CN86107242A - 新颖的微生物 - Google Patents
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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Abstract
本发明提供了新颖的微生物——它们含有苏云金菌柯氏变种δ-内毒素编码的基因和嗜暗变种δ-内毒素的编码基因，并提供了它们的制备方法及含有这种微生物的杀虫剂组分和应用。
Description
本发明是涉及苏云金芽孢杆菌（Bacillus thurimgiensis，下文称B.T）杂交菌株、它们的制备、含有杂交菌株的杀虫剂成分以及它们的使用。
更明确地说，本发明涉及带有苏云金菌柯氏变种（B.T.Kurs-taki）δ-内毒素编码基因的一种质粒和苏云金菌嗜暗变种（B.T.tenebrionis）δ-内毒素编码基因的一种质粒的杂交细菌细胞;通过产生内毒素晶体的柯氏变种和产生内毒素晶体的嗜暗变种之间的接合作用而获得这种杂交株，并且杂交株还能够产生柯氏变种和嗜暗变种各自典型的δ-内毒素晶体。柯氏变种的δ-内毒素晶体具有特有的双锥形，而嗜暗变种晶体典型的平行六面体（或正方体）外形。本发明的B.T杂交菌株可以对其亲代菌株柯氏变种和嗜暗变种所控制的害虫进行生物防治。
以常规方式通过产生δ-内毒素的柯氏变种和产生δ-内毒素的嗜暗变种的接合，以及用完全知道的分离技术分离杂交株，来获得本发明的B.T.杂菌菌株。接合技术在遗传工程领域内也已有很好的了解（如见Gonzatez J.M.and Carlton B.C.，1982.Plasmid transfer in Bacillus thuringiensis，85-95.in Genetic and Cellular Technology一书，L.P.Gape编Genetic Exchange vol.1.U.N.Streips等编，Marcel Dekker，Inc.New York and Basel）。
常规来说，接合作用是由接合质粒传递的。这种接合质粒的一个合适例子是从粪链球菌（Streptococcus faecalis）得到的pAMβ1，一般称为β-质粒。然后这质粒用已知技术（例如由H.-M.Fisher发明的技术，1983，Plasmid und Plasmidübertragung bei Bacillus thuringiensis.博士论文ETH Nr.7375，Swiss Federal Institute of Technology，ADAG Administration and Druck AG.共83页。或按照上述的Gonzalez等一文）引入到B.T.菌株内。这样获得的含有β-质粒的B.T菌株在柯氏变种和嗜暗变种菌株之间的接合反应中作为给体株。
为了有利于接合体（如本发明的杂交菌株）的检测和分离，给体菌株和受体菌株都作了遗传标记。这些标记能以完全了解的方式被引入菌内。例如已经知道在格蓝氏阴性细菌中，β-质粒给以抗红霉素。这一特性能用来消除接合反应后不具有此特性的所有菌株。
当另外的接合反应的匹配株也作标记时，例如经过诱导或自发对四环素等某个抗菌素有抗性，通过把这微生物培养在含有红霉素和四环素的适当培养基上，将有可能除去那些不带这两种遗传标记的所有菌株（即所有非接合菌株）。唯有在这种培养基上能生长的微生物将是携带两种标记（即抗红霉素和四环素）的菌株。
然后可以以完全已知的方式，如通过肉眼鉴定含有B.T.柯氏变种和嗜暗变种两种晶体类型的菌落或者文献中已知的生物学/生物化学技术，实现最终分离本发明的B.T.杂交株。制备本发明杂交株的一个方便步骤是柯氏变种用嗜暗变种进行接合反应，其中给体菌株含有接合质粒，给体和受体菌株都带有适当的遗传选择标记。一般来说，在本发明的接合反应过程中更倾向于用柯氏变种作为给体。
接合反应一般是在含有氮源、碳水化合物和生长因子的培养基中，20-40℃范围的某一温度下适当地进行的。
起始材料可以常规方式得到，从已知的柯氏变种和嗜暗变种开始。
对液体培养基来说，通常给以通气或振荡，因此培养时间一般为4至96小时。
在下面非限定的接合反应实例中，含β-质粒的柯氏变种用作给体，该质粒作为接合质粒，具有红霉素抗性。受体嗜暗变种是自发的四环素抗性实变株。
温度以摄氏表示。份数以重量表示。
实例1：接合反应
