球形芽孢杆菌LP1-G的发酵特性及杀蚊活性*

球形芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐｈａｅｒｉｃｕｓ）ＬＰ１－Ｇ菌株在ＭＢＳ培养基上能正常生长、发育,产生位于芽孢孢外膜外的伴孢晶体。其产生的４１．９和５１．２ｋＤ二元毒素蛋白合成于芽孢形成期,另一分子量约为４９ｋＤ蛋白和二元毒素同期合成,并随着芽孢的释放而被降解。生物测定结果表明该菌株在营养体生长阶段对致倦库蚊幼虫无毒,孢子囊初期毒力较高,并在整个芽孢形成期都维持较高的毒力水平。其全发酵液对敏感和抗性库蚊幼虫都具有中等的毒杀作用,对３～４龄幼虫４８ｈ的ＬＣ_５０。值分别为０．１１３和０．１     

球形芽孢杆菌LP1—G的发酵特性及杀蚊活性

球形芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐｈａｅｒｉｃｕｓ）ＬＰ１－Ｇ菌株在ＭＢＳ培养基上能正常生长、发育,产生位于芽孢孢外膜外的伴孢晶体。其产生的４１．９和５１．２ｋＤ二元毒素蛋白合成芽孢形成期,另一分子量约为４９ｋＤ蛋白和二元毒素同期全成,并随着芽孢的释放而被降解。生物测定结果表明该菌株在营养体生长阶段对致倦库蚊幼虫无毒,孢子种初期毒力较高,关在整个芽孢形成期都维持较高的毒力水平。其全发酶液对敏感和     

球形芽孢杆菌C3—41二元毒素基因在苏云金芽孢杆菌以色列亚种中的？…

球形芽孢杆菌Ｃ３－４１是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于２３６２菌株,Ｓｏｕｔｈｅｒｎ杂交证明Ｃ３－４１总ＤＮＡ中３．５ＫｂＨｉｎｄＩＩＩ片段上带有４１．９和５１．４ｋＤ二元毒素基因。     

球形芽孢杆菌Mtx1杀蚊毒素在苏云金芽孢杆菌以色列亚种中的表达*

将来源于球形芽孢杆菌SSII 1的mtx1毒素基因克隆至穿梭载体 pBU4上 ,得到mtx1插入方向相反的重组质粒 pMT9和pMT4。含有 pMT9和 pMT4的大肠杆菌转化子能表达产生Mtx1毒素 ,发酵液对敏感和抗性致倦库蚊幼虫具有中度毒杀作用 ;含有pMT9和pMT4的苏云金芽孢杆菌转化子B pMT9和B pMT4在营养体生长阶段对敏感蚊幼和抗性幼虫也具有毒性 ,毒力与野生型SSII 1相当 ,而不同转化子在芽孢形成期的毒力因插入的mtx1基因转录方向不同而表现出差异 ,其中B pM     

球形芽孢杆菌Mtx1杀蚊毒素在苏云金芽孢杆菌以色列亚种中的表达

将来源于球形芽孢杆菌SSII-1的mtx1毒素基因克隆至穿梭载体pBU4上,得到mtx1插入方向相反的重组质粒pMT9和pMT4.含有pMT9和pMT4的大肠杆菌转化子能表达产生Mtx1毒素,发酵液对敏感和抗性致倦库蚊幼虫具有中度毒杀作用;含有pMT9和pMT4的苏云金芽孢杆菌转化子B-pMT9和B-pMT4在营养体生长阶段对敏感蚊幼和抗性幼虫也具有毒性,毒力与野生型SSII-1相当,而不同转化子在芽孢形成期的毒力因插入的mtx1基因转录方向不同而表现出差异,其中B-pMT4对目标蚊幼毒力极低(LC50＞100mg/mL),而B-pMT9对蚊幼虫具有毒性(LC50=2.49mg/mL).     

球形芽孢杆菌C_3－41对致倦库蚊的毒效及在蚊体内的再循环

球形芽抱杆菌Ｃ３－４１菌株（Ｂａｃｉｌｌｕｓｓｐｈａｅｒｉｃｕｓ　Ｃ３－４１）对致倦库蚊（Ｃｕｌｅｘｑｕｉｎｑｕｅｆａ－ｓｃｉａｔｕｓ）幼虫有很高毒效,对２龄和３—４龄幼虫的半致死剂量（ＬＤ５０）分别为６３．１和８９．７芽孢／蚊幼虫。处理浓度越高,取食时间越长,蚊幼虫取食到的杀蚊活性物质量越多,死亡率越高。当蚊幼虫取食互致死剂量杀蚊活性物质后,球形芽孢杆菌在感染的活幼虫体内不增殖;但当蚊幼虫取食致死剂量杀蚊活性物质后,蚊幼死亡,球形芽抱杆菌在死蚊幼虫体内增殖明显,６天内芽抱从感染初期的１．８６×１０２／蚊幼虫增加到１．５９×１０６／蚊幼虫。芽抱在死蚊幼虫体内能正常荫发、生长、产孢和形成毒素。增殖的芽抱同样对致倦库蚊幼虫有较高毒力。     

