSY204 .
  • the band type of parasporal crystal was analyzed by SDS-PAGE electrophoresis, and it was found that the strain HSY204 had similar bands with the standard strain, as shown in Figure 3.
  • the Illumina pair-end library and SMRT bell library were constructed with the genomic DNA fragment of strain HSY204, which were used for whole-genome sequencing by Illumina Hiseq and PacBio RSII, respectively.
  • the original sequencing data of PacBio was assembled with Canu software, and the software Pilon corrected the assembly results of Canu with the original sequencing data of Illumina, and finally obtained the whole genome of HSY204 with a length of 6,035,572 bp, 0 gap, consisting of one chromosome and four Plasmid composition.
  • Gene prediction was performed using Glimmer software, and it was obtained that HSY204 contained 6102 CDSs. These genes were annotated in six databases (NR, Swiss-Prot, Pfam, EggNOG, GO and KEGG) to further explore potential functions.
  • rRNA and tRNA were predicted with the software RNAmmer-1.2 and tRNAscan-SE, respectively.
  • the genomic DNA of strain HSY204 was extracted by alkaline lysis, primers were designed according to the coding sequences of the genes orf5878, orf5055, and orf5877, and the genes were inserted into the expression vector pET-30a by the technical means of seamless cloning, and a successful recombinant transfection was constructed. into BL(DE3) for induction expression.
  • the seamless cloning (SeamLess cloning) technology is specifically:
  • the vector was linearized by double-enzyme digestion method or PCR amplification method
  • the linear vector is recovered by gel, and both blunt ends and sticky ends can be used;
  • a linear vector is obtained by PCR amplification. If it is a single band, the PCR product is directly purified, otherwise, the gel is recovered and purified.
  • Table 1 Primer sequence information table of three genes in HSY204 bacteria
  • the primers cannot be phosphorylated
  • the amplification product is a high-fidelity DNA polymerase with blunt ends
  • the cloning primers include the target fragment-specific primer sequence (22nt) and the vector overlapping sequence (15-25nt; for the recombination of more than 3 fragments, increase the homology region to 30nt);
  • the target DNA fragment and the linearized vector are added in an amount of 0.01-0.25pmol (estimated 1kb 20ng, 1.5kb 30ng), and the optimal molar ratio is 2:1, add to the centrifuge tube and mix;
  • reaction time 50 °C of reaction 15min to carry out the recombination reaction, after the reaction is placed on ice to cool for several seconds (for the recombination of more than 3 fragments, the reaction time is extended to 60min);
  • the recombinant product is transformed into E.coli competent cells by rapid or traditional methods
  • Protein expression was induced by IPTG, expressed in small amounts, disrupted by sonication, and the expressed protein was analyzed by SDS-PAGE electrophoresis. Affinity chromatography was performed on a metal-chelated nickel column for mass expression, and a chelating agent (nitrotriacetic acid or NTA) was covalently coupled to agarose medium (4% cross-linking) using a high-affinity Ni-NTA purification medium. Then it is prepared by chelating Ni 2+ . NTA can firmly chelate Ni 2+ through four sites, thereby reducing the leakage of Ni 2+ into protein samples during purification.
  • a chelating agent nitrogen triacetic acid or NTA
  • the specific method for expressing the insecticidal gene of strain HSY204 in Escherichia coli is as follows:
  • the constructed expression engineering strain is inoculated into 2-5 mL of LB medium (Amp 100Kan 30 ⁇ g/mL) containing the corresponding antibiotic to activate the expression strain overnight;
  • the prepared HSY204 protein was used for biological activity assay, the expressed protein was quantitatively analyzed by Bio-Rad densitometric scanning method, the protein sample was serially diluted, 2-3 instar target insects were prepared, blank control was set, bioassay was performed, and the death was observed. The number of insects was analyzed by probability regression using SPSS13.0 software to obtain the LC50 value and 95% confidence interval value, and the insecticidal activity was determined.
