苏云金芽胞杆菌资源多样性分析与杀虫基因发掘
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摘要
苏云金芽胞杆菌（Bacillus thuringiensis， Bt），革兰氏阳性细菌，在芽胞形成期可产生具有杀虫活性的伴胞晶体。Bt晶体主要由cry基因编码的杀虫晶体蛋白组成，在害虫的防治中具有重要作用，有巨大的商业价值，因此发掘新基因并进行知识产权保护是Bt资源研究的重要内容。知识产权作为综合竞争力的重要体现，而目前我国在Bt杀虫基因资源的知识产权方面处于劣势，大部分Bt杀虫基因的专利权掌握在西方各大型农业生物技术企业手中，我国掌握的Bt新的基因发明专利不足60项。高通量测序技术的发展为发掘杀虫基因提供了新思路，成为国内外发掘Bt杀虫基因的主要手段。但是测序价格较高，Bt资源库中存在的冗余菌株会造成较大的资金浪费，目前尚没有便捷的方法去除重复菌株。因此，迫切需要建立可以快速分析比较大量样品的方法，来排除重复菌株，进一步提高杀虫基因发掘效率。本论文在分离1500株Bt菌株的基础上，进一步建立了菌株多样性分析方法，对菌株多样性进行研究，去除冗余菌株，对其中90株菌株进行了杀虫基因测序筛选，主要结果如下：
Bt菌株分离与鉴定：杀虫活性评估结果显示多数菌株对鳞翅目害虫有较高活性，1500株测试菌株中，对棉铃虫有活性的菌株有1346株，对玉米螟有活性菌株有813株，而对叶甲类害虫大猿叶甲有活性的菌株较少，只有42株；镜检结果显示不同菌株产生的晶体形状、大小、种类有较大差异，晶体种类主要为菱形，部分为球形和不规则的晶体形状；杀虫晶体蛋白图谱显示大部分菌株表达了130kD蛋白，部分菌株还表达了100Kd、60kD蛋白，具有典型Bt杀虫晶体蛋白的特征；质粒图谱显示不同菌株质粒图谱条带数量和大小都有所不同，并且有部分菌株图谱相似，说明资源库中有冗余菌株。
建立Bt菌株多样性分析方法：首先，设计了可以扩增多个基因家族的兼并引物，fastPCR软件分析结果显示该引物可以对超过30种的杀虫基因家族进行较好的扩增，PCR实验检测显示，该引物可以扩增出预期条带但是条带较弱。进一步通过增加侧翼特异序列的办法对兼并引物进行改良，改良后的引物显著提高了扩增效率。同时，还对引物浓度与退火温度进行了优化，利用72个不同血清型的标准菌对优化后的PCR体系进行了评估，结果显示95%以上的菌株都可以获得预期条带；利用含有多个基因的HD12菌株评估了优化后体系对多个基因同时扩增的能力，结果显示优化后的PCR可以从HD12获得9种不同的RFLP类型。这些结果说明优化后的PCR体系具备了cry基因指纹图谱的基本要求。此外，本论文还对Bt基因组提取方法进行了改良，改良后的方法可以提取并获得质量均一的基因组DNA。用改良的PCR体系对Bt基因组进行扩增，并用HinfI对PCR产物进行消化，利用LabChip GX电泳系统进行分析，获得了888个菌株的RFLP图谱。进一步对电泳图谱进行数字化、计算相似性、构建系统发生树分析。结果显示，该方法可以有效分析菌株多样性，去除冗余菌株，获得可用于进一步基因发掘的菌株。
90株Bt菌株杀虫基因测序筛选：利用高通量测序技术分析了90株不同的Bt菌株的序列，进一步通过SOAPdenovo软件拼接、GeneMark基因预测、模式杀虫基因数据库及专利数据库blast比对，最终获得全新杀虫基因21个。
总之，本论文建立菌株多样性研究方法，解决了冗余菌株会造成资金的浪费的问题，最终完成了“菌株分离去除冗余基因测序筛选”这一杀虫基因高通量发掘平台的构建，为提升我国在Bt资源发掘中的竞争力提供了重要保障。
Bacillus thuringiensis，gram positive bacteria, it can produce parasporal crystal during spore periodand play an important role in the pest control. The crystal mainly consist by the insecticidal crystalproteins which encode by cry genes, because the commercial value of Bt insecticidal gene, andexploring new toxin genes and protecting intellectual property rights is one of the important Bt resourceresearch content.