含有粪链球菌接合片段pAMβ1的柯氏变种HD-73（NRRL B-4488）培养物2.5毫升和嗜暗变种（Deutsch Sammlung für Microorgamismen DSM 2803）一株四环素抗性的自发突变体的培养物2.5毫升，每一培养物都处于对数生长期（约5×107细胞/毫升）、混合。在醋酸纤维素滤膜（O×oid/Nuflow）（0.45微米，25毫米直径）上过滤。过滤物在Lurla培养基（LA）平板上（1升Luria培养液含有15克琼脂、10克酪蛋白、5克醇母浸出物、10克NaCl、0.02克胸苷），在37℃培养16-24小时。
过滤物悬浮在0.5毫升液体LA（没有琼脂）中，用力摇匀，它的10°和10-1稀释度加在含有50微克/毫升红霉素和5微克/毫升四环素的LA平板上，选择接合体。
这样获得的红霉素和四环素抗性接合体放在允许产生芽孢的培养基上，用显微镜检查δ-内霉素的存在和性质。在杂交株里，裂介之前孢子除了有嗜暗变种典型的平行六面体（立方体）晶体之外，还含有柯氏变种特有的双锥形晶体。这些晶体的大小约2微米，在相差显微镜下很容易看见。
杂交株照例用肉眼鉴定很容易分离。杂交株的菌落（在LA平板上30℃培养16小时后）具有与嗜暗变种相同的菌落形态，而很容易与柯氏变种的菌落区分开。
嗜暗变种
菌落 或杂交株 柯氏变种
形状 不规则 圆形
高度 凸起 凸面
边缘 波纹形 完整
表面 粗糙 光滑
琼脂糖凝胶电泳分析从杂交种提取的DNA。也可以具体地证明质粒从给体株导入到了受体株内。质粒DNA分子经溴化乙啶染色后用紫外光照射可以观察到，并照相（见Lereclus D.Menou Gand Lecadet M-M.，1983，Mol.Gen.Genet.191，307-313）。
尤其本发明的大多数杂交株具有受体株嗜暗变种的典型的10兆道尔顿（Md）的质粒，给体菌柯氏变种具有5.6Md的质粒。携带柯氏变种和嗜暗变种的δ-内毒素编码基因的质粒有大致相同的分子量，因而不容易在杂交株的质粒图谱（plasmadogram）中区分开来。
本发明的B.T.杂交株具有杀昆虫的有用特性。它们对易受柯氏变种以及嗜暗变种感染的害虫都具有活性。一般来说，在活性的水平上或活性的范围，或者两方面，杂交种的杀虫性能都超过接合亲本菌株简单混合所能观察到的性能。
本发明B.T杂交选择到的菌株具有十分令人惊奇的杀虫性能，在下面生物测定中描述了它们对粉纹夜蛾Trichoplusia ni（苷兰菜的尺蠖虫）、海灰翅夜蛾Spodoptera Litteralis（埃及棉叶虫）和辣根猿叶甲Phaedon cochleariae（芥菜甲虫）等昆虫幼虫的活性。
生物测定
108孢子/毫升的水悬浮液用微型喷枪喷洒到中国甘兰的叶子上，直到液珠掉下来的程度。当喷洒液干燥时，每张叶子都摊在直径9厘米的塑料培养皿中潮湿滤纸上，每皿放进10个昆虫（处于二龄期的幼虫）。每种昆虫各用三个培养皿。
测定在25℃的气候小室中进行，相对湿度65%，光照时间16小时。
在这种条件下，幼虫在5天期间喂饲处理过的叶子。然后计算幼虫的死亡率，用%表示。
所得结果总结于下面的表：
5天后的死亡率（%）
B.T.杂交菌株 海灰翅夜蛾 粉纹夜蛾 辣根猿叶甲
L21001 87 100 100
L21004 90 100 100
L21016 97 100 100
L21017 97 100 100
L21019 93 100 100
柯氏变种HD73 <1> 3 100 0
嗜暗变种 <2> 0 0 100
“K+t”混合物<3> 0 100 43
注：<1>：具有β-质粒（在接合反应实例中所使用的）
<2>：四环素抗性菌株（在接合反应实例中所使用的）
<3>：只在喷洒使用之前制备的混合物，即柯氏变种
（1）和嗜暗变株（2）以等比例悬浮，调到总浓度为108孢子/毫升。
上述有些杂交株已在1985年10月15日委托给农业研究培养物收集中心（NRRL）（Peoria，Illinois，61604，美国）保管。菌株L21004，L21017和L21019已分别定名为NRRL编号B-18011，B-18012和B-18013。
前面表中所列数据表明，杂交株L21001，L21004，L21016，L21017和L21019不仅仅对它们“亲本”菌株敏感的昆虫种类有活性，而且很奇怪，杂交株的活性一直扩大到对“亲本”株不敏感的昆虫种类，如海灰翅夜蛾。这些结果是特别令人惊讶的，因为“亲本”株的简单混合比单独使用嗜暗变种对辣根猿叶甲的作用活性要小，并对海灰翅夜蛾根本没有活性。
