萝卜抗真菌蛋白Rs-AFP_2基因PCR定点突变及在E.coli中的表达
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摘要
萝卜抗真菌蛋白Rs-AFP_2是从萝卜（Raphanus sativus）种子中分离到的一
种6KD富含半胱氨酸的抗真菌蛋白（RaPhnus sativus-antifingal protein）。其
cDNA全长有243bp，共编码80个氨基酸。它的N端的29个氨基酸为信号肽，
余下的51个氨基酸为成熟肽。
本研究根据Rs-AFP_2基因的序列，结合基因及其表达调控中遗传密码的偏
爱性，人工合成了7条引物。利用PCR重叠延伸技术，把萝卜抗真菌蛋白
Rs-AFP_2基因的编码序列第9号氨基酸Arg密码AGG改为细菌偏爱密码CGA，
同时对编码序列起始区的部分核苷酸进行突变，又采用嵌套PCR，迅速地克隆
到目的基因。将之插入pGEM-5Zf/EcoRV-T easy克隆载体，得到重组质粒
pGEM-T_m。用其转化宿主菌XL1，筛选阳性菌落。通过重组质粒的PCR鉴定、
双酶切及全序列测定，其结果表明已完成了对该基因的改造。
为了除去cDNA上29个氨基酸信号肽编码区，重新合成了2条上游PCR
引物，5'端具有NdeⅠ识别位点，并分别与原Rs-AFP_2基因和突变后Rs-AFPm
基因的序列互补，与上游引物匹配的是原PCR反应的下游引物（5'端具XhoⅠ
位点）。以含Rs-AFP_2和Rs-AFPm基因的克隆载体为模板，扩增Rs-AFP_2和
Rs-AFPm成熟肽的编码序列。PCR产物经NdeⅠ和XhoⅠ双酶切后，插入
pET-2lb，构建成不带信号肽的非融合表达载体pETAFPo和pETAFPm。
将表达载体分别导入E.coli BL21，经IPTG诱导，Rs-AFP_2和Rs-AFPm基
因均得到表达。表达产物经低分子量蛋白质电泳系统检测，在6KD左右位置上
出现了一条空白载体受体菌缺乏的表达蛋白带。该表达蛋白以包含体的形式存
在于细胞中。
体外抑菌实验表明，Rs-AFPm表达产物的抑菌活性明显高于Rs-AFP_2表达
产物的抑菌活性。这说明突变后的基因有效地提高了表达效率。
Abstract
Raphnus sativus -antifun gal protein2 (Rs-AFP,) was isolated from the seeds of radish. It was a small , cysteine-rich, defense-related antifungal protein. The encoding sequence was 243 bp. It encoded 80 amino acids which including signal peptide of 29 amino acids and mature peptide of 51 amino acids.
Seven primers had been synthesized according to the Rs-AFP2 gene sequences and preference of the genetic code in the gene expressions and regulations. The splicing by overlap extension was applied to change the bare codon AGG into favored codon CGA, which encoded the ninth amino acid in the gene encoding Rs-AFP2. Some other bases were also mutated in the beginnig of the encoding parts. The mutated gene was quickly cloned by nested PCR. PCR product of Rs-AFP2 gene was retrieved by low melting agaroses(1 .5% wt/vol) gel electrophoresis, then was inserted into pGEM-5Zf/EcoRV-T easy vector. The recombination plasmid was named pOEM-Tm and was identified by digestions with EcoR I, PCR and sequence analysis, thereby proving that the gene alteration had been completed successfully.
In order to remove the signal peptide sequences of Rs-AFP2 eDNA, two different upstream primers, both of which had a 5'-terminal Nde I site, were designed and synthesized. Each could match with sequences either Rs-AFP2 or Rs-AFPm. The downstream primmer had a 5'-terminal Xho I site. The coding sequences of the mature peptides were amplified by using the respective recombinant cloning vectors as templates. Then the digested products were inserted into pET2lb vectors at the site of Nde I and Xho I to construct expression vectors pETAFP,~ffl pETAFPm, which had not the signal peptide at all.
The two expression vectors, which were constructed by the way of the mature
peptides, were expressed in E. coli BL2 1 strain, and the products were identified by SDS-Ticine-PAGE. The inhabition experimelfls showed that the inhibition activity of pETAFPm expression product was much stronger than that of pETAFP2 one. Therefore the mutated gene had improved the expression efficiency validly as a result.
