黑曲霉N25植酸酶phyA基因表达片段的克隆及pPIC9K重组酵母表达载体的构建
摘要
采用改良二次沉淀法从黑曲霉N25中抽提出细胞总DNA。根据本课
题组已经测定出的，并在GenBank中注册的黑曲霉N25植酸酶phyA基
因的全序列（Accession No. AF218813），从信号肽序列切割位点序列之
后（第19个氨基酸之后）设计上游引物（设计一个EcoRI酶切位点）；
在终止密码子处设计下游引物（设计两个酶切位点KpnⅠ和NotⅠ）。用高
保真度的聚合酶Advantage-HF PCR Kit进行表达片段扩增，通过对PCR
条件的优化，得到了大小约1.4kb的单一条带产物。将该产物经EcoRⅠ
和KpnⅠ酶切处理后连接到pUC18载体上，转化大肠杆菌JM109菌株，
在含Amp和x-gal的LB平板上筛选到了白色的阳性菌落。质粒酶切分
析结果表明获得了pUC18-phyA重组质粒（命名为pANP-1）；测序结果
表明，表达片段除不含有信号肽和内含子外，其序列与黑曲霉N25植酸
酶phyA基因相对应的序列完全一致。采用EcoRⅠ和NotⅠ内切酶从pANP-1
质粒中酶切出phyA基因表达片段，插入到毕赤巴斯德表达载体pPIC9K
的多克隆位点，并转化大肠杆菌DH5a菌株，用光生物素标记的phyA基
因探针进行原位杂交，筛选到了阳性转化菌落，质粒酶切分析结果表明
获得了pPIC9K—phyA重组表达载体，命名为pPNP-1。pPNP-1重组表
达载体的构建为phyA基因在毕赤巴斯德酵母中的高效表达奠定了基础。
By using modified method of two-step precipitation, the total DNA of
chromosom~(vas extracted from an Aspergiiius Niger (N25, China strain). A pair of
wiv~S
primers were designed and synthesized according to the sequence of phyA gene
(GenBank Accession No. AF2 18813) registered by Wang Hong-ning. The upstream
primer started with signal cleavage site (EcoR I site was designed), and the downstream
primer ended at stop codon (Kpn I and Not I were designed). By using a high-fidelity
polyerase (Advantage-HF), the DNA of encoding region (1.4kb) was amplified by PCR,
inserted into EcoR I and Kpn I sites of pUC 18 vector, and transformed into JM 109 strain.
As a result, white colonies were screened on LB plate (Amp and x-gal added). The result
of enzyme digestion of vector proved that the recombination vector (named pANP-1) was
obtained , and the sequence of interesting DNA showed that it was consistent with total
DNA of phyA gene without signal peptide and intron sequence. pANP- 1 vector was
digested by EcoR I and Not I , and the DNA of encoding region of phyA gene was taken
out, inserted into the multi-cloning site of pPIC9K vector, and transformed into E. coil
DH5a strain finally. Positive colonies 搼~~ selected by situ-hybridization with photo-biotin
labeled phyA gene. The result of enzyme digestion of vector proved that recombinati~i~
vector was obtained (named pPNP- 1). The successful construction of pPNP- 1 vector is
the key foundation of phyA gene expressed in Pichia pastoris.
题组已经测定出的，并在GenBank中注册的黑曲霉N25植酸酶phyA基
因的全序列（Accession No. AF218813），从信号肽序列切割位点序列之
后（第19个氨基酸之后）设计上游引物（设计一个EcoRI酶切位点）；
在终止密码子处设计下游引物（设计两个酶切位点KpnⅠ和NotⅠ）。用高
保真度的聚合酶Advantage-HF PCR Kit进行表达片段扩增，通过对PCR
条件的优化，得到了大小约1.4kb的单一条带产物。将该产物经EcoRⅠ
和KpnⅠ酶切处理后连接到pUC18载体上，转化大肠杆菌JM109菌株，
在含Amp和x-gal的LB平板上筛选到了白色的阳性菌落。质粒酶切分
析结果表明获得了pUC18-phyA重组质粒（命名为pANP-1）；测序结果
表明，表达片段除不含有信号肽和内含子外，其序列与黑曲霉N25植酸
酶phyA基因相对应的序列完全一致。采用EcoRⅠ和NotⅠ内切酶从pANP-1
质粒中酶切出phyA基因表达片段，插入到毕赤巴斯德表达载体pPIC9K
的多克隆位点，并转化大肠杆菌DH5a菌株，用光生物素标记的phyA基
因探针进行原位杂交，筛选到了阳性转化菌落，质粒酶切分析结果表明
获得了pPIC9K—phyA重组表达载体，命名为pPNP-1。pPNP-1重组表
达载体的构建为phyA基因在毕赤巴斯德酵母中的高效表达奠定了基础。
By using modified method of two-step precipitation, the total DNA of
chromosom~(vas extracted from an Aspergiiius Niger (N25, China strain). A pair of
wiv~S
primers were designed and synthesized according to the sequence of phyA gene
(GenBank Accession No. AF2 18813) registered by Wang Hong-ning. The upstream
primer started with signal cleavage site (EcoR I site was designed), and the downstream
primer ended at stop codon (Kpn I and Not I were designed). By using a high-fidelity
polyerase (Advantage-HF), the DNA of encoding region (1.4kb) was amplified by PCR,
inserted into EcoR I and Kpn I sites of pUC 18 vector, and transformed into JM 109 strain.