因而，本发明也提供了消灭昆虫的一种方法，包括把杀虫有效量的本发明的杂交菌株施加到昆虫上或它们的环境中。
本发明的B.T.杂交株以浓缩的悬浮液或粉末等杀虫剂混合物的形式常规地进行使用。这种混合物适当地含有稀释剂，最好有杀虫上可接受的添加剂，如UV保护剂、表面活性剂和乳化剂。它们根据含有生物杀虫剂的已知B.T.菌株，用常规方式制备。
这里所用的稀释剂一词是指液体或固体，农业上可以接受的物质，加到B.T.杂交株里可以使它们分别成为一种更容易或更佳的使用形式，把有活性的制剂稀释成为通常的或所需要的活性强度。
值得称赞的是本发明的B.T.杂交株，一旦用接合反应获得，就可以在含有氮源（如干鱼粉）、碳水化合物（如淀粉）和无机盐，并调到pH7左右的适当的营养培养基中进行发酵，将更加经济地生长和繁殖。
这种发酵在20-40℃（如30℃）的温度下常规地进行。合适的发酵时间是24到72小时，如36小时。
把发酵液蒸发到需浓度，可以得到本发明的B.T.杂交株适当的悬浮浓度。
类似地，发酵液喷雾干燥，得到的固体经过粉碎，获得可湿性粉末的配方，任意地加乳化剂和UV防护剂之类的东西。
这样得到的配方里含有相当大一部分营养培养基的成分，成为稀释剂。
或者，发酵液可以经过离心把营养培养基的较大颗粒分离开，然后如上面所指出的转变成适当的悬浮浓度和可湿性粉末的配方。合适的配方是配制的产品每毫克或每毫升含有1000-40000IU。
可溶浓缩液
含水的营养培养基配成含有1.86%左右的甜菜糖蜜，1.4%无油的棉籽胚乳粉，1.7%玉米浸出液的固形物和0.1%碳酸钙，pH调到7.2-7.6范围内，在121℃成批灭菌20-30分钟，然后接种5%培养基体积的B.T.杂交株。培养过程加表压为5磅/英寸2的反压进行通气，培养温度为30℃。培养液冷却到18-20℃，pH用浓硫酸调到5.5左右，用每平方英寸150目的筛过滤，过筛的培养液经离心除去一部分不纯物质，并蒸发到具有所需效能的浓缩体积。在这浓缩物里加入按重量计算0.4%的乳化剂（如异辛基苯基聚氧化乙醇（isooctyl phenyl polyoxyethanol）和重量计算的0.8%的UV吸收剂。
可湿性粉末
按上面实例获得的蒸发了的浓缩体积进行喷雾干燥，这样得到的技术浓缩物同10%硅胶和90%大豆蛋白构成的“载体混合物”混杂，加入载体混合物的量是根据可湿性粉末的技术浓缩物的效能和可湿性粉末所需的效能而定。
这可湿性粉末中加入0.4%（重量）的乳化剂和0.8%（重量）的UV吸收剂。
本发明中较优的B.T.杂交株是用嗜暗变种作受体菌，柯氏变种作给体菌进行接合得到的。
Claims (6)
1、杂交株的苏云金芽孢杆菌(Bacillus thuringie nsis)细胞，含有给体B.T.柯氏变种δ-内毒素编码的基因和嗜暗变种δ-内毒素编码的基因。
2、按照权利要求1的杂交株细菌细胞，能够产生B.T.柯氏变种各自典型的δ-内毒素晶体。
3、杂交株细菌细胞，是通过产生δ-内毒素晶体的柯氏变种和产生δ-内毒素晶体的嗜暗变种的接合反应产生的，并能产生δ-内毒素，这些内毒素允许对柯氏变种和嗜暗变种亲代菌株可以控制的害虫进行生物学防治。
4、按照权利要求1至3之一的杂交株细胞的制备方法，包括：
a）使柯氏变种和嗜暗变种接合，或者
b）按a）所获得的杂交株细胞，在发酵条件下的营养培养基中进行生长。
5、含有按照权利要求1-3的B.T.杂交株的杀虫剂成分。
6、消灭昆虫的方法，包括把权利要求1-3中所述的B.T.杂交株的杀虫有效的量，施于昆虫或它们的环境之中。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB858526774A GB8526774D0 (en) | 1985-10-30 | 1985-10-30 | Bacillus thuringiensis hybrids |
| GB8526774 | 1985-10-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN86107242A true CN86107242A (zh) | 1987-05-13 |
Family
ID=10587498
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN198686107242A Pending CN86107242A (zh) | 1985-10-30 | 1986-10-29 | 新颖的微生物 |