  • mosquitoes has 4 stages: egg, larva, swarm and adult; larvae grow in dechlorinated tap water, and are continuously fed with artificial feed (flour 9: yeast powder 1);
  • Mosquitoes used for protein virulence determination are sensitive strains artificially raised in the laboratory, and the calibrated concentration protein solution is diluted to a certain concentration by dechlorinated tap water, and placed in sterilized transparent drinking plastic cups respectively;
  • Each plastic cup holds 10ml of protein solution, and uses pasteur pipette to transfer 10 mosquito larvae of 3rd instar;
  • HSY204 insecticidal protein has obvious effect on the growth of mosquitoes, but has no effect on the growth of diamondback moth and cotton bollworm.
  • Insecticidal gene protein Molecular weight of insecticidal protein Insecticidal protein solubility Aedes aegypti lethality orf5055 76KDa ⁇ 200mg/mL 67% orf5878 45KDa ⁇ 200mg/mL 100% orf5877 94KDa ⁇ 200mg/mL 33.33%
  • insecticidal genes orf5055, orf5878 and Orf5877 have high lethality to Aedes aegypti, and the lethality of the protein of gene orf5878 to Aedes aegypti can reach 100%.
  • test results of the insecticidal activity of HSY204 insecticidal gene protein 5878 against Aedes mosquitoes at different concentrations are as follows:

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Abstract

提供了一种芽孢杆菌新菌株HSY204及其杀虫基因和应用。该芽孢杆菌新菌株HSY204的分类命名为蜡样芽孢杆菌(Bacillus cereus)HSY204，其包括3个新型杀虫基因；经过生物活性测定表明菌株HSY204及其杀虫基因对埃及伊蚊幼虫具有很高的毒性，对蚊子的生长具有明显影响，可应用于防控蚊虫。

Description

一种芽孢杆菌新菌株HSY204及其杀虫基因和应用 技术领域
本发明涉及环境微生物领域和分子生物学技术领域，特别涉及一种芽孢杆菌新菌株HSY204及其杀虫基因和应用。
背景技术
蚊虫是我们熟悉的一种害虫，它的种类繁多，已知全世界有3200多种，其中我国已知蚊类达18个属48个亚属和371种或亚种。蚊虫不仅严重影响我们的生活，更重要的是它是传播多种疾病的媒介。目前在我国吸血并能传播疾病的蚊种有10多种。以蚊子为媒介传播的病在预防医学中占有重要地位，因为蚊类传播的转播力强、发病率高、危害性大。其传播的疾病主要有疟疾、登革热、丝虫病、流行性乙型脑炎和寨卡等，严重地危害着人民群众的身体健康。
