青贮用纤维素分解工程乳酸菌的构建
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摘要
青贮是一种以新鲜的青绿饲料为原料,利用植物表面自然附生的乳酸菌,在密闭条件下,通过厌氧发酵,将植物表面的可溶性碳水化合物转化为有机酸,导致饲料pH值降低,来抑制腐败微生物菌群的生长繁殖,从而达到保持作物的营养特性的目的的技术。其有着其独特的,其它种类饲料所无法比及的优点:青贮饲料能够较好的保存原料的蛋白、可溶性碳水化合物、小分子维生素和矿物质的含量,便于牲畜吸收利用,提高青贮饲料的消化率的同时也提高了生产率;其适口性好,利于牲畜采食;青贮过程中有益微生物的活动,如乳酸菌产生的乳酸可以杀死很多牲畜的病害微生物、寄生虫卵等。人为的添加一些工程乳酸菌作为青贮剂,能促进有益微生物的活动,可以进一步降解原料中的纤维素成分转化为小分子的单糖、寡糖或者低聚糖,便于牲畜吸收利用,提高青贮饲料的消化率。
本实验以在模式大肠杆菌菌株Rosetta和乳酸菌菌株NZ9000中表达瑞氏木霉(Trichoderma reesei)CBHⅡ基因的方法,得到两株能表达外源纤维素酶的工程菌;然后,利用REAL-TIME RT-PCR对外源基因在工程乳酸菌中的转录水平表达进行准确的绝对定量分析,建立一种新的REAL-TIME RT-PCR分析方法,同时利用pNPC法测定了工程菌的酶活力,为构建青贮用工程乳酸菌提供了一种技术路线和对其进行相关指标评价的方法。
通过实验得到如下结果:将纤维素酶基因CBHⅡ与表达载体pET30a重组,构建了重组质粒pETCBH。将重组质粒转化Rosetta宿主菌,获得了重组菌株Rosetta(pETCBH);利用IPTG诱导重组菌株Rosetta(pETCBH),并通过pNPC法测定了重组纤维素酶CBHⅡ的活性,其活性为2.677U/mL。将纤维素酶基因CBHⅡ与表达载体pNZ8148重组,构建了重组质粒pNZCBH。将重组质粒转化NZ9000宿主菌,获得了重组菌株NZ9000(pNZCBH)。建立了利用基于DNA扣除法的REAL-TIME RT PCR对基因进行定量分析的方法,该方法平行性好,可信度高,可以在转录水平分析目的基因表达情况;同时,此方法从理论上解决了传统定量法结果不准确的问题。最后,通过两种聚丙烯酰胺凝胶银染色方法的比较,证明了硝酸银染色法的效果大大优于传统的考马斯亮蓝染色法。
本实验在以下方面有所创新:
1.建立了一种较准确的在转录水平检测外源基因在原核载体中表达的绝对量的方法。
2.为构建青贮用工程乳酸菌提供了一条可以借鉴的技术路线。
Silage is that,in hermetic containers,fresh green material is fermented by plant surface of naturally occurring lactic acid bacteria. The result is soluble carbohydrates is transformed into organic acids, which leads to the lower pH. With inhibiting the growing of harmful micro-organisms, the nutritional characteristics of crops can be maintained. Compared with other types of feed, silage has unique advantages. First of all, protein, soluble carbohydrates, small molecules, vitamin and mineral of raw materials are maintained, and absorption and utilization of livestock are promoted. Silage improves the digestibility, but also increases productivity. The second one, during the activities of beneficial micro-organisms, the products such as the lactic acid produced by lactic acid bacteria, can kill a lot of disease micro-organism, parasite eggs, etc. Adding some gene-modified lactic acid bacteria as additive agents furtherly degradate the cellulose component of raw materials into small molecules of simple sugars, oligosaccharides, or oligosaccharides, at the same time the specific engineering bacteria increases productivity.
In this experiment, the model E. coli strains and lactic acid bacteria strains NZ9000 were used to express exogenous cellulase by transfected CBHⅡg ene of Trichoderma reesei. The application of REAL-TIME RT-PCR was used to analysis the transcription of foreign gene in the engineered lactic acid bacteria. Then, method of pNP is devoted to measure the enzyme activity of engineering bacteria. The experiment provided a technical route to construct gene-modified lactic acid bacteria for silage, and with methods providing by this project, associated indicators can be evaluated properly.
