HEREDITAS (Beijing) 2007 12, 29(12): 1533 1537 ISSN 0253-9772 www.chinagene.cn 技术与方法 DOI: 10.1360/yc-007-1533 涂知明, 陈泠, 杨广笑, 何光源,, 430074 : 采用碱性磷酸酶标记 DNA 制备分子探针, 并首次在植物中应用 酶在苯醌作用下与单链 DNA 联结, 形成 DNA 和酶的共价复合物即酶标探针 此探针通过分子杂交与待测 DNA 结合, 再与酶的底物作用显色, 3~6h 内可观察结果 用此探针检测转基因植物中的 UidA 基因, 点杂交和 Southern 杂交结果表明, 所合成的酶标探针具有快速 准确 安全而经济的优点 点杂交证明外源 UidA 基因被成功转化到受体植物中, Southern 杂交对转基因的材料检测的结果证明, 该材料包含多个外源 UidA 基因拷贝, 初步确定其外源 UidA 基因拷贝数在 5 个以上 : 组织特异性启动子 ;UidA 基因 ; 杂交 ; 酶标探针 Application of enzyme-labeled probe in testing of transgenic plant TU Zhi-Ming, CHEN Lin, YANG Guang-Xiao, HE Guang-Yuan Key Laboratory of Molecular Biophysics of the Ministry of Education, China-UK Joint Lab, College of life Science & Technology, Huazhong University of Science & Technology Wuhan, 430074, China Abstract: Used alkaline-phosphatase-labeled DNA as a probe to examine the expression of foreign UidA gene in transgenic plants. Alkaline phosphatase coupled with polyethyleneimine (PEI) using P-benzoquine as the cross-linking reagent was covalently linked to single-stranded DNA via glutraldehyde. Such DNA-enzyme complexes were used as a probe for dot hybridization and Southern blot. After hybridization and incubation with a substrate solution, results can be observed directly in three to six hours and the results showed that it was a sensitive, specific, rapid, safe and economical probe. Dot hybridization analysis showed that the UidA gene was transformed into the target plants and southern blot showed that there were at least 5 copies of UidA gene in transgenic plants. Keywords: tissue-specific promoter; UidA gene; hybridization; enzyme-labeled probe, UidA tritordeum, tritordeum [1], X-gluc UidA, UidA, UidA tritordeum, ( ), [2],, UidA 收稿日期 : 2007 04 26; 修回日期 : 2007 07 08 基金项目 : (973 )( 2002CB111302) ( 30370807) [Supported by National Basic Research Program of China (973 Program)(No.2002CB111302), National Natural Science Foundation of China(No.30370807) and PhD dissertation Foundation of Huazhong University of Science & Technology] 作者简介 : (1965 ),,,,, Tel: 027-62128955 通讯作者 : (1958 ),,,,, E-mail: hegy@mail.hust.edu.cn
1534 HEREDITAS (Beijing) 2007 29 Southern,, [3] 10 DNA [4],, DNA,, Renz [5,6], c RNA, Renz DNA, HBV DNA [7],, UidA, 1 1.1 PEG 8000 Sigma ;, (NBT) X- (BCIP) PEG 6000 phbv pahc25 1.2 : 0.5 mol/l NaOH; 1.5 mol/l NaCl : 1 mol/l Tris-HCl (ph 7.4); 1.5 mol/l NaCl (20 SSC): NaCl 175.3 g; 82.2 g, NaOH ph 7.0, ddh 2 O 1 000 ml 2 2.1 DNA phbv pahc25 Sambrook [9],, S.O.C, OD 600 0.4 0.5, DNA, TE DNA EcoR,, DNA DNA CTAB [9] 2.2 [6] 2.3 DNA Sambrook [9] DNA (50%, 2 Denhardt s, 4 SET, 0.1% SDS, 30 μg ml trna), 38 42 l h, 6% PEG 8000 PEG 6000 2 24 h, (50%, 0.4%SDS, 0.5 SSC) 38 42 2, 2 SSC 0.5 SSC 1, 0.1 mo1 L NaCl, 5 mmol L MgCl 2, 0.1 mo1 L Tris-HCl (ph9.5) 2 2.4 DNA 1.5 ml DNA(1 μg /μl)20 μg, 10 buffer 4.0 μl, (10 U/μL) 5.0 μl, ddh 2 O 500 μl, 3 h 2.5 DNA 0.8% 2.6 DNA DNA (NC ) DNA (1) : 30 min (2) : 15 min (3) : NC,, 5 min Whatman 3 mm, 3 5 8 24 h, 20 SSC (4) NC, 6 SSC 3 5 min,,, 80 C 2 h, NC, 2.7 Southern NC 2 SSC 5 min,
12 : 1535 (8 cm 8 cm 5 ml ),, 42, 42 (50%, 2 Denhardt s, 4 SET, 0.1% SDS, 30 μg ml trna), 42 l h, 6% PEG 8000 PEG 6000 42 (50%, 0.4% SDS, 0.5 SSC) 38 42 2, 2 SSC 0.5 SSC 1, 0.1 mo1 L NaCl, 5 mmol L MgCl 2, 0.1 mo1 L Tris-HCl (ph9.5) 2 [9] 2.8, 2, (NBT) X (BCIP) Renz [5] ; Boehringer Mannheim Dig DNA 37 30 min 2 d, TE 5 min,, TE, TE 42, 68, SDS, 68, 38 50 1 h,, 3,,, 1 3 6,, 6 3.2 HBV DNA,, 50 pg DNA 30 min, 1 h Renz [5], 6 PEG 6000 PEG 8000, 10 pg, 32 P 1 10 ng, 1 2.9 UidA DNA 5 μl, 150 μl 0.5 mol L NaOH, 20 min, 0.5 mol L NaOH, 1.5 mol L NaCl 10 min, 1 mol L Tris-HCl, ph 7.0, 3 mol l NaCl 15 min, 50 mmo1 L Tris-HCl, ph 8.0, 125 mmol L NaCl, 10 mmol l EDTA, 0.5% SDS(buffer B) 10 min, 2 ml 0.4 mg K buffer B, 50 30 min 2 h, 10% 4 SSC 10 min, 80 5 min, 20 min, 95% 2 min, 80 2 h 3 3.1, 38 42 50 68 4 图 1 酶标探针与 32 P 标记探针灵敏度的比较 32 P 1 h, 1 h 1~5 100 ng 10 ng 1 ng 0.1 ng 0.05 ng Fig. 1 A sensitivity comparison between enzyme-labeled probe and 32 P-labeled probe The above line demonstrated the autoradiograph result after exposure to X-film for 1 h using 32 P-labeled probe; the below line demonstrated the result of color development reaction for 1 h using enzyme-labeled probe; The amounts of HBV DNA samples from 1 to 5 were as follows: 100 ng 10 ng 1 ng 0.1 ng and 0.05 ng., HBV DNA HBV DNA, phbv, pahc25, DNA, trna, DNA, Tritordeum DNA HBV DNA phbv,