近年来我国一些老的以蚊子为媒介传播的疾病不断复发，并且新的以蚊子为媒介传播不时出现，因此对蚊子的防治受到广泛关注。蚊子防控的主要策略有环境防治、化学防治和生物防治等。环境防治效率较低；化学防治效率高、见效快但污染环境，短时间内会导致抗性产生、高残，甚至直接导致人畜中毒。而生物防治既是用一种生物控制另一种生物或者利用生物本身的特点来控制，其最大的优点是比环境比较友好、成本低。
目前随着对杀蚊活性的菌株中晶体蛋白结构和有关分子作用机理的不断广泛而深入研究，并通过利用现代生物技术手段提高杀蚊效果。无疑，研究高效的菌株生物杀虫剂，对控制蚊虫具有非常重要的现实意义。研究杀蚊作用机理的关键是受体。Pootanakit等已从埃及伊蚊(Aedes aegypti)中肠中鉴定出两种晶体蛋白作用受体——氨肽酶N异构体。Charles等发现以色列亚种晶体蛋白作用于伊蚊的肠道上皮细胞导致其组织病理学变化与鳞翅目昆虫中的情况相似。芽孢杆菌是一类兼性厌氧或好氧，革兰氏阳性杆菌的总称，产芽孢，生理特征丰富多样，分布极其广泛，是植物体表、根际和土壤、空气的重要微生物种群。芽孢杆菌是一类产生大量的不同化学结构的多肽抗菌物质的物种。蜡样芽孢杆菌(Bacillus cereus)是环境中常见的食源性致病菌之一，兼性好氧。生长温度范围20～45℃，10℃以下生长缓慢或不生长，存在于土壤、水、空气以及动物肠 道等处，可产生抗菌物质，抑制有害微生物的繁殖，降解土壤中的营养成分，改善生态环境，但目前对于蜡样芽孢杆菌在杀虫剂方面中的应用未见报导。
发明内容
鉴以此，本发明提出一种芽孢杆菌新菌株HSY204及其杀虫基因和应用，蜡样芽孢杆菌(Bacillus cereus)HSY204是海森元生物科技有限公司从海南热带雨林分离一株菌。蜡样芽孢杆菌(Bacillus cereus)HSY204进行生物测定结果表明该菌对蚊子具有高毒力，明显对蚊子的生长具有影响，预示着该菌在防治蚊虫方面有着良好的应用前景。
本发明的技术方案是这样实现的：
本发明提供一种芽孢杆菌新菌株HSY204，其分类命名为蜡样芽孢杆菌(Bacillus cereus)HSY204，于2020年9月21日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO.M2020523。保藏地址为：中国.武汉.武汉大学。
进一步说明，蜡样芽孢杆菌(Bacillus cereus)HSY204包括3个新型杀虫基因orf5878、orf5055和orf5877；其中，orf5878、orf5055和orf5877的DNA序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示的核苷酸序列。
进一步说明，所述3个杀虫基因orf5878、orf5055和orf5877的氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示的氨基酸序列。
进一步说明，所述3个杀虫基因orf5878、orf5055和orf5877的克隆方法为：以蜡样芽孢杆菌(Bacillus cereus)HSY204的基因组为模板，采用PCR扩增方法，分别获得orf5878、orf5055和orf5877的完整杀虫基因片段。
进一步说明，所述PCR扩增的引物序列与限制性内切酶包括：
orf5878引物F：taagaaggagatatacatatgaaaataatgggggaatatatat，R：gtggtggtggtggtgctcgagccaccaaactccctcttttac，限制性内切酶为Nde Ⅰ和Xho Ⅰ；
orf5055引物F：taagaaggagatatacatatgaattcaaatattcaaaattcta，R：gtggtggtggtggtgctcgagccggtaaggtaccaattcaat，限制性内切酶为Nde Ⅰ和Xho Ⅰ；
Orf5877引物F：taagaaggagatatacatatggttactacaaaaatgatacctag，R：gtggtggtggtggtgctcgagaatttgatccgattcgtataact，限制性内切酶为Nde Ⅰ和Xho Ⅰ；