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| EP (1) | EP0221024B1 (zh) |
| JP (1) | JPS62107782A (zh) |
| KR (1) | KR870004134A (zh) |
| CN (1) | CN86107242A (zh) |
| AT (1) | ATE73846T1 (zh) |
| AU (1) | AU596125B2 (zh) |
| BR (1) | BR8605301A (zh) |
| CA (1) | CA1325393C (zh) |
| CS (1) | CS270443B2 (zh) |
| DE (1) | DE3684406D1 (zh) |
| DK (1) | DK516386A (zh) |
| ES (1) | ES2035823T3 (zh) |
| GB (1) | GB8526774D0 (zh) |
| GR (1) | GR3004102T3 (zh) |
| HU (1) | HU201802B (zh) |
| IE (1) | IE59560B1 (zh) |
| IL (1) | IL80441A0 (zh) |
| NZ (1) | NZ218078A (zh) |
| PL (1) | PL153465B1 (zh) |
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1985
- 1985-10-30 GB GB858526774A patent/GB8526774D0/en active Pending
-
1986
- 1986-10-21 DE DE8686810469T patent/DE3684406D1/de not_active Expired - Lifetime
- 1986-10-21 EP EP86810469A patent/EP0221024B1/en not_active Expired - Lifetime
- 1986-10-21 AT AT86810469T patent/ATE73846T1/de not_active IP Right Cessation
- 1986-10-21 ES ES198686810469T patent/ES2035823T3/es not_active Expired - Lifetime
- 1986-10-28 AU AU64509/86A patent/AU596125B2/en not_active Ceased
- 1986-10-28 PT PT83634A patent/PT83634B/pt not_active IP Right Cessation
- 1986-10-28 IL IL80441A patent/IL80441A0/xx not_active IP Right Cessation
- 1986-10-28 HU HU864519A patent/HU201802B/hu not_active IP Right Cessation
- 1986-10-28 DK DK516386A patent/DK516386A/da not_active Application Discontinuation
- 1986-10-28 IE IE283086A patent/IE59560B1/en not_active IP Right Cessation
- 1986-10-28 NZ NZ218078A patent/NZ218078A/xx unknown
- 1986-10-28 US US06/924,372 patent/US4797279A/en not_active Expired - Fee Related
- 1986-10-29 CA CA000521701A patent/CA1325393C/en not_active Expired - Fee Related
- 1986-10-29 CS CS867829A patent/CS270443B2/cs unknown
- 1986-10-29 KR KR860009056A patent/KR870004134A/ko not_active Application Discontinuation
