有益农用微生物资源鉴定和相关基因的克隆及其功能验证
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摘要
本文的研究主旨是鉴定并开发有益农用微生物资源,并对相关功能基因进行克隆和功能验证研究；本文的研究内容主要包括两部分：第一部分是苏云金芽胞杆菌(Bacillus thuringiensis, Bt)营养期杀虫蛋白基因(Vegetative insecticidal protein, Vip)资源的鉴定及功能验证,这是分子育种的上游工作,即发掘新的高效的杀虫基因资源,为农作物抗虫育种提供新的高效的基因资源；第二部分是大豆慢生根瘤菌属新种的鉴定研究。
苏云金芽胞杆菌是目前研究最深入、历史最悠久、使用最广泛的生物防治微生物。为了提高生防制剂的杀虫活性、扩大杀虫谱和解决害虫的抗性问题,开发新的、高效杀虫蛋白的基因资源是解决这些问题的有效途径,这对我国的生物防治领域的发展有着重要的意义。Vip作为第二代生物杀虫剂,其与ICPs蛋白的氨基酸序列无同源性,两者的杀虫机理完全不同；Vip对某些害虫的杀虫活性比ICPs的杀虫活性还高,还对ICPs不能杀死的昆虫具有较好的活性。因此,分析不同生态环境下vip,基因资源分布情况,并开发新的、高效的vip基因是解决长期主要使用ICPs所带来的抗性问题的有效途径。根据以上研究背景,这部分的研究主要包括以下三个方面：
第一,四川地区苏云金芽胞杆菌vip基因资源的评价。从四川盆地不同生态环境的14个地区采集的1789个土样中分离了2134个Bt菌株,因此证明了四川地区蕴藏着丰富的Bt菌株,且不同生态环境的Bt分布不同。本研究使用vipls-vipla, vip2s-vip2a和vip3s-vip3a引物对分别鉴定了2134个Bt菌株vip1、vip2和vip3基因资源情况,其中有8.6%和14.6%的Bt菌株分别含有vip1和vip2基因,67.4%的Bt菌株含有vip3基因。与vp1和vip2基因资源分布相比,四川地区vp3基因资源相对较为丰富。由于巴郎山和峨嵋山独特的生态环境,例如：高海拔、低温、氧压低、积雪覆盖,这两个地方的Bt菌株不含有vip1和vip2基因,且只有9.2%和8.4%的Bt菌株含有vip3基因。Vip1和Vip2是二元毒素,但是有些地方的Bt菌株只含有其中之一,例如：白马寺和梁山。不产生任何阳性PCR产物的Bt菌株可能不含有vip基因或者含有vip突变基因。尤其是能够产生Vip3的阳性蛋白带的22个Bt菌株,其很有可能含有新的的vip基因。
第二,挖掘新的、高效的Vip,及生物活性功能验证。本研究采用PCR-RFLP鉴定的新的vip1基因,开发出了新的的二元毒素基因viplAcl/vip2Ae3;并建立了一个仅使用限制性内切酶AciI的PCR-RFLP鉴定体系,该方法简单、快速、有效。二元毒素基因viplAcl和vip2Ae3基因共表达于载体pCOLADuet-1,共表达蛋白Vip1Ac1/Vip2Ae3对同翅目害虫棉蚜(Aphis gossypii)有较好的杀虫活性,其致死中浓度LC50为87.5(34.2-145.3)ng mL-1。本研究建立的鉴定体系为挖掘更多新的、高效的Vip蛋白有着重要的意义；本研究挖掘的新的的Vip1Ac1-Vip2Ae3二元毒素为Vip资源库提供了新的资源,对于害虫的生物防治和昆虫的抗药性有着重要的意义。
第三,构建多价效工程菌株,提高其杀虫活性、扩大杀虫谱和解决昆虫的抗性问题。Vip3主要对鳞翅目害虫有毒杀活性,Cyt2主要对蚊子幼虫有杀虫活性,并具有溶细胞和溶血作用,且对一些Cry蛋白具有协同增效作用。本研究将cyl2Aa3与vip3Aa29基因表达于pCOLADuet-1载体上,并分析了Cyt2Aa3和Vip3Aa29蛋白的协同作用。Vip3Aa29和Cyt2Aa3的共表达蛋白对同翅目害虫倦库蚊和稻瘿蚊的LC50分别为1.10μg mL-1and39μg mL-1,且对倦库蚊的毒杀活性存在较小的抑制作用。Vip3Aa29和Cyt2Aa3的共表达蛋白对水稻二化螟和甜菜夜蛾的LC5o分别为50.2μg mL-1和59.0μgmL-1,且存在协同增效作用。Vip3Aa29和Cyt2Aa3共表达蛋白对棉铃虫有明显的抑制作用,其抑制中浓度IC50为162.5μg mL-1,且SF接近于1,因此Vip3Aa29和Cyt2Aa3蛋白对棉铃虫不存在协同作用。本研究首次发现Vip3Aa29与Cyt2Aa3之间存在协同作用,为Vip3杀虫蛋白活性的提高提供了理论依据。