Through the experiment we owned the following results: the cellulose of CBHⅡgene and expression vector pET30a constructed the recombinant plasmid pETCBH. The recombinant plasmid was transformed into Rosetta host bacteria obtained recombinant strain Rosetta (pETCBH).IPTG induced recombinant strain Rosetta (pETCBH), and through determination of the restructuring pNPC cellulase CBHⅡactivity, its activity was 2.677U/mL. In the experiment, cellulose of CBHⅡgene and expression vector pNZ8148 constructed the recombinant plasmid pNZCBH. The recombinant plasmid was transformed into NZ9000 host bacteria obtained recombinant strain NZ9000 (pNZCBH). Also, the project established the method of REAL-TIME RT-PCR based on DNA subtraction for absolute quantification of gene expression in engineered lactic acid bacteria. Method with good parallelism, high reliability can be analyzed target gene expression at the transcriptional level. This method is in theory to solve the traditional quantitative method without an accurate result. The comparison of two methods to stain Polyacrylamide gel confirmes that the effect of silver nitrate staining method is much better than the traditional coomassie brilliant blue.
The innovative points of this test are as follows:
1. A more accurate way to detect the absolute volume of transcription and the expression of foreign genes in prokaryotic absolute volume was established.
2. The project provided a method of recombinant lactic acid bacteria strain, which can draw the draft of the technical route.
本实验以在模式大肠杆菌菌株Rosetta和乳酸菌菌株NZ9000中表达瑞氏木霉(Trichoderma reesei)CBHⅡ基因的方法,得到两株能表达外源纤维素酶的工程菌;然后,利用REAL-TIME RT-PCR对外源基因在工程乳酸菌中的转录水平表达进行准确的绝对定量分析,建立一种新的REAL-TIME RT-PCR分析方法,同时利用pNPC法测定了工程菌的酶活力,为构建青贮用工程乳酸菌提供了一种技术路线和对其进行相关指标评价的方法。
通过实验得到如下结果:将纤维素酶基因CBHⅡ与表达载体pET30a重组,构建了重组质粒pETCBH。将重组质粒转化Rosetta宿主菌,获得了重组菌株Rosetta(pETCBH);利用IPTG诱导重组菌株Rosetta(pETCBH),并通过pNPC法测定了重组纤维素酶CBHⅡ的活性,其活性为2.677U/mL。将纤维素酶基因CBHⅡ与表达载体pNZ8148重组,构建了重组质粒pNZCBH。将重组质粒转化NZ9000宿主菌,获得了重组菌株NZ9000(pNZCBH)。建立了利用基于DNA扣除法的REAL-TIME RT PCR对基因进行定量分析的方法,该方法平行性好,可信度高,可以在转录水平分析目的基因表达情况;同时,此方法从理论上解决了传统定量法结果不准确的问题。最后,通过两种聚丙烯酰胺凝胶银染色方法的比较,证明了硝酸银染色法的效果大大优于传统的考马斯亮蓝染色法。
本实验在以下方面有所创新:
1.建立了一种较准确的在转录水平检测外源基因在原核载体中表达的绝对量的方法。
2.为构建青贮用工程乳酸菌提供了一条可以借鉴的技术路线。
Silage is that,in hermetic containers,fresh green material is fermented by plant surface of naturally occurring lactic acid bacteria. The result is soluble carbohydrates is transformed into organic acids, which leads to the lower pH. With inhibiting the growing of harmful micro-organisms, the nutritional characteristics of crops can be maintained. Compared with other types of feed, silage has unique advantages. First of all, protein, soluble carbohydrates, small molecules, vitamin and mineral of raw materials are maintained, and absorption and utilization of livestock are promoted. Silage improves the digestibility, but also increases productivity. The second one, during the activities of beneficial micro-organisms, the products such as the lactic acid produced by lactic acid bacteria, can kill a lot of disease micro-organism, parasite eggs, etc. Adding some gene-modified lactic acid bacteria as additive agents furtherly degradate the cellulose component of raw materials into small molecules of simple sugars, oligosaccharides, or oligosaccharides, at the same time the specific engineering bacteria increases productivity.