一种芽孢杆菌新菌株HSY204的应用为，蜡样芽孢杆菌(Bacillus cereus)HSY204在杀伊蚊方面中的应用。
进一步说明，所述蜡样芽孢杆菌(Bacillus cereus)HSY204在制备杀蚊剂中的应用。
与现有技术相比，本发明的有益效果是：本发明通过对蜡样芽孢杆菌(Bacillus cereus)HSY204进行筛选、鉴定、营养期杀虫蛋白的杀虫活性以及其基因的克隆表达等实验，经过生物活性测定表明，蜡样芽孢杆菌(Bacillus cereus)HSY204菌株及其杀虫蛋白对埃及伊蚊幼虫具有很高的毒性，对蚊子的生长具有明显影响，对小菜蛾和棉铃虫的生长没有影响，可以应用防控蚊虫，有效降低蚊媒疾病大规模传播的风险。
附图说明
图1为本发明蜡样芽孢杆菌(Bacillus cereus)HSY204不同视野倍数下的光学显微镜图；
图2为本发明蜡样芽孢杆菌(Bacillus cereus)HSY204培养到芽孢期时的不同视野倍数下的扫描电镜图；
图3为本发明蜡样芽孢杆菌(Bacillus cereus)HSY204的SDS-PAGE分析，图中，1：HSY204；2：Bti(Bacillus thuringiensis israelensis)
图4为本发明蜡样芽孢杆菌(Bacillus cereus)HSY204杀虫基因PCR扩增产物琼脂糖电泳分析图，图中，A为5877基因；B为5878基因；C为5055基因；
图5为本发明蜡样芽孢杆菌(Bacillus cereus)HSY204杀虫基因5055、5877、5878蛋白的SDS-PAGE分析图，图中，A为5877蛋白；B为5878蛋白；C为5055蛋白。
具体实施方式
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明，均为常规方法。
以下实施例中所用的酶,试剂盒及其他试剂均购自上海生物工程有限公司。
以下实施例中所用引物由上海生物工程有限公司代为合成。
实施例1-菌株HSY204的筛选与鉴定
(1)土壤样品采集：采样地点选择在海南省热带雨林，为了增加获取新菌株的概率，采样点选择植被保护完整、生态环境没有人为干预少、土壤腐殖质丰富的地方；
(2)菌株HSY204筛选：采用醋酸钠-温度筛选法，称取5g左右土壤放入20ml BPA培养基中，充分振荡，30℃摇床培养4-5h，75℃水浴15min，取1ml上清稀释至10 -3、10 -4和10 -5，各吸取200μL NB平板，30℃倒置培养3d，挑取形态各异的单菌落划平板。
(3)芽孢杆菌培养3-5天形成芽孢后，进一步利用石炭酸复红染色和光学显微镜分离株的是否含有伴胞晶体蛋白，光学显微镜下观察到无色芽孢为芽孢杆菌，产生伴胞晶体的芽孢杆菌，命名为蜡样芽孢杆菌HSY204，然后进一步用电子显微镜观察芽孢的形状，如图1和图2所示，可以出菌和晶体的具体形状。为了明确筛选得到野生菌株的杀虫活性，以埃及伊蚊作为靶标昆虫进行生物测定，筛选一株菌株HSY204对伊蚊表现出很好的杀虫效果，命名为蜡样芽孢杆菌(Bacillus cereus)HSY204。为了进一步鉴定该菌株是否为新型芽孢菌株，通过SDS-PAGE电泳分析其伴胞晶体带型，发现菌株HSY204与标准菌株有类似的带，如图3所示。
实施例2-菌株HSY204的DNA提取及杀虫基因鉴定
通过利用MiSeq Illumina下一代测序和PacBio技术对菌株HSY204的基因组序列进行分子和生物信息学分析。
1.菌株HSY204基因组DNA提取
(1)HSY204单菌落接种于2-5mL左右的LB液体培养基中，28℃，220rpm培养过夜活化；
(2)第二天按1％量转接到新鲜的LB液体培养基中，28℃，220rpm继续培养4～6小时至OD600＝2.0左右；
(3)以8000rpm离心2min收集1-3mL菌体；
(4)1mL J Buffer(0.1M Tris HCl(pH 8.0)、0.1M EDTA(pH 8.0)、0.15M Nacl)洗涤沉淀，8000rpm离心2min，弃上清；
(5)移液枪吹打将沉淀重新悬浮于500μL J Buffer(J Buffer使用前预先加入溶菌酶至20mg/mL)中，37℃温育30min，期间振荡3-5次；