在大豆根瘤菌资源分析方面,随着分类方法的不断发展,根瘤菌新种群越来越多地被发现,从而完善了根瘤菌的种群分类系统。本研究通过对16个大豆根瘤菌与慢生根瘤菌属所有种标准菌株的16S rRNA、结瘤基因nodC、固氮基因nifH以及六个持家基因(atpD、glnll、recA、gyrB、rpoB、dnaK)构建了系统发育树,结果发现这16个根瘤菌在系统发育树上处于一个独立的分支上面,是慢生根瘤菌属的一个新种。然后,从这个新种群的三个亚群中分别选择了009、085和099三个菌株进行DNA-DNA杂交,其gDNA与其他种的gDNA同源性明显小于70%,这进一步证明了这个种群是慢生根瘤菌的新种；这三个菌株的G+C含量是62-64%。最后,将这个新种命名为渥太华慢生根瘤菌(Bradyrhizobium ottawense),并将099菌株作为标准菌株保存于HAMBI,其保藏号为HAMBI3284,其表型特征描述如下：B. ottawense sp. gov.为革兰氏阴性菌株,菌株大小为0.4-0.9μm×1.7-2.3μm,形状为杆状,带有极生或亚极生鞭毛：最适生长温度为28℃,最适生长盐分是1%NaCl,最适生长pH是7.2；在YEM平板培养基上,28℃条件下培养7天,单菌落为白色、圆形且光滑,大小都是小于1mm,其世代生长时间为10.7-12.3h,且能产碱；对卡拉霉素(100(100μg/mL)、四环素(20μg/mL)、氯霉素(100μg/mL)、头孢霉素(<5μg/mL)盘尼西林(<10μg/mL)和红霉素(100μg/mL)六种抗生素具有抗性；能利用D-甘露糖、L-树胶醛糖做为碳源,对麦芽糖和L-鼠李糖的利用是微弱的；能将苏氨酸作为氮源来利用,且对L-脯氨酸和L-谷氨酸的利用是十分微弱的；其能与豆科植物Glycine max, Glycine soja, Macroptilium atropurpurem, Vigna unguiculata, Phaseolus vulgaris, Desmodium canadense, Amphicarpa bracteata共生结瘤,且大部分根瘤的固氮效率较高,但与Amphicarpa bracteata和Phaseolus vulgaris的共生结瘤而不能有效地固氮。
The main subject of the thesis is the identification and exploitation for beneficial and agricultural Bacteria and the clone and function test of some useful genes from the Bacteria. The thesis inculds two parts. The fist part is the identification and function test of Vegetative insecticidal protein (Vip) genes from Bacillus thuringiensis (Bt). The part is the research on the upstream work of molecular breeding, which is identification of novel and high activity genes of biological control. The research can provide novel and high activity gene sources for molecular breeding. The second part is the characterization for a new species of Rhizobium geneus.