- 1986-10-29 PL PL1986262107A patent/PL153465B1/pl unknown
- 1986-10-29 JP JP61258064A patent/JPS62107782A/ja active Pending
- 1986-10-29 CN CN198686107242A patent/CN86107242A/zh active Pending
- 1986-10-29 BR BR8605301A patent/BR8605301A/pt not_active Application Discontinuation
- 1986-10-30 ZA ZA868281A patent/ZA868281B/xx unknown
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|---|---|---|---|---|
| CN103497907A (zh) * | 2013-08-02 | 2014-01-08 | 国家海洋局第三海洋研究所 | 高空芽孢杆菌在对虾养殖中的应用 |
| CN103497907B (zh) * | 2013-08-02 | 2015-11-18 | 国家海洋局第三海洋研究所 | 高空芽孢杆菌在对虾养殖中的应用 |
| CN103805535A (zh) * | 2013-11-28 | 2014-05-21 | 中国农业科学院农业资源与农业区划研究所 | 一种同温层芽胞杆菌及微生物菌剂和它们的应用 |
| CN103805535B (zh) * | 2013-11-28 | 2016-06-22 | 中国农业科学院农业资源与农业区划研究所 | 一种同温层芽胞杆菌及微生物菌剂和它们的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| BR8605301A (pt) | 1987-07-28 |
| ZA868281B (en) | 1988-06-29 |
| CA1325393C (en) | 1993-12-21 |
| DE3684406D1 (de) | 1992-04-23 |
| US4797279A (en) | 1989-01-10 |
| CS782986A2 (en) | 1989-11-14 |
| PT83634A (en) | 1986-11-01 |
| KR870004134A (ko) | 1987-05-07 |
| DK516386D0 (da) | 1986-10-28 |
| IL80441A0 (en) | 1987-01-30 |
| EP0221024A2 (en) | 1987-05-06 |
| GR3004102T3 (zh) | 1993-03-31 |
| IE59560B1 (en) | 1994-03-09 |
| AU596125B2 (en) | 1990-04-26 |
| AU6450986A (en) | 1987-05-07 |
| DK516386A (da) | 1987-05-01 |
| ATE73846T1 (de) | 1992-04-15 |
| HU201802B (en) | 1990-12-28 |
| CS270443B2 (en) | 1990-06-13 |
| PL262107A1 (en) | 1987-12-28 |
| PL153465B1 (en) | 1991-04-30 |
| EP0221024A3 (en) | 1988-06-01 |
| EP0221024B1 (en) | 1992-03-18 |
| IE862830L (en) | 1987-04-30 |
| ES2035823T3 (es) | 1993-05-01 |
| PT83634B (pt) | 1989-05-31 |
| HUT45557A (en) | 1988-07-28 |
| NZ218078A (en) | 1989-08-29 |
| GB8526774D0 (en) | 1985-12-04 |
| JPS62107782A (ja) | 1987-05-19 |
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