(6)加入15μLRNase(10mg/mL)，50℃作用15min；
(7)再加入80μL SDS(10％)，70℃处理20min，室温冷却至室温；
(8)等体积酚：氯仿：异戊醇(25：24：1)氯仿：异戊醇(24：1)各抽提一次(上下剧烈倒置10-30次，12,000rpm离心10min，小心吸取上清)；
(9)加入等体积的异丙醇混匀，12,000rpm离心10min，倾去上清；
(10)加入500μL的70％乙醇(勿剧烈震荡)，12,000rpm离心2min；
(11)风干后溶50μL TE Buffer中，取5μL DNA溶液在1.0％的胶电泳检测。
2.菌株HSY204杀虫基因鉴定
用菌株HSY204的基因组DNA片段构建了Illumina pair-end文库和SMRT bell文库，分别用于Illumina Hiseq和PacBio RSII进行全基因组测序。PacBio的原始测序数据用Canu软件进行组装，软件Pilon用Illumina的原始测序数据对Canu的组装结果进行了较正，最终得到了HSY204全基因组长度为6,035,572bp，0个gap，由一个染色体和四个质粒组成。使用Glimmer软件进行 基因预测，得到HSY204包含6102个CDS。将这些基因在六个数据库(NR,Swiss-Prot,Pfam,EggNOG,GO and KEGG)中进行了注释，以进一步发掘出潜在功能。
rRNA和tRNA分别用软件RNAmmer-1.2和tRNAscan-SE进行预测。
实施例3-菌株HSY204杀虫基因克隆表达
通过采用碱裂解的方法提取菌株HSY204的基因组DNA，根据基因orf5878、orf5055、和orf5877的编码序列设计引物，并采用无缝克隆的技术手段将基因插入表达载体pET-30a，构建成功的重组子转入BL(DE3)，进行诱导表达。
无缝克隆(SeamLess cloning)技术具体为：
A.采用双酶切法者PCR扩增方法将载体线性化
(1)酶切后线性载体进行胶回收，平末端和粘性末端均可；
(2)PCR扩增获得线性载体，如果是单一带，PCR产物直接纯化，否则进行胶回收纯化。
B.使用设计好的引物进行目的DNA片段的PCR扩增；
表1：HSY204菌中的三个基因引物序列信息表
Figure PCTCN2021075231-appb-000001
(1)引物不能磷酸化；
(2)扩增产物为平末端的高保真DNA聚合酶；
(3)电泳检测扩增产物的完整性，有杂带，凝胶回收目的片段；
(4)克隆引物包括目的片段特异引物序列(22nt)和载体重叠序列(15-25nt；对于3个以上片段的重组，将同源区域增加至30nt)；
(5)对酶切位点有严格要求，选择合适的酶切位点，正向和反向引物增加缺少碱基恢复原有的酶切位点；
(6)按照线性载体末端结构5`突出、3`突出、平末端引物设计。
C.克隆反应体系
Component Volume
PCR product 1-4μL
Linearized Vector       1-2μL
2×Assembly Mix 5μL
H 2O                     xμL
Total 10μL
(1)将目的DNA片段和线性化载体加入量为0.01-0.25pmol(估算1kb 20ng、1.5kb 30ng)，最佳摩尔比为2:1，加到离心管混匀；
(2)50℃反应15min进行重组反应，反应结束置于冰上冷却数秒(对于3个以上片段的重组，反应时间延长至60min)；
(3)重组产物保存-20℃或直接用于转化。
重组产物通过快速或传统方法转化E.coli感受态细胞
(4)将感受态细胞从-70℃冰箱取出后，置于冰浴中融化；
(5)在50μL感受态细胞中加入5μL重组产物，轻轻弹动离心管壁混匀(禁止涡旋)后，在冰上放置30min，期间不要振动离心管；
(6)将离心管置于42℃恒温水浴热激30-90sec，取出管后立即置于冰浴中静 置2～3min；
(7)向离心管中加入500μL 37℃预热的SOC或LB培养基(不含抗生素)，200rpm、37℃振荡培养1h；
(8)将离心管中的菌液轻弹或吸吹混匀，吸取200μL加到含相应抗生素的LB固体培养基(最好提前37℃预热)过夜培养。