Bacillus thuringiensis (Bt) is the biological control Bacteria of the most thorough resrarch, the oldest history and the widest application. To enhance insecticidal activity, enlarge insecticidal spectrum and solve the resistance problem of insects, the effective method is the exploitation of novel and high activity insecticidal protein genes, which is beneficial for the biological control in China. As the second generation toxin of biological control, VIPs show no holomogy of amino acid with ICPs, and their insecticidal mechanism is different. VIPs show higher toxic activity to some insects than ICPs, and VIPs can show pesticidal activity to some insects that ICPs have no activity against. Therefore, the effective ways of solving the problem of using ICPs long time is the analysis of distribution of vip genes in different entiroment and the exploitation of novel and high activity toxin genes. According to the above reasons, the research results of the part mainly focused on the three points behind:
Firstly, the distrubtion of vip genes from B. thuringiensis in Sichuan was evaluated.2134Bt strains were isolated from1789soil samples of14sites with different environments in Sichuan, which demonsteated abundant Bt strains in Sichuan and different distrubtion of Bt in diverse environments. Vip1, vip2and vip3genes of2134Bt strains were identified by primers of vipls-vipla, vip2s-vip2a and vip3s-vip3a, respectively. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vipl-type genes (8.1%). Comparing with vipl and vip2genes, vip3genes were more abundant in Sichuan. Because of unique environment with high altitude, low temperature, insufficient oxygen, and abundant snow, Bt srtains coming from the two sites had no vipl and vip2genes, and only9.2%and8.4%Bt strains contained vip3genes. Although Vipl and Vip2were binary toxins, there were only one of the two genes in some sites, such as Bi Ma Si and Liang Shan. Bt strains without any PCR products with the three pairs of primer may had no vip genes, or contained mutational vip genes. Especially, the22Bt strains with vip3genes PCR product may contain novel vip3genes.
Secondly, novel and high activity VIPs were exploited, and their biological activity were tested. A novel vipl gene were identified with the method of PCR-RFLP, and a novel binary toxin gene of viplAcl/vip2Ae3were exploited. A identification system only with enduclease Acil was built, and the method are simple, fast and effective. ViplAcl and vip2Ae3were co-expressioned in pCOLADuet-1. The co-expression proteins showed high toxicity against Aphis gossypii, the50%lethal concentration (LC50) was87.5(34.2-145.3) ng mL-1. It is beneficial to use the identification system to exploite more novel and high activity Vip. As a novel binary toxin, ViplAcl-Vip2Ae3applied one more binary toxin into Vip resource libiary, which is beneficial for biological control and the resistance of insects.
Thirdly, engineering bacteria with one more genes were constructed, which may be helpful for enhancing insecticidal activity, amplifying toxicity spectrum and solveing the problem of insect resistance. Vip3mainly shows toxicity to Lepidoptera. Cyt2maily has insecticidal activity against mosquito larvae, haemolysis and cytolytic function and synergism with some Cry proteins. Cyl2Aa3and vip3Aa29genes were co-expressioned in pCOLADuet-1, and the synergism Cyt2Aa3and Vip3Aa29proteins were analysised. The LC50of co-expression proteins against Culex quinquefasciatus and Chironomus tepperi respectively were0.53μg mL-1and36μg mL-1, and Vip3Aa29and Cyt2Aa3showed a little of antagonistic effect against Cx. quinquefasciatus. The LC50of the co-expression proteins against Chilo suppressalis and Spodoptera respectively were50.2μg mL-1and59.0μg mL-1, and Vip3Aa29and Cyt2Aa3had synergistic effect against the two insects. The co-expression protein of Vip3Aa29and Cyt2Aa3could inhibit Helicoverpa armigera growth, with50%inhibition concentration at22.6μg mL-1. Its SF was close to1, so there was not synergistic effect between Vip3Aa29and Cyt2Aa3. It was the first time to report that Vip3Aa29and Cyt2Aa3had synergism between each other, which applied theory basis for enhancing the insecticidal activity of Vip3.