In this experiment, the model E. coli strains and lactic acid bacteria strains NZ9000 were used to express exogenous cellulase by transfected CBHⅡg ene of Trichoderma reesei. The application of REAL-TIME RT-PCR was used to analysis the transcription of foreign gene in the engineered lactic acid bacteria. Then, method of pNP is devoted to measure the enzyme activity of engineering bacteria. The experiment provided a technical route to construct gene-modified lactic acid bacteria for silage, and with methods providing by this project, associated indicators can be evaluated properly.
Through the experiment we owned the following results: the cellulose of CBHⅡgene and expression vector pET30a constructed the recombinant plasmid pETCBH. The recombinant plasmid was transformed into Rosetta host bacteria obtained recombinant strain Rosetta (pETCBH).IPTG induced recombinant strain Rosetta (pETCBH), and through determination of the restructuring pNPC cellulase CBHⅡactivity, its activity was 2.677U/mL. In the experiment, cellulose of CBHⅡgene and expression vector pNZ8148 constructed the recombinant plasmid pNZCBH. The recombinant plasmid was transformed into NZ9000 host bacteria obtained recombinant strain NZ9000 (pNZCBH). Also, the project established the method of REAL-TIME RT-PCR based on DNA subtraction for absolute quantification of gene expression in engineered lactic acid bacteria. Method with good parallelism, high reliability can be analyzed target gene expression at the transcriptional level. This method is in theory to solve the traditional quantitative method without an accurate result. The comparison of two methods to stain Polyacrylamide gel confirmes that the effect of silver nitrate staining method is much better than the traditional coomassie brilliant blue.
The innovative points of this test are as follows:
1. A more accurate way to detect the absolute volume of transcription and the expression of foreign genes in prokaryotic absolute volume was established.
2. The project provided a method of recombinant lactic acid bacteria strain, which can draw the draft of the technical route.
引文
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关海涛,徐世昌,郭玉华. 2006.两种聚丙烯酰胺凝胶银染方法的比较[J].沈阳农业大学学报,37(1): 86?87.
林春健. 2003.生物技术在畜禽营养和饲养上的应用-青贮保存法[M].国外畜禽生产新技术.北京:中国农业大学出版社,
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相丽,姜毓君,霍贵成. 2008.金黄色葡萄球菌基因表达的DNA扣除法REAL-TIME RT PCR相对定量分析[J].微生物学报Acta Microbiologica Sinica ,48(4):526~531.
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张继泉,王瑞明,孙玉英. 2003.瑞氏木霉生产纤维素酶的研究进展[J].饲料工业,24(1):9-13.
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戴忠民,高凤菊,王友平等. 2004.青贮玉米的育种及发展趋势[J].玉米科学,12(4): 9-11.
关海涛,徐世昌,郭玉华. 2006.两种聚丙烯酰胺凝胶银染方法的比较[J].沈阳农业大学学报,37(1): 86?87.
林春健. 2003.生物技术在畜禽营养和饲养上的应用-青贮保存法[M].国外畜禽生产新技术.北京:中国农业大学出版社,
阮孟斌,朱明月,周鹏. 2004.外源基因在乳酸菌中的表达[J].应用与环境生物学报,10(4):526-529.
孙冬霞. 2007.玉米秸秆工厂化青贮的社会经济效益分析[J].现代农业科技,20:190-193.
孙磊,孔健. 2005.乳酸菌青贮剂的研究[D].山东大学硕士论文.
苏俊. 2006.黑龙江玉米育种50年回顾[J].黑龙江农业科学,5: 8-12.
相丽,姜毓君,霍贵成. 2008.金黄色葡萄球菌基因表达的DNA扣除法REAL-TIME RT PCR相对定量分析[J].微生物学报Acta Microbiologica Sinica ,48(4):526~531.
杨洁彬,郭兴华,张旎等. 1996.乳酸菌一生物学基础及应用[M].中国轻工业出版社,46:187-191.
杨雪霞,陈洪章,李佐龙. 2001.添加纤维素酶的青贮研究进展[J].生物技术通报,1: 37-41.
张晶,刘敬忠,谭淑珍等. 2002.苯丙氨酸脱氨酶在乳酸乳球菌NICE系统的高效表达及其实验动物研究[J].生物工程学报,18 (6):713-717.
张贺,李波. 2006.实时荧光定量PCR技术研究进展及应用[J].动物医学进展,27(增):5-12.
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