阳性克隆鉴定
(9)菌落PCR或提取重组质粒PCR验证；
(10)重组质粒酶切验证；
(11)测序鉴定，初步鉴定后，将所有新构建的载体选取1～2个阳性克隆进行测序鉴定。
实施例4-制备毒素蛋白
通过IPTG诱导蛋白表达，小量表达，超声波破碎，利用SDS-PAGE电泳分析表达蛋白。大量表达采用金属螯合镍柱进行亲和层析，利用高亲和Ni-NTA纯化介质把螯合剂(氮川三乙酸或NTA)共价偶联到琼脂糖介质(4％交联)上，然后再螯合Ni 2+制备而成。NTA能够通过四个位点牢固的螯合Ni 2+，从而减少纯化过程中Ni 2+泄露到蛋白样品中。
菌株HSY204杀虫基因在大肠杆菌中表达方法具体如下：
(1)构建好表达工程菌株接种到2-5mL含有对应的抗生素的LB培养基(Amp 100Kan 30μg/mL)中过夜活化表达菌株；
(2)第二天按1％量转接到含相对应抗生素的新鲜LB培养基中培养2h至OD600为0.6左右，加入2-8μl IPTG(500mM)至终浓度为0.1-0.5mM培养8-20h(其中，空载用IPTG诱导作对照；表达菌株转接2份，一份加入IPTG，另为一份作为阴性对照不加IPTG)；
(3)以不同的温度、IPTG溶度和表达时间，优化表达体系，实现可溶性表达；
(4)8000rpm离心，5min收集菌体，用0.5M NaCl或ddH2O或PBS缓冲液洗涤1-3次；
(5)加入800μl适量PBS缓冲液，超声波破碎处理5-20min，至液体澄清(Sonics VCX750S参数：小探头功率20％，工作10s，间隔10s)；
(6)10000rpm离心5min，分别收集上清，沉淀用1mL PBS缓冲液洗涤1-2次，然后用50μl PBS缓冲液吹打悬浮；
(7)表达菌株和参照菌株蛋白产物上清和沉淀进行SDS-PAGE分析，结果如图5所示。
实施例5-HSY204蛋白生物活性测定
将制备获得的HSY204蛋白用于生物活性测定，Bio-Rad光密度扫描法对表达蛋白进行定量分析，蛋白样品做梯度稀释，准备2-3龄靶标昆虫，设置空白对照，进行生物测定，观察死虫的数量，利用SPSS13.0软件进行概率回归分析，获得LC50值和95％置信区间值，明确杀虫活性。
1.棉铃虫、斜纹夜蛾人工生物测定
(1)称取10g人工饲料置一灭菌培养皿中，加入1000μl待测样品溶液，用药匙充分搅拌均匀，室温放置，使饲料多余水分蒸发；
(2)分装于经消毒的24孔细胞培养板中，用毛笔轻轻接入初孵幼虫，每孔一头，每处理重复3次，严格密封，防止幼虫逃逸；
(3)放置于光照培养箱(25℃，湿度为65％，光周期为12h∶12h)中培养，第4天或6天分别调查死活虫数，计算死亡率和LC50。
2.埃及伊蚊饲养及蚊子的生物测定
(1)蚊幼虫采集于夏季静止小溪流；
(2)在人工室内环境，温度为26度，相对空气湿度为80％和光照周期为14小时的条件下饲养；
(3)蚊的生长周期为4个阶段：卵、幼虫、涌和成虫；幼虫生长在去氯自来水中，不断加入人工饲料(面粉9：酵母粉1)喂食；
(4)成虫放置在玻璃虫笼中，放入10％蔗糖溶液(或者蜂蜜溶液)的脱脂棉球喂食成虫，放入小老鼠让蚊吸血后进入繁殖阶段；
(5)因雌性蚊要将虫卵产在静止的水中，在成虫笼中放入一个盛水的开放容器收集虫卵；
(6)3-4龄的幼虫用于生物测定实验，每天观察，记录其死亡数，计算死亡率和LC50；
(7)用于蛋白毒力测定的蚊虫为实验室人工饲养的敏感品系，标定好浓度蛋白溶液由去氯自来水稀释成一定的浓度，分别置于消毒透明饮水塑料杯里；
(8)每个塑料杯盛10ml蛋白溶液，用pasteur pipette移入10头3龄的蚊幼虫；
(9)不加入任何蛋白的去氯自来水作为阴性对照，每个蛋白浓度设置3个重复。
3.菌株HSY204杀虫蛋白的生物活性测定结果如下表：
表2 HSY204杀虫蛋白的生物活性测定
Figure PCTCN2021075231-appb-000002
Figure PCTCN2021075231-appb-000003
(其中，ND:未检测到。>300：300μg/g时无致死作用和体重抑制。)
由上表可以看出，HSY204杀虫蛋白对蚊子的生长具有明显影响，对小菜蛾和棉铃虫的生长没有影响。
4.菌株HSY204中杀虫基因的蛋白对埃及伊蚊杀虫活性测定结果如下表：