The second part is the analysis of rhizobium sources. With the development of classification method, more and more groups of rhizobium have been identified, which perfect the classification system of rhizobium. Phylogentic trees of nine genes (16S rRNA, nodC, nifH, atpD, glnⅡ, recA, gyrB, rpoB, dnaK) were built for16rhizobium isolated from soybean and all of the type strains of Bradyrhizobium. As a result, the16rhizboubium strains was a independent group on phylogentic tree, which suggested that the group was a novel species of Bradyrhziboium. Then, three rhizobium strains of009,085and099were selected for DNA-DNA hybridization. The homology of gDNA between the three selected strain's gDNA and other type strain's gDNA was obviously less than70%, and the results also proved that the group was a novel species of Bradyrhizobium. The G+C contents of the three rhizobium strains (009,085and099) were between62%and64%. The novel species was named as Bradyrhizobium ottawense, and099was selected as the type strain.099were preserved in HAMBI, and its accession number is HAMB13284.
Pheotype of Bradyrhizobium ottwaense:Gram negative rods as for the novel species of Bradyrhizobium genus. The size of cells is0.4-0.9μm×1.7-2.3μm, and the shape is ellipse with polar or subpolar flagellum. The optium growth temperature, salt and pH of tested strains were28℃,1%NaCl and7.2, respectively. Colonies are white, roundness and smooth in YMA at28℃for7days, and the size of colony was less than1mm. The generation time of the species was10.7-12.3hours, and the strains can product alkali. The strains showed resistance to kanamycin sulfate (100μg/mL), tetracycline (20μg/mL), chloramphenicol (100μg/mL), erythromycin (<5μg/mL), cefuroxime sodium (<10μg/mL), penicillin G sodium (100ug/mL). Assimilation of D-mannose, L-arabinose as carbon source is positive. Assimilation of maltose and L-rhamnose as carbon is weak. Assimilation of L-threonine as nitrogen source is positive. Assimilation of L-proline and L-glutamic acid as nitrogen source is weak. The type strain nodulate leguminous plants of Glycine max, Glycine soja, Macroptilium alropurpurem, Vigna unguiculata, Phaseolus vulgaris, Desmodium canadense, Amphicarpa bracteata, and the fixing nitrogen efficiency of most nod is high. Although the type strain nodulates Amphicarpa bracteata and Phaseolus vulgaris, it can not fix nitrogen for the two leguminous plants.
苏云金芽胞杆菌是目前研究最深入、历史最悠久、使用最广泛的生物防治微生物。为了提高生防制剂的杀虫活性、扩大杀虫谱和解决害虫的抗性问题,开发新的、高效杀虫蛋白的基因资源是解决这些问题的有效途径,这对我国的生物防治领域的发展有着重要的意义。Vip作为第二代生物杀虫剂,其与ICPs蛋白的氨基酸序列无同源性,两者的杀虫机理完全不同；Vip对某些害虫的杀虫活性比ICPs的杀虫活性还高,还对ICPs不能杀死的昆虫具有较好的活性。因此,分析不同生态环境下vip,基因资源分布情况,并开发新的、高效的vip基因是解决长期主要使用ICPs所带来的抗性问题的有效途径。根据以上研究背景,这部分的研究主要包括以下三个方面：