As the intellectual property rights reflect the comprehensive competitiveness, our country at adisadvantage situation now. At present, most of Bt insecticidal gene patents were controlled by foreignagricultural biotechnology companies, and our country contoled less than60new gene with patents. Thedevelopment of high-throughput sequencing technologies provides new idea for insecticidal genemining and became the principal means to find new Bt insecticidal genes. But the sequencing price isrelative higher and the redundancy in Bt resource will be a big waste; and now there was no method canbe applied in strain comparison. Therefore, there is an urgent need.to set up an efficient method that cananalyzing and comparing large scale sample, removing redundant strains, to improve the insecticidalgene discovery efficiency. This thesis, beyond isolating1500strains Bt strains, setting up Bt diversityanalysis method, analyzing the strains diversity, removing redundant strains, sequencing screening90strains, and the results are:
Isolation and analysis of Bt strains: The toxic activity evaluation results indicated that most strainshave highly toxicity against lepidoptera pests, in1500tested strains, there are1346strains toxic toHelicoverpa armigera,813strains toxic to Ostrinia furnacalis and only a few strains have toxic to leafbeetle, just42strains. Microscopic examination results shown that crystals are different in shape, size,and types between strains, most of them were rhombus, also contains spherical or irregular shapecrystals. Insecticidal crystal protein profiles analysis indicated that most strains expressing130kDprotein, and few of strains have100kD、60kD bands. With typical characteristic of Bt insecticidalcrystal proteins. Plasmid profile analysis indicated that the bands size and number is different betweenstrains and some strains’ plasmid profile are identical, this result indicated that they are redundant strainin the collection.
Set up Bt stain diversity analysis method. Firstly, a pair degenerated primer were designed toamplify multi-cry genes. The fastPCR software analysis results showed that the primer set can amplifymore than30types insecticidal gene families. The following PCR results shown that the primer canwork but the amplicon bands produced were very week. Then, the primer set was improved by adding flank specific sequence, the new primer set were significantly improved the amplification efficiency.Furthermore, the primer concentrations and annealing temperature of PCR system were optimized; andthe testing of72different serotype standard Bt strains shown that the optimized PCR system canamplify expect bands from more than95%of the strains. The multiple genes amplification ability of theoptimized PCR system was evaluate by HD12that containing multiple genes, the results indicated thatit amplified9different cry gene fragments from the HD12strain. These results state that the optimizedPCR system with the basic requirements for Bt strains cry gene fingerprinting for its widelyamplification spectrum. In this thesis, Bt genome DNA extraction method also improved, the newmethod can get homogeneous quality genome DNA. The strains genome DNA were amplified by theoptimized PCR system and the PCR products were digested by Hinf I. the digested PCR products wereanalyzed by LabChip GX Electrophoresis system. Totally, in this thesis,888strains PCR-RFLP patternwere analyzed. The strains similarity and phylogenetic tree were constructed by PCR-RFLP patternsdigitize and computing. The results indicated that the method described here can effectively remove theredundant strain and yield valuable strains for next toxin gene finding.
Screening insecticidal gene by sequencing of90Bt strains:90different strains sequence wereanalyzed by HiSeqTM2000and assembled by SOAPdenovo, then, the nsecticidal genes were predictby GeneMark, the insecticidal genes were screen out by blast software packge; and the NCBI patentdatabase were used to get rid of these already pattened genes. Finaly,21new insecticidal genes havebeen revealed in this study.
In conclusion, a Bt strain diversity analysis method set up, and it can be used in removing theredundancy strains in collection, finally completed the “strain isolationg redundant removingtoxin gene sequencing screening” high throughput insecticidal gene screening platform construction.This work provides important guarantee to enhance competitiveness of China in the Bt resourcesdiscovery.
Bt菌株分离与鉴定：杀虫活性评估结果显示多数菌株对鳞翅目害虫有较高活性，1500株测试菌株中，对棉铃虫有活性的菌株有1346株，对玉米螟有活性菌株有813株，而对叶甲类害虫大猿叶甲有活性的菌株较少，只有42株；镜检结果显示不同菌株产生的晶体形状、大小、种类有较大差异，晶体种类主要为菱形，部分为球形和不规则的晶体形状；杀虫晶体蛋白图谱显示大部分菌株表达了130kD蛋白，部分菌株还表达了100Kd、60kD蛋白，具有典型Bt杀虫晶体蛋白的特征；质粒图谱显示不同菌株质粒图谱条带数量和大小都有所不同，并且有部分菌株图谱相似，说明资源库中有冗余菌株。
建立Bt菌株多样性分析方法：首先，设计了可以扩增多个基因家族的兼并引物，fastPCR软件分析结果显示该引物可以对超过30种的杀虫基因家族进行较好的扩增，PCR实验检测显示，该引物可以扩增出预期条带但是条带较弱。进一步通过增加侧翼特异序列的办法对兼并引物进行改良，改良后的引物显著提高了扩增效率。同时，还对引物浓度与退火温度进行了优化，利用72个不同血清型的标准菌对优化后的PCR体系进行了评估，结果显示95%以上的菌株都可以获得预期条带；利用含有多个基因的HD12菌株评估了优化后体系对多个基因同时扩增的能力，结果显示优化后的PCR可以从HD12获得9种不同的RFLP类型。这些结果说明优化后的PCR体系具备了cry基因指纹图谱的基本要求。此外，本论文还对Bt基因组提取方法进行了改良，改良后的方法可以提取并获得质量均一的基因组DNA。用改良的PCR体系对Bt基因组进行扩增，并用HinfI对PCR产物进行消化，利用LabChip GX电泳系统进行分析，获得了888个菌株的RFLP图谱。进一步对电泳图谱进行数字化、计算相似性、构建系统发生树分析。结果显示，该方法可以有效分析菌株多样性，去除冗余菌株，获得可用于进一步基因发掘的菌株。
90株Bt菌株杀虫基因测序筛选：利用高通量测序技术分析了90株不同的Bt菌株的序列，进一步通过SOAPdenovo软件拼接、GeneMark基因预测、模式杀虫基因数据库及专利数据库blast比对，最终获得全新杀虫基因21个。
总之，本论文建立菌株多样性研究方法，解决了冗余菌株会造成资金的浪费的问题，最终完成了“菌株分离去除冗余基因测序筛选”这一杀虫基因高通量发掘平台的构建，为提升我国在Bt资源发掘中的竞争力提供了重要保障。
Bacillus thuringiensis，gram positive bacteria, it can produce parasporal crystal during spore periodand play an important role in the pest control. The crystal mainly consist by the insecticidal crystalproteins which encode by cry genes, because the commercial value of Bt insecticidal gene, andexploring new toxin genes and protecting intellectual property rights is one of the important Bt resourceresearch content.