球形芽孢杆菌二元毒素基因的定位有无芽孢突变株的生物学特性

ＤＥＳ诱变得到了三株球形芽孢杆菌无芽孢突变株Ｇ５,Ｃ４,Ｌ５,经显微观察,超微结构分析,生物测定,蛋白持ＳＤＳ－ＰＡＧＥ分析及质粒检测,观察到突变株Ｃ４,Ｌ５阻碍于芽孢怕牝Ⅱ期,突变株Ｇ５阻碍芽孢形成的第Ⅲ期,其细胞中已有晶体形成,它对致倦库蚊幼虫的毒力明显高于仅有二元毒素蛋白合成,而无毒素晶体形成的突变株Ｃ４和Ｌ５。     

cry4Aa、cry4Ba和cryllAa基因在苏云金杆菌无晶体型菌株中的表达

利用穿梭载体pBU4,将苏云金杆菌以色列亚种(Bti)的cry4Aa、cry4Ba和cry11Aa基因分别转入Bti无晶体突变株4Q7中,获得了转化菌株Bt-B601、Bt-B611和Bt-B640.SDS-PAGE结果显示Cry4Aa、Cry4Ba和Cry11Aa蛋白均分别获得了表达.透射电镜下观察,转化菌株能产生球形或菱形伴胞晶体.转化菌株对敏感和抗性致倦库蚊及白纹伊蚊幼虫的生物测定结果显示Cry4Aa、Cry4Ba和Cry11Aa蛋白对库蚊和伊蚊的毒力较低,二元毒素抗性库蚊幼虫对Bti杀蚊毒素蛋白无明显的交叉抗性.     

球形芽孢杆菌二元毒素基因的定位及无芽孢突变株的生物学特性

DES诱变得到了三株球形芽孢杆菌无芽孢突变株G5、C4、L5,经显微观察、超微结构分析、生物测定、蛋白质SDSPAGE分析及质粒检测,观察到突变株C4、L5阻碍于芽孢形成的第Ⅱ期,突变株G5阻碍于芽孢形成的第Ⅲ期,其细胞中已有晶体形成,它对致倦库蚊幼虫的毒力明显高于仅有二元毒素蛋白合成、而无毒素晶体形成的突变株C4和L5。突变株L5消除了质粒,仍有二元毒素蛋白合成。Southern blot分析表明含二元毒素基因部分DNA片段的探针仅与菌株C341、BS10的染色体具有同源性,因此证明供试菌株的二元毒素基因定位于染色体上。     

cry4Aa、cry4Ba和cry11Aa基因在苏云金杆菌无晶体型菌株中的表达

利用穿梭载体pBU4,将苏云金杆菌以色列亚种（Bti)的cry4Aa、cry4Ba和cry11Aa基因分别转入Bti无晶体突变株4Q7中,获得了转化菌株Bt-B601、Bt-B611和Bt-B640。SDS－PAGE结果显示：cry4Aa、cry4Ba和cry11Aa蛋白均分别获得了表达。透射电镜下观察,转化菌 有产生球形或菱形伴胞晶体。转化菌株对敏感和抗性致倦库蚊及白纹伊蚊幼虫的生物测定结果显示：cry4Aa、cry4Ba和cry11Aa蛋白对库蚊和伊蚊的毒力较低,二元毒素抗性库蚊幼虫对Bti杀蚊毒素蛋白无明显的交叉抗性。     

Toxicity of Bacillus sphaericus LP1-G against susceptible and resistant Culex quinquefasciatus and the cloning of the mosquitocidal toxin gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.     

Toxicity of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> LP1-G Against Susceptible and Resistant <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> and the Cloning of the Mosquitocidal Toxin Gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.Received: 7 October 2002 / Accepted: 4 November 2002     

High-Level Field Resistance to Bacillus sphaericus C3-41 in Culex quinquefasciatus from Southern China

A flowable mosquito-larvicidal formulation of Bacillus sphaericus strain C3-41 has been continuously used for 8 years to control Culex quinquefasciatus larvae in Dongguan, Guangdong Province, China. This formulation had high efficacy against the target larvae during the first 6 years of treatment. However, under this high selection pressure, C. quinquefasciatus showed a significant level of resistance to C3-41 from years seven to eight. The resistance ratio of field-collected larvae at LC 50 was calculated to be 22 672-fold in comparison with the susceptible laboratory colony. Interestingly, no cross-resistance was observed to B. sphaericus strain LP1-G which had the same toxicity against both susceptible and resistant larvae, and B. thuringiensis subsp. israelensis was found to be more active to the latter than the former. After six months treatment with the B. thuringiensis subsp. israelensis formulation in the B. sphaericus resistant population area, the mosquito population recovered its susceptibility to B. sphaericus C3-41, with the resistance ratio of field-collected larvae dropping from 22 672- to 5.67-fold.     