第一,四川地区苏云金芽胞杆菌vip基因资源的评价。从四川盆地不同生态环境的14个地区采集的1789个土样中分离了2134个Bt菌株,因此证明了四川地区蕴藏着丰富的Bt菌株,且不同生态环境的Bt分布不同。本研究使用vipls-vipla, vip2s-vip2a和vip3s-vip3a引物对分别鉴定了2134个Bt菌株vip1、vip2和vip3基因资源情况,其中有8.6%和14.6%的Bt菌株分别含有vip1和vip2基因,67.4%的Bt菌株含有vip3基因。与vp1和vip2基因资源分布相比,四川地区vp3基因资源相对较为丰富。由于巴郎山和峨嵋山独特的生态环境,例如：高海拔、低温、氧压低、积雪覆盖,这两个地方的Bt菌株不含有vip1和vip2基因,且只有9.2%和8.4%的Bt菌株含有vip3基因。Vip1和Vip2是二元毒素,但是有些地方的Bt菌株只含有其中之一,例如：白马寺和梁山。不产生任何阳性PCR产物的Bt菌株可能不含有vip基因或者含有vip突变基因。尤其是能够产生Vip3的阳性蛋白带的22个Bt菌株,其很有可能含有新的的vip基因。
第二,挖掘新的、高效的Vip,及生物活性功能验证。本研究采用PCR-RFLP鉴定的新的vip1基因,开发出了新的的二元毒素基因viplAcl/vip2Ae3;并建立了一个仅使用限制性内切酶AciI的PCR-RFLP鉴定体系,该方法简单、快速、有效。二元毒素基因viplAcl和vip2Ae3基因共表达于载体pCOLADuet-1,共表达蛋白Vip1Ac1/Vip2Ae3对同翅目害虫棉蚜(Aphis gossypii)有较好的杀虫活性,其致死中浓度LC50为87.5(34.2-145.3)ng mL-1。本研究建立的鉴定体系为挖掘更多新的、高效的Vip蛋白有着重要的意义；本研究挖掘的新的的Vip1Ac1-Vip2Ae3二元毒素为Vip资源库提供了新的资源,对于害虫的生物防治和昆虫的抗药性有着重要的意义。
第三,构建多价效工程菌株,提高其杀虫活性、扩大杀虫谱和解决昆虫的抗性问题。Vip3主要对鳞翅目害虫有毒杀活性,Cyt2主要对蚊子幼虫有杀虫活性,并具有溶细胞和溶血作用,且对一些Cry蛋白具有协同增效作用。本研究将cyl2Aa3与vip3Aa29基因表达于pCOLADuet-1载体上,并分析了Cyt2Aa3和Vip3Aa29蛋白的协同作用。Vip3Aa29和Cyt2Aa3的共表达蛋白对同翅目害虫倦库蚊和稻瘿蚊的LC50分别为1.10μg mL-1and39μg mL-1,且对倦库蚊的毒杀活性存在较小的抑制作用。Vip3Aa29和Cyt2Aa3的共表达蛋白对水稻二化螟和甜菜夜蛾的LC5o分别为50.2μg mL-1和59.0μgmL-1,且存在协同增效作用。Vip3Aa29和Cyt2Aa3共表达蛋白对棉铃虫有明显的抑制作用,其抑制中浓度IC50为162.5μg mL-1,且SF接近于1,因此Vip3Aa29和Cyt2Aa3蛋白对棉铃虫不存在协同作用。本研究首次发现Vip3Aa29与Cyt2Aa3之间存在协同作用,为Vip3杀虫蛋白活性的提高提供了理论依据。
在大豆根瘤菌资源分析方面,随着分类方法的不断发展,根瘤菌新种群越来越多地被发现,从而完善了根瘤菌的种群分类系统。本研究通过对16个大豆根瘤菌与慢生根瘤菌属所有种标准菌株的16S rRNA、结瘤基因nodC、固氮基因nifH以及六个持家基因(atpD、glnll、recA、gyrB、rpoB、dnaK)构建了系统发育树,结果发现这16个根瘤菌在系统发育树上处于一个独立的分支上面,是慢生根瘤菌属的一个新种。然后,从这个新种群的三个亚群中分别选择了009、085和099三个菌株进行DNA-DNA杂交,其gDNA与其他种的gDNA同源性明显小于70%,这进一步证明了这个种群是慢生根瘤菌的新种；这三个菌株的G+C含量是62-64%。最后,将这个新种命名为渥太华慢生根瘤菌(Bradyrhizobium ottawense),并将099菌株作为标准菌株保存于HAMBI,其保藏号为HAMBI3284,其表型特征描述如下：B. ottawense sp. gov.为革兰氏阴性菌株,菌株大小为0.4-0.9μm×1.7-2.3μm,形状为杆状,带有极生或亚极生鞭毛：最适生长温度为28℃,最适生长盐分是1%NaCl,最适生长pH是7.2；在YEM平板培养基上,28℃条件下培养7天,单菌落为白色、圆形且光滑,大小都是小于1mm,其世代生长时间为10.7-12.3h,且能产碱；对卡拉霉素(100(100μg/mL)、四环素(20μg/mL)、氯霉素(100μg/mL)、头孢霉素(<5μg/mL)盘尼西林(<10μg/mL)和红霉素(100μg/mL)六种抗生素具有抗性；能利用D-甘露糖、L-树胶醛糖做为碳源,对麦芽糖和L-鼠李糖的利用是微弱的；能将苏氨酸作为氮源来利用,且对L-脯氨酸和L-谷氨酸的利用是十分微弱的；其能与豆科植物Glycine max, Glycine soja, Macroptilium atropurpurem, Vigna unguiculata, Phaseolus vulgaris, Desmodium canadense, Amphicarpa bracteata共生结瘤,且大部分根瘤的固氮效率较高,但与Amphicarpa bracteata和Phaseolus vulgaris的共生结瘤而不能有效地固氮。
The main subject of the thesis is the identification and exploitation for beneficial and agricultural Bacteria and the clone and function test of some useful genes from the Bacteria. The thesis inculds two parts. The fist part is the identification and function test of Vegetative insecticidal protein (Vip) genes from Bacillus thuringiensis (Bt). The part is the research on the upstream work of molecular breeding, which is identification of novel and high activity genes of biological control. The research can provide novel and high activity gene sources for molecular breeding. The second part is the characterization for a new species of Rhizobium geneus.