As a result, white colonies were screened on LB plate (Amp and x-gal added). The result
of enzyme digestion of vector proved that the recombination vector (named pANP-1) was
obtained , and the sequence of interesting DNA showed that it was consistent with total
DNA of phyA gene without signal peptide and intron sequence. pANP- 1 vector was
digested by EcoR I and Not I , and the DNA of encoding region of phyA gene was taken
out, inserted into the multi-cloning site of pPIC9K vector, and transformed into E. coil
DH5a strain finally. Positive colonies 搼~~ selected by situ-hybridization with photo-biotin
labeled phyA gene. The result of enzyme digestion of vector proved that recombinati~i~
vector was obtained (named pPNP- 1). The successful construction of pPNP- 1 vector is
the key foundation of phyA gene expressed in Pichia pastoris.
引文
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2．王红宁，黄勇，陈慧等．饲用微生物植酸酶的研究进展(综述)．国外畜牧学——饲料，1999，(3)：9-10
3．Cantor，A．H．家禽日粮中添加植酸酶可提高磷利用率和降低动物粪便磷环境污染．中国饲料，1993，(1)：40-43
4．罗绪刚，刘彬．荷兰猪饲粮种微生物植酸酶应用研究及相关环境问题．见：饲料营养研究进展．亚洲中医药杂志社，1998，77-86
5．黄遵锡，章克昌．植酸酶基础与应用研究概况．食品与发酵工业．1999，25(2)：54-58
6．蒋守群，吴天星．植酸酶的研究进展．动物营养学报，1999，11(3)：1-11
7．武素平．植酸酶在动物饲料磷营养中的作用．中国饲料，1996，(18)：25-26
8．张若寒等．植酸酶替代蛋鸡饲料磷酸氢钙的研究．中国饲料，1996，21：12-15
9. Lei, X. G., P. K. Ku, E. R. Miller, et al. Supplemental microbial phytase improves bioavailability of dietary zinc to weanling pigs. J. Nutr. 1993, 123:1117
10. Lei, X. G., P. K. Ku, E. R. Miller, et al. Supplementing corn-soybean meal diets with microbial phytase maximizes phytase phosphorus utilization by weanling pig. J. Anim. Sci. 1993b, 71:3368
11. Lei, X.G., Ku, P. K., Miller, E. R., et al. Calcium level affects the efficacy of supplemental microbial phytase in corn-soybean mael diets of weanling pigs. J. Anim. Sci. 1994, 72:139
12. Han, Y. M., Yang, F., Zhou, A. G., et al. Supplemental Phytase of Microbial and cereal Sources improve dietary phytase phosphorus utilization by pigs from weaning through finishing. J. Anim. Sci, 1997, 75:1017-1025
13. Han, Y. M., Roneker, K. R., et al. Adding wheat middling, microbial phytase, and citric acid to corn-soybean meal diets for growing pigs may replace inorganic phosphorus supplementation. J. Anim. Sci. 1998, 76:2649-2656
14. Nelson, T. S., Shieh, T. R., Wodzinski, R. J., et al. Effect of supplemental phytase on the utilization of phytase phosphorus by chicks. J. Nutr. 1971, 101:1289-1294
15. Simons, P.C.M., Versteegh, H.A.J., and Jongbloed, A.W., et al. Improvement of phosphorus avaiablity by microbial phytase in broiler and pigs. Br. J. Nutr. 1990,64:525-540
16. Rudy, J. Wodzinski, and Ullah, A.H.J.. Phytase. Advances in applied microbiology. 1996, (42): 263-302
17. Shieh, T.R. and Ware, J.H. Survey of microorganisms for the production of extracellular phytase. Appl. Microbiol. 1968, 16:1348-1351
18. Simons, P.C.M., Versteegh, H.A.J., and Jongbloed, A.W., et al. Improvement of phosphorus avaiablity by microbial phytase in broiler and pigs. Br. J. Nutr. 1990,64:525-540
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22．陈惠，王红宁．植酸酶产酶条件及热稳定性的研究．四川农业大学学报，1999，17(3)：291-294