表3 HSY204杀虫基因对埃及伊蚊杀虫活性测定
杀虫基因蛋白 杀虫蛋白分子量 杀虫蛋白溶度 埃及伊蚊致死率
orf5055 76KDa ≥200mg/mL 67％
orf5878 45KDa ≥200mg/mL 100％
orf5877 94KDa ≥200mg/mL 33.33％
由上表可以看出杀虫基因orf5055、orf5878和0rf5877对埃及伊蚊具有较高的致死率，其中，基因orf5878的蛋白对埃及伊蚊的致死率可达100％。
5.HSY204杀虫基因蛋白5878不同浓度下对伊蚊杀虫活性测试结果如下表：
表4 HSY204杀虫基因5878不同蛋白浓度下对伊蚊杀虫活性测试
Figure PCTCN2021075231-appb-000004
由上表可知，随着杀虫基因5878的蛋白浓度的增加，其对伊蚊的致死率增加，当杀虫基因5878蛋白的浓度为22.845μg/mL时，其对伊蚊的致死率可达到 40％；经SPSS计算得出杀虫基因5878对库蚊的LC50为27.115μg/m(19.381-61.199μg/mL)。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (7)

  1. 一种芽孢杆菌新菌株HSY204，其特征在于：其分类命名为蜡样芽孢杆菌(Bacillus cereus)HSY204，保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO.M2020523。
  2. 如权利要求1所述的一种芽孢杆菌新菌株HSY204的杀虫基因，其特征在于：蜡样芽孢杆菌(Bacillus cereus)HSY204包括3个新型杀虫基因orf5878、orf5055和orf5877；其中，orf5878、orf5055和orf5877的DNA序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示的核苷酸序列。
  3. 如权利要求2所述的一种芽孢杆菌新菌株HSY204的杀虫基因，其特征在于：所述3个杀虫基因orf5878、orf5055和orf5877的氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示的氨基酸序列。
  4. 如权利要求3所述的一种芽孢杆菌新菌株HSY204的杀虫基因，其特征在于：所述3个杀虫基因orf5878、orf5055和orf5877的克隆方法为：以蜡样芽孢杆菌(Bacillus cereus)HSY204的基因组为模板，采用PCR扩增方法，分别获得orf5878、orf5055和orf5877的完整杀虫基因片段。
  5. 如权利要求4所述的一种芽孢杆菌新菌株HSY204的杀虫基因，其特征在于：所述PCR扩增的引物序列与限制性内切酶包括：
    orf5878引物F：taagaaggagatatacatatgaaaataatgggggaatatatat，R：gtggtggtggtggtgctcgagccaccaaactccctcttttac，限制性内切酶XhoⅠ和NdeⅠ；
    orf5055引物F：taagaaggagatatacatatgaattcaaatattcaaaattcta，R：gtggtggtggtggtgctcgagccggtaaggtaccaattcaat，限制性内切酶XhoⅠ和NdeⅠ；
    Orf5877引物F：taagaaggagatatacatatggttactacaaaaatgatacctag，R：gtggtggtggtggtgctcgagaatttgatccgattcgtataact，限制性内切酶XhoⅠ和NdeⅠ。
  6. 如权利要求1所述的一种芽孢杆菌新菌株HSY204的应用，其特征在于：所述蜡样芽孢杆菌(Bacillus cereus)HSY204在杀伊蚊方面中的应用。
  7. 如权利要求1所述的一种芽孢杆菌新菌株的应用，其特征在于：所述蜡样芽孢杆菌(Bacillus cereus)HSY204在制备杀蚊剂中的应用。
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