Bacillus thuringiensis (Bt) is the biological control Bacteria of the most thorough resrarch, the oldest history and the widest application. To enhance insecticidal activity, enlarge insecticidal spectrum and solve the resistance problem of insects, the effective method is the exploitation of novel and high activity insecticidal protein genes, which is beneficial for the biological control in China. As the second generation toxin of biological control, VIPs show no holomogy of amino acid with ICPs, and their insecticidal mechanism is different. VIPs show higher toxic activity to some insects than ICPs, and VIPs can show pesticidal activity to some insects that ICPs have no activity against. Therefore, the effective ways of solving the problem of using ICPs long time is the analysis of distribution of vip genes in different entiroment and the exploitation of novel and high activity toxin genes. According to the above reasons, the research results of the part mainly focused on the three points behind:
Firstly, the distrubtion of vip genes from B. thuringiensis in Sichuan was evaluated.2134Bt strains were isolated from1789soil samples of14sites with different environments in Sichuan, which demonsteated abundant Bt strains in Sichuan and different distrubtion of Bt in diverse environments. Vip1, vip2and vip3genes of2134Bt strains were identified by primers of vipls-vipla, vip2s-vip2a and vip3s-vip3a, respectively. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vipl-type genes (8.1%). Comparing with vipl and vip2genes, vip3genes were more abundant in Sichuan. Because of unique environment with high altitude, low temperature, insufficient oxygen, and abundant snow, Bt srtains coming from the two sites had no vipl and vip2genes, and only9.2%and8.4%Bt strains contained vip3genes. Although Vipl and Vip2were binary toxins, there were only one of the two genes in some sites, such as Bi Ma Si and Liang Shan. Bt strains without any PCR products with the three pairs of primer may had no vip genes, or contained mutational vip genes. Especially, the22Bt strains with vip3genes PCR product may contain novel vip3genes.
Secondly, novel and high activity VIPs were exploited, and their biological activity were tested. A novel vipl gene were identified with the method of PCR-RFLP, and a novel binary toxin gene of viplAcl/vip2Ae3were exploited. A identification system only with enduclease Acil was built, and the method are simple, fast and effective. ViplAcl and vip2Ae3were co-expressioned in pCOLADuet-1. The co-expression proteins showed high toxicity against Aphis gossypii, the50%lethal concentration (LC50) was87.5(34.2-145.3) ng mL-1. It is beneficial to use the identification system to exploite more novel and high activity Vip. As a novel binary toxin, ViplAcl-Vip2Ae3applied one more binary toxin into Vip resource libiary, which is beneficial for biological control and the resistance of insects.