种6KD富含半胱氨酸的抗真菌蛋白（RaPhnus sativus-antifingal protein）。其
cDNA全长有243bp，共编码80个氨基酸。它的N端的29个氨基酸为信号肽，
余下的51个氨基酸为成熟肽。
本研究根据Rs-AFP_2基因的序列，结合基因及其表达调控中遗传密码的偏
爱性，人工合成了7条引物。利用PCR重叠延伸技术，把萝卜抗真菌蛋白
Rs-AFP_2基因的编码序列第9号氨基酸Arg密码AGG改为细菌偏爱密码CGA，
同时对编码序列起始区的部分核苷酸进行突变，又采用嵌套PCR，迅速地克隆
到目的基因。将之插入pGEM-5Zf/EcoRV-T easy克隆载体，得到重组质粒
pGEM-T_m。用其转化宿主菌XL1，筛选阳性菌落。通过重组质粒的PCR鉴定、
双酶切及全序列测定，其结果表明已完成了对该基因的改造。
为了除去cDNA上29个氨基酸信号肽编码区，重新合成了2条上游PCR
引物，5'端具有NdeⅠ识别位点，并分别与原Rs-AFP_2基因和突变后Rs-AFPm
基因的序列互补，与上游引物匹配的是原PCR反应的下游引物（5'端具XhoⅠ
位点）。以含Rs-AFP_2和Rs-AFPm基因的克隆载体为模板，扩增Rs-AFP_2和
Rs-AFPm成熟肽的编码序列。PCR产物经NdeⅠ和XhoⅠ双酶切后，插入
pET-2lb，构建成不带信号肽的非融合表达载体pETAFPo和pETAFPm。
将表达载体分别导入E.coli BL21，经IPTG诱导，Rs-AFP_2和Rs-AFPm基
因均得到表达。表达产物经低分子量蛋白质电泳系统检测，在6KD左右位置上
出现了一条空白载体受体菌缺乏的表达蛋白带。该表达蛋白以包含体的形式存
在于细胞中。
体外抑菌实验表明，Rs-AFPm表达产物的抑菌活性明显高于Rs-AFP_2表达
产物的抑菌活性。这说明突变后的基因有效地提高了表达效率。
Abstract
Raphnus sativus -antifun gal protein2 (Rs-AFP,) was isolated from the seeds of radish. It was a small , cysteine-rich, defense-related antifungal protein. The encoding sequence was 243 bp. It encoded 80 amino acids which including signal peptide of 29 amino acids and mature peptide of 51 amino acids.
Seven primers had been synthesized according to the Rs-AFP2 gene sequences and preference of the genetic code in the gene expressions and regulations. The splicing by overlap extension was applied to change the bare codon AGG into favored codon CGA, which encoded the ninth amino acid in the gene encoding Rs-AFP2. Some other bases were also mutated in the beginnig of the encoding parts. The mutated gene was quickly cloned by nested PCR. PCR product of Rs-AFP2 gene was retrieved by low melting agaroses(1 .5% wt/vol) gel electrophoresis, then was inserted into pGEM-5Zf/EcoRV-T easy vector. The recombination plasmid was named pOEM-Tm and was identified by digestions with EcoR I, PCR and sequence analysis, thereby proving that the gene alteration had been completed successfully.
In order to remove the signal peptide sequences of Rs-AFP2 eDNA, two different upstream primers, both of which had a 5'-terminal Nde I site, were designed and synthesized. Each could match with sequences either Rs-AFP2 or Rs-AFPm. The downstream primmer had a 5'-terminal Xho I site. The coding sequences of the mature peptides were amplified by using the respective recombinant cloning vectors as templates. Then the digested products were inserted into pET2lb vectors at the site of Nde I and Xho I to construct expression vectors pETAFP,~ffl pETAFPm, which had not the signal peptide at all.
The two expression vectors, which were constructed by the way of the mature
peptides, were expressed in E. coli BL2 1 strain, and the products were identified by SDS-Ticine-PAGE. The inhabition experimelfls showed that the inhibition activity of pETAFPm expression product was much stronger than that of pETAFP2 one. Therefore the mutated gene had improved the expression efficiency validly as a result.
引文
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[2] Terras FRG, Torrekens S, Van Leuven F, et al. A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae-species.FEBS.lett. 1993,316:233-240
[3] Terras FRG, Goderis IJ, Van Leuven F, et al. In vitro antifungal activity of a radish (Raphanus sativus) seed protein homologous to nonspecific lipid transfer proteins. Plant Physiol. 1992a, 100:1055-1058
[4] Terras FRG, Schoofs HME, De Bolle,et al.Analysis of two novel classes of antifungal proteins from radish (Raphanus sativus) seed. J Biol Chem. 1992b,267:15301-15309
[5] Parijs J V, Broekaeit W F, Goldstein I J, et al. Hevein: an antifungal protein from rubber tree.Planta,1991,183:258-264
[6] Broekaett WF. Antimicrobial peptides from Amaranthus caudatus seeds with sequnce homology to the cysteine/glycine-rich domain of chitinbinding proteins.Biochemistry. 1992,31:4308-4316
[7] Shishi H. Structure of a tabacco endochitinase gene: evidence that differenct chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain. Plant Mol Bio1.1990, 14:357-368
[8] 姚泉洪。植物抗真菌病毒基因工程的研究进展。植物生理学通讯。1995，31(4)：303-307
[9] 但汉鸿。植物几丁质酶与病害防治的研究进展。生物技术通报。1995，2：1-3
[10] Brogile K. Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science, 1991,254:1194-1197
[11] Vierheilig H. Colonization of transgenic Nicotiana sylvestris plants, expressing different forms of Nicotiana tabacum chitinase, by the root pathogen Rhazoctonia solani and by the mycorrhizal symbiont Glomus mosseae. Mol Plant-Microbe Interact. 1993, 6:261-270
[12] Kless H.Cloning of the gene coding for chitobiase of serratia marcescens. Mol Gen Genet. 1989, 217:471-476
[13] Howie W. Transgenic tobacco plants which express the ChiA gene from Serratia marcescens have enhanced tolerance to Rhizoctonia. Tansgenetic Research. 1994,3:90-96
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