1536 HEREDITAS (Beijing) 2007 29 图 2 酶标探针的特异性检测 1: HBV DNA; 2: phbv ; 3: pahc25 ; 4: DNA; 5: trna; 6: DNA; 7: Tritordeum DNA Fig. 2 A specificity detection of enzyme-labeled HBV DNA probe 1: HBV DNA; 2: phbv plasmid; 3: pahc25 plasmid; 4: Cattle thymus DNA; 5: Yeast trna; 6: Human genomic DNA; 7: Tritordeum DNA. DNA Southern DNA DNA PCR Southern pahc25, L88-6, Southern UidA, UidA 5,,, 3.3, 3 UidA pahc25 B73-6-1, L88-6, UidA 图 3 点杂交对转基因植物材料中的 UidA 基因检测结果 1A: (pahc25); 1B: (L88-6); 1C: (B73-6-1); 7A 7B 7C ( ) 2A~6A: ; 2B~6B: ; 2C~6C: Fig.3 Dot hybridization analysis of the UidA gene in transgenic tritordeum using enzyme-labeled probe 1A: Positive control (Plasmid pahc25); 1B: Negative control (L88-6); 1C: Positive control (B73-6-1); 7A.7B.7C: Negative control (water), 2A 6A: Anther-primordea-specific promoter labeled transgenic tritordeum; 2B 6B: Short-cell-specific promoter labeled transgenic tritordeum; 2C 6C,: Pollen-grain-specific promoter labeled transgenic tritordeum. 3.4 Southern Southern DNA,, DNA 图 4 Southern 杂交对转基因材料中 UidA 基因拷贝数检测的结果 1: ( L88-6); 2: ; 3: (pahc25 ) Fig. 4 Southern blot analysis of the UidA gene in transgenic tritordeum 1: Negative control (L88-6); 2: Transgenic tritordeum; 3: Positive control (Plasmid pahc25)., [10] HBV DNA, UidA,,,,, (References): [1] HE Guang-Yuan, WU Xu-Qian, TU Zhi-Ming, CHEN
12 : 1537 Ming-Jie. Genetic analysis of the transgenic tritordeum. Journal of Huazhong University of Science and Technology(Nature Science), 2005, 33(8): 94 96.,,,. tritordeum. ( ), 2005, 33(8): 94 96. [2] CHEN Ln, HE Guang-Yuan, TU Zhi-Ming, CHEN Ming- Jie. Pollen specific expression analysis of the transgenic tritordeum. Journal of Huazhong University of Science and Technology(Nature Science), 2006, 34(4): 116 118.,,,. tritordeum. ( ), 2006, 34(4): 116 118. [3] LIU Zhong, YANG Gui-Zhen, WANG Wen-Yu. Exploring of hybridization condition of the biotin labeled DNA probe. Progress of Biochemistry and Biophysics, 1992, 19(6): 478 481.,,. DNA., 1992, 19(6): 478 481. [4] WU Yun-Tao, FANG Qing. Progress of the preparation techniques of nucleic acid probes. Foreign Medicine (Molecular Biology Edition), 1989, 11(3): 101 104.,.., 1989, 11(3): 101 104. [5] Renz M. Polynucleotide-histone H1 complexes as probes for blot hybridization, EMBO J, 1983, 2: 817 822. [6] Renz M, Kurz C. A colorimetric method for DNA hybridi- zation. Nucleic Acids Research, 1984, 12: 3435 3438.[DOI] [7] TU Zhi-Ming, KE Li-Hua. Study of the enzyme labeled HBV DNA probe. Virologica Sinica, 1991, 6(2): 132 137.,. HBV DNA., 1991, 6(2): 132 137. [8] CHEN Ming-Jie, LIU Yong, TU Zhi-Ming, HE Guang-Yuan. Rapid confirmation of the transgenic wheat lines by multiplex polymerase chain reaction (MPCR). Journal of Huazhong University of Science and Technology(Nature Science), 2004, 32(9): 105 107.,,,. PCR. ( ), 2004, 32(9): 105 107. [9] Sambrook J, Fritsch EF, Maniatis T. Molecular Clone: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, 1995. J., E.F., T. (,, ). ( ). :, 2002. [10] ZM Tu, LH Ke, GY He. The application of alkaline phosphatase labeled HBV probe in serum detection. Virus Genes, 2004, 28(2): 153 158. ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ 中国遗传学会植物遗传和基因组学专业委员会 2007 年学术研讨会 成功召开 2007 10 12~14 170 Hong Ma 12 30