Thirdly, engineering bacteria with one more genes were constructed, which may be helpful for enhancing insecticidal activity, amplifying toxicity spectrum and solveing the problem of insect resistance. Vip3mainly shows toxicity to Lepidoptera. Cyt2maily has insecticidal activity against mosquito larvae, haemolysis and cytolytic function and synergism with some Cry proteins. Cyl2Aa3and vip3Aa29genes were co-expressioned in pCOLADuet-1, and the synergism Cyt2Aa3and Vip3Aa29proteins were analysised. The LC50of co-expression proteins against Culex quinquefasciatus and Chironomus tepperi respectively were0.53μg mL-1and36μg mL-1, and Vip3Aa29and Cyt2Aa3showed a little of antagonistic effect against Cx. quinquefasciatus. The LC50of the co-expression proteins against Chilo suppressalis and Spodoptera respectively were50.2μg mL-1and59.0μg mL-1, and Vip3Aa29and Cyt2Aa3had synergistic effect against the two insects. The co-expression protein of Vip3Aa29and Cyt2Aa3could inhibit Helicoverpa armigera growth, with50%inhibition concentration at22.6μg mL-1. Its SF was close to1, so there was not synergistic effect between Vip3Aa29and Cyt2Aa3. It was the first time to report that Vip3Aa29and Cyt2Aa3had synergism between each other, which applied theory basis for enhancing the insecticidal activity of Vip3.
The second part is the analysis of rhizobium sources. With the development of classification method, more and more groups of rhizobium have been identified, which perfect the classification system of rhizobium. Phylogentic trees of nine genes (16S rRNA, nodC, nifH, atpD, glnⅡ, recA, gyrB, rpoB, dnaK) were built for16rhizobium isolated from soybean and all of the type strains of Bradyrhizobium. As a result, the16rhizboubium strains was a independent group on phylogentic tree, which suggested that the group was a novel species of Bradyrhziboium. Then, three rhizobium strains of009,085and099were selected for DNA-DNA hybridization. The homology of gDNA between the three selected strain's gDNA and other type strain's gDNA was obviously less than70%, and the results also proved that the group was a novel species of Bradyrhizobium. The G+C contents of the three rhizobium strains (009,085and099) were between62%and64%. The novel species was named as Bradyrhizobium ottawense, and099was selected as the type strain.099were preserved in HAMBI, and its accession number is HAMB13284.
Pheotype of Bradyrhizobium ottwaense:Gram negative rods as for the novel species of Bradyrhizobium genus. The size of cells is0.4-0.9μm×1.7-2.3μm, and the shape is ellipse with polar or subpolar flagellum. The optium growth temperature, salt and pH of tested strains were28℃,1%NaCl and7.2, respectively. Colonies are white, roundness and smooth in YMA at28℃for7days, and the size of colony was less than1mm. The generation time of the species was10.7-12.3hours, and the strains can product alkali. The strains showed resistance to kanamycin sulfate (100μg/mL), tetracycline (20μg/mL), chloramphenicol (100μg/mL), erythromycin (<5μg/mL), cefuroxime sodium (<10μg/mL), penicillin G sodium (100ug/mL). Assimilation of D-mannose, L-arabinose as carbon source is positive. Assimilation of maltose and L-rhamnose as carbon is weak. Assimilation of L-threonine as nitrogen source is positive. Assimilation of L-proline and L-glutamic acid as nitrogen source is weak. The type strain nodulate leguminous plants of Glycine max, Glycine soja, Macroptilium alropurpurem, Vigna unguiculata, Phaseolus vulgaris, Desmodium canadense, Amphicarpa bracteata, and the fixing nitrogen efficiency of most nod is high. Although the type strain nodulates Amphicarpa bracteata and Phaseolus vulgaris, it can not fix nitrogen for the two leguminous plants.
引文
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