Cytolytic toxin Cyt1Aa of Bacillus thuringiensis synergizes the mosquitocidal toxin Mtx1 of Bacillus sphaericus

Using the shuttle vector pBU4, the mosquitocidal toxin gene mtx1 from Bacillus sphaericus strain SSII-1 was introduced into an acrystalliferous strain of B. thuringiensis both individually and in combination with the accessory protein gene p20 and the cytolytic protein gene cyt1Aa from B. thuringiensis subsp. israelensis. Bioassay results indicated that the recombinants B-pMT4(Mtx1) and B-pMT9(Mtx1), both individually containing mtx1, had moderate toxicities to binary toxin susceptible and binary toxin resistant Culex quinquefasciatus larvae during the vegetative growth stage, but that their toxicities declined rapidly during the sporulation phase. The LC50 values were 2.5 and 4.8 mg/ml respectively, against 3-4 instar susceptible and resistant larvae for the final sporulated cultures of recombinants B-pMT9(Mtx1), and little toxicity was detected for B-pMT4(Mtx1). Meanwhile, the recombinant B-pMPX2(Mtx1+Cyt1Aa) expressing Mtx1, P20 alone, and Cyt1Aa in combination had stable toxicities during both the vegetative phase and the sporulation phase, with a LC50 ranging from 0.45-0.58 mg/ml. Furthermore, expression of Cyt1Aa appeared to enhance the activity of Mtx1 to target mosquito larvae, suggesting a synergism between Cyt1Aa and Mtx1 toxins.     

几株球形芽孢杆菌二元毒素基因的检测及对敏感和抗性尖音库蚊的毒性

根据Bacillus sphaericus 2362二元毒素基因核苷酸序列合成的一组寡聚核苷酸为引物,通过PCR扩增出1.1 kb的DNA片段作探针检测了C₃-41、IAB881、IAB872、BS-197和lPl-G菌株中二元毒素基因。Southern杂交表明C₃-41、IAB881、IAB872和BS-197菌株中3.5kb Hind III及LPl-G中4.7kb Hind III的酶切片段分别带有与探针有高度同源性的二元毒素基因。SDS-PAGE和Western印迹杂交表明所有菌株都能产生41.9kD和51.4kD的毒素蛋白。C₃.41、IAB881、BS.197和2362的全发酵液和碱抽提液对敏感尖音库蚊Culex pipienssubsp.Pipiens幼虫毒性高,但对抗性幼虫几乎无毒,LPl-G对敏感和抗性蚊幼虫具有相同的中度毒杀作用;IAB872对敏感幼虫毒性高,对抗性幼虫的毒力同LPl-G相似。这两株菌对抗性蚊幼虫的毒性可能是由Mtx毒素蛋白所导致的。     

Cross-resistance between strains of Bacillus sphaericus but not B. thuringiensis israelensis in colonies of the mosquito Culex quinquefasciatus

Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3-41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145,000- and 48.3-fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross-resistance (8500- to 145,000-fold) to B. sphaericus strains C3-41, 1593, 2297 and 2362. They were relatively susceptible to B. sphaericus strains LP1-G and 47-6B (only 0.8- to 2.8-fold tolerance), with 24.8- to 48.3-fold cross-resistance to strain IAB59. B. sphaericus-resistant mosquito colonies remained highly susceptible to B. thuringiensis israelensis, suggesting that B.t.i. would be of value in the management of B. sphaericus-resistant Cx. quinquefasciatus colonies. The demonstration of low or no cross-resistance of two selected resistant Cx. quinquefasciatus colonies to IAB59, LP1-G and 47-6B strains of B. sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains.     

球形芽孢杆菌C3-41二元毒素基因在苏云金芽孢杆菌以色列亚种中的克隆和表达

球形芽孢杆菌C3-41是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于2362菌株,Southern杂交证明C\-3\|41总DNA中35Kb HindIII片段上带有419和514kD二元毒素基因,该片段由3479个核苷酸组成,核苷酸序列同2362菌株的二元毒素基因序列完全相同。含二元毒素基因的重组质粒pCW\|1和pCW\|2能在大肠杆菌中表达产生二元毒蛋白,但表达量低,重组子杀蚊毒性低。无晶体型苏云金芽孢杆菌以色列亚种重组子在其芽孢形成中能产生以晶体形式存在的二元毒素蛋白,其全发酵液和纯化晶体蛋白的杀蚊活性与C\-3\|41相近。