酸性植酸酶基因的克隆及其在大肠杆菌和毕赤酵母中的表达
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摘要
植酸酶是一种新型的可作为动物饲料添加剂的重要酶制剂，是一种能水解植酸的磷酸酶类。在动物饲料中富含磷的谷物、豆类和油料等作物中，磷的基本贮存形式是植酸磷，因单胃动物体内缺少能分解植酸的酶很难利用这些植酸中的磷，其利用率极低。植酸酶对提高饲料中磷的利用率以及减轻因动物高磷粪便所导致的环境污染有很大的作用。
植酸酶主要来自于植物和微生物，来源于植物的植酸酶均属于6—植酸酶，最适pH范围在5.0—7.5，不适合在单胃动物中起作用的，而且在植物中含量极低，因此植酸酶的研究方向转向最适pH值为酸性、酶含量较高的丝状真菌。
本实验成功从丝状真菌中提取出总DNA，通过PCR扩增，将此酸性植酸酶全基因通过pPIC9K vector转化酵母细胞，提取酵母转化子基因组进行PCR验证，结果表明外源基因整合到了酵母的染色体上。
提取未降解的、不含DNA与其他杂质的RNA是进行分子克隆和基因表达分析等研究的基础。由于复杂的细胞壁和内源酶活性的存在，所以要从丝状真菌中提取和纯化出有生物活性的RNA比较困难。为获得高质量的RNA，本实验比较了TRIZOL提总RNA、异硫氰酸胍法提取菌株总RNA、热硼酸法提取菌株总RNA三种不同的提取方法，得出热硼酸法适合于富含Rnase和多糖的丝状真菌的RNA的提取，该方法可得到完整、均一的RNA样品，并且得到的RNA可用于RT-PCR等分子生物学实验操作。
本实验以丝状真菌黑曲霉的总cDNA为模板，通过PCR方法扩增到理想大小的DNA片段，分别将其与质粒pBV220连接，将正确构建的重组质粒pBV220-PhyB转化大肠杆菌D(?)5α，在温度诱导下，该基因在大肠杆菌中得到了表达，SDS-PAGE分析表达产生的蛋白约60kD，表达产生的植酸酶其最适反应PH为2.5，最适反应温度为55℃。
Phytase is a new-type important enzyme product as animal feed additives, which can catalyze phytic acid. Phosphorus presented in crops such as grains, beans, oil-bearing crops is in the form of phytate-phosphorus. The utilization rate of phosphorus in phytate by monogastric animals was very low because of the lack of phytase. Supplementation with phytase is an effective way to increase utilization rate of phosphorus in seed-based animal feed and to reduce phosphate pollution in the environment.
Phytase mainly comes from plant and organism, phytase of plant belongs to 6-phytase and it' s optimum pH ranges from 5.0 to 7. 5. Plant phytase can not function effectively in monogastric animals and it is not rich in plant. So research direction about phytase is turned to Aspergillus niger which is rich in acid phytase.
This experiment extracted total DNA from Aspergillus niger, using the DNA as template,we get complete phytase gene by PCR amplification. We cloned this gene into pPIC9K vector and transformed this express vector into yeast.
Extracting RNA which is not degraded and contain no DNA is basis to do research about molecular cloning and gene expressing analysis. It is very difficult to extract active RNA from Aspergillus niger because of the existance of complex cell wall and endo-Rnase activity. To obtain RNA with high quality, we compared three such different extraction methods as TRIZOL, Isothiocyanate Method and Hot Borate Method. Experiements show that Hot Borate Method is best for the RNA extraction from Aspergillus
niger that is rich in Rnase and polysaccharide,and we can get pure and integrate RNA sample which can be used in RT-PCR experiment.
The phytase gene was amplified by PCR using Aspergillus niger cDNA as template, and the gene was inserted into the vector pBV220 and pPIC9K. The recombinant vector containing acid phosphorutase gene was transformed into ? coli DH5 a strain. Induced by temperature the acid phosphorutase gene was expressed to be a protein of about 60kD by SDS-PAGE analysis, its optimun reaction pH is 2.5 and its optimum reaction temperature is 55C.
Zhang Jianying(Biochemistry and Molecular Biology) Directed by professor Xie Daping Jiao Qinghua
植酸酶主要来自于植物和微生物，来源于植物的植酸酶均属于6—植酸酶，最适pH范围在5.0—7.5，不适合在单胃动物中起作用的，而且在植物中含量极低，因此植酸酶的研究方向转向最适pH值为酸性、酶含量较高的丝状真菌。
本实验成功从丝状真菌中提取出总DNA，通过PCR扩增，将此酸性植酸酶全基因通过pPIC9K vector转化酵母细胞，提取酵母转化子基因组进行PCR验证，结果表明外源基因整合到了酵母的染色体上。
提取未降解的、不含DNA与其他杂质的RNA是进行分子克隆和基因表达分析等研究的基础。由于复杂的细胞壁和内源酶活性的存在，所以要从丝状真菌中提取和纯化出有生物活性的RNA比较困难。为获得高质量的RNA，本实验比较了TRIZOL提总RNA、异硫氰酸胍法提取菌株总RNA、热硼酸法提取菌株总RNA三种不同的提取方法，得出热硼酸法适合于富含Rnase和多糖的丝状真菌的RNA的提取，该方法可得到完整、均一的RNA样品，并且得到的RNA可用于RT-PCR等分子生物学实验操作。
本实验以丝状真菌黑曲霉的总cDNA为模板，通过PCR方法扩增到理想大小的DNA片段，分别将其与质粒pBV220连接，将正确构建的重组质粒pBV220-PhyB转化大肠杆菌D(?)5α，在温度诱导下，该基因在大肠杆菌中得到了表达，SDS-PAGE分析表达产生的蛋白约60kD，表达产生的植酸酶其最适反应PH为2.5，最适反应温度为55℃。
Phytase is a new-type important enzyme product as animal feed additives, which can catalyze phytic acid. Phosphorus presented in crops such as grains, beans, oil-bearing crops is in the form of phytate-phosphorus. The utilization rate of phosphorus in phytate by monogastric animals was very low because of the lack of phytase. Supplementation with phytase is an effective way to increase utilization rate of phosphorus in seed-based animal feed and to reduce phosphate pollution in the environment.