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24. Howson, S. J., Davis, R. P. Production of phytase hydrolyzing enzyme by some fungi. Enzyme Microb. Technol, 1983, 5:377-382
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2．王红宁，黄勇，陈慧等．饲用微生物植酸酶的研究进展(综述)．国外畜牧学——饲料，1999，(3)：9-10
3．Cantor，A．H．家禽日粮中添加植酸酶可提高磷利用率和降低动物粪便磷环境污染．中国饲料，1993，(1)：40-43
4．罗绪刚，刘彬．荷兰猪饲粮种微生物植酸酶应用研究及相关环境问题．见：饲料营养研究进展．亚洲中医药杂志社，1998，77-86
5．黄遵锡，章克昌．植酸酶基础与应用研究概况．食品与发酵工业．1999，25(2)：54-58
6．蒋守群，吴天星．植酸酶的研究进展．动物营养学报，1999，11(3)：1-11
7．武素平．植酸酶在动物饲料磷营养中的作用．中国饲料，1996，(18)：25-26
8．张若寒等．植酸酶替代蛋鸡饲料磷酸氢钙的研究．中国饲料，1996，21：12-15
9. Lei, X. G., P. K. Ku, E. R. Miller, et al. Supplemental microbial phytase improves bioavailability of dietary zinc to weanling pigs. J. Nutr. 1993, 123:1117
10. Lei, X. G., P. K. Ku, E. R. Miller, et al. Supplementing corn-soybean meal diets with microbial phytase maximizes phytase phosphorus utilization by weanling pig. J. Anim. Sci. 1993b, 71:3368
11. Lei, X.G., Ku, P. K., Miller, E. R., et al. Calcium level affects the efficacy of supplemental microbial phytase in corn-soybean mael diets of weanling pigs. J. Anim. Sci. 1994, 72:139
12. Han, Y. M., Yang, F., Zhou, A. G., et al. Supplemental Phytase of Microbial and cereal Sources improve dietary phytase phosphorus utilization by pigs from weaning through finishing. J. Anim. Sci, 1997, 75:1017-1025
13. Han, Y. M., Roneker, K. R., et al. Adding wheat middling, microbial phytase, and citric acid to corn-soybean meal diets for growing pigs may replace inorganic phosphorus supplementation. J. Anim. Sci. 1998, 76:2649-2656
14. Nelson, T. S., Shieh, T. R., Wodzinski, R. J., et al. Effect of supplemental phytase on the utilization of phytase phosphorus by chicks. J. Nutr. 1971, 101:1289-1294
15. Simons, P.C.M., Versteegh, H.A.J., and Jongbloed, A.W., et al. Improvement of phosphorus avaiablity by microbial phytase in broiler and pigs. Br. J. Nutr. 1990,64:525-540
16. Rudy, J. Wodzinski, and Ullah, A.H.J.. Phytase. Advances in applied microbiology. 1996, (42): 263-302
17. Shieh, T.R. and Ware, J.H. Survey of microorganisms for the production of extracellular phytase. Appl. Microbiol. 1968, 16:1348-1351
18. Simons, P.C.M., Versteegh, H.A.J., and Jongbloed, A.W., et al. Improvement of phosphorus avaiablity by microbial phytase in broiler and pigs. Br. J. Nutr. 1990,64:525-540
19．郑常文，刘惠侠，董长江等．米曲霉(Aspergillus oryzae)植酸酶的分离纯化及性质研究．四川大学学报(自然科学版)，1993，30(2)：253-259
20．赵允麟等．无花果曲霉(NRRL3135)与黑曲霉(AsN70)植酸酶产酶条件的研究．酶食品与发酵工业，1996(3)：42-45
21．苗雪霞，贾新成．根霉植酸酶的研究I．菌株的分离筛选及发酵条件研究．菌物系统，1997，16(1)：70-73
22．陈惠，王红宁．植酸酶产酶条件及热稳定性的研究．四川农业大学学报，1999，17(3)：291-294
23. Yamada, K., Minoda, Y., Yamamoto, S. Phytase from Aspergillus terrus. production and some general properties of the enzyme. Agric. Biol. Chem, 1968, 32:1275-1282
24. Howson, S. J., Davis, R. P. Production of phytase hydrolyzing enzyme by some fungi. Enzyme Microb. Technol, 1983, 5:377-382
25．王红宁，吴琦，谢晶等．真菌植酸酶phyA朋基因研究进展．四川农业大学学报，2000，18(1)：84-88
26. Piddington, C.S., Houston, C.S., and Paloheimo, M., et al. The cloning and sequencing of the genes encoding phytase (phy) and pH2.5-optimum acid phosphatase (apb) from Aspergillus niger var. awamori, Gene, 1993,(133):55-62
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