As the intellectual property rights reflect the comprehensive competitiveness, our country at adisadvantage situation now. At present, most of Bt insecticidal gene patents were controlled by foreignagricultural biotechnology companies, and our country contoled less than60new gene with patents. Thedevelopment of high-throughput sequencing technologies provides new idea for insecticidal genemining and became the principal means to find new Bt insecticidal genes. But the sequencing price isrelative higher and the redundancy in Bt resource will be a big waste; and now there was no method canbe applied in strain comparison. Therefore, there is an urgent need.to set up an efficient method that cananalyzing and comparing large scale sample, removing redundant strains, to improve the insecticidalgene discovery efficiency. This thesis, beyond isolating1500strains Bt strains, setting up Bt diversityanalysis method, analyzing the strains diversity, removing redundant strains, sequencing screening90strains, and the results are:
Isolation and analysis of Bt strains: The toxic activity evaluation results indicated that most strainshave highly toxicity against lepidoptera pests, in1500tested strains, there are1346strains toxic toHelicoverpa armigera,813strains toxic to Ostrinia furnacalis and only a few strains have toxic to leafbeetle, just42strains. Microscopic examination results shown that crystals are different in shape, size,and types between strains, most of them were rhombus, also contains spherical or irregular shapecrystals. Insecticidal crystal protein profiles analysis indicated that most strains expressing130kDprotein, and few of strains have100kD、60kD bands. With typical characteristic of Bt insecticidalcrystal proteins. Plasmid profile analysis indicated that the bands size and number is different betweenstrains and some strains’ plasmid profile are identical, this result indicated that they are redundant strainin the collection.
Set up Bt stain diversity analysis method. Firstly, a pair degenerated primer were designed toamplify multi-cry genes. The fastPCR software analysis results showed that the primer set can amplifymore than30types insecticidal gene families. The following PCR results shown that the primer canwork but the amplicon bands produced were very week. Then, the primer set was improved by adding flank specific sequence, the new primer set were significantly improved the amplification efficiency.Furthermore, the primer concentrations and annealing temperature of PCR system were optimized; andthe testing of72different serotype standard Bt strains shown that the optimized PCR system canamplify expect bands from more than95%of the strains. The multiple genes amplification ability of theoptimized PCR system was evaluate by HD12that containing multiple genes, the results indicated thatit amplified9different cry gene fragments from the HD12strain. These results state that the optimizedPCR system with the basic requirements for Bt strains cry gene fingerprinting for its widelyamplification spectrum. In this thesis, Bt genome DNA extraction method also improved, the newmethod can get homogeneous quality genome DNA. The strains genome DNA were amplified by theoptimized PCR system and the PCR products were digested by Hinf I. the digested PCR products wereanalyzed by LabChip GX Electrophoresis system. Totally, in this thesis,888strains PCR-RFLP patternwere analyzed. The strains similarity and phylogenetic tree were constructed by PCR-RFLP patternsdigitize and computing. The results indicated that the method described here can effectively remove theredundant strain and yield valuable strains for next toxin gene finding.
Screening insecticidal gene by sequencing of90Bt strains:90different strains sequence wereanalyzed by HiSeqTM2000and assembled by SOAPdenovo, then, the nsecticidal genes were predictby GeneMark, the insecticidal genes were screen out by blast software packge; and the NCBI patentdatabase were used to get rid of these already pattened genes. Finaly,21new insecticidal genes havebeen revealed in this study.
In conclusion, a Bt strain diversity analysis method set up, and it can be used in removing theredundancy strains in collection, finally completed the “strain isolationg redundant removingtoxin gene sequencing screening” high throughput insecticidal gene screening platform construction.This work provides important guarantee to enhance competitiveness of China in the Bt resourcesdiscovery.
引文
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