Phytase mainly comes from plant and organism, phytase of plant belongs to 6-phytase and it' s optimum pH ranges from 5.0 to 7. 5. Plant phytase can not function effectively in monogastric animals and it is not rich in plant. So research direction about phytase is turned to Aspergillus niger which is rich in acid phytase.
This experiment extracted total DNA from Aspergillus niger, using the DNA as template,we get complete phytase gene by PCR amplification. We cloned this gene into pPIC9K vector and transformed this express vector into yeast.
Extracting RNA which is not degraded and contain no DNA is basis to do research about molecular cloning and gene expressing analysis. It is very difficult to extract active RNA from Aspergillus niger because of the existance of complex cell wall and endo-Rnase activity. To obtain RNA with high quality, we compared three such different extraction methods as TRIZOL, Isothiocyanate Method and Hot Borate Method. Experiements show that Hot Borate Method is best for the RNA extraction from Aspergillus
niger that is rich in Rnase and polysaccharide,and we can get pure and integrate RNA sample which can be used in RT-PCR experiment.
The phytase gene was amplified by PCR using Aspergillus niger cDNA as template, and the gene was inserted into the vector pBV220 and pPIC9K. The recombinant vector containing acid phosphorutase gene was transformed into ? coli DH5 a strain. Induced by temperature the acid phosphorutase gene was expressed to be a protein of about 60kD by SDS-PAGE analysis, its optimun reaction pH is 2.5 and its optimum reaction temperature is 55C.
Zhang Jianying(Biochemistry and Molecular Biology) Directed by professor Xie Daping Jiao Qinghua
引文
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2 Rudy J. Wodzinski A.H.J. Ullah. Phytase [J]. Advances in applied microbiology,1996, (42): 263～302
3 Ostanin K, Saeed A, Robert L van Ettenl Heterologous expression of human prostatic acid phosphatase and site-directedmutagenesis of the enzyme active site [J]. J Biol Chem,1994,269(12): 8971-8978
4 WYSS M, PASAMONTES L, FRIEDLEIN A, et al. Biophysical characterization of fungal phytase (myo-inositol hexakisphosphate phosphohydrolase): molecular size, glycosylation pattern and engineering of proteolytic resistance [J]. Appl Environ Microbiol, 1999,65 (2): 359-366.
5 WYSSM, BRUGGER R, KROENBERGER A, et al. Biochemical of fungal phytase (myo-inositol hexakisphosphate phosphohydrolase): catalytic properties[J]. Appl Environ Microbiol, 1999,65(2): 367-373.
6 RODRIGUEZ E, MULLANEY E J, LEI X G. Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme [J]. Biochem Biophys Res Commun ,2000,268 (2) :373-378.
7 MULLANEY E J, DALY C B, SETHUMADHAVAN K, et al. Phytase activity in Aspergillusfumigatus isolates [J]. Biochem Biophys Res Commun, 2000,275 (3) :759-763.
8 姚斌，范云六，张春义，等，来源于Bacillus subtilis的中性植酸酶基因的克隆及其在大肠杆菌中表达[J]．生物工程学报，2001，97(1)：5-11
9 屈健，饲用酶制剂—植酸酶[J]．饲料博览，1998，10(5)：10-12
10 KEROVUO J ,LAURAEUS M, NURINEN P, et al. Isolation, characterization ,molecular gene cloning and sequencing of a novel phytase from Bacillus. subtilis [J]. Appl Environ Microbiol ,1998,64(6): 2079-2085.
11 PASAMONTES L, HAIKER M,WYSS M, et al. Gene cloning, purification, and characterization of a heat-stable phytase from the fungus: Aspergillusfumigatus [J]. Appl Environ Microbiol, 1997 63(5): 1696-1700.
12 彭日荷，熊爱生，李贤，等．应用毕氏酵母高效表达耐高溫植酸酶[J]．生物化学和生物物理学报，2002，34(6)：725-730．
13 BERKA R M, REY M W, BROWN K M, et al.
Molecular charterization and expression of phytase gene from thermomyces lanuginosus [J]. Appl Environ Microbiol, 1998,64 (1): 4423-4427.
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