美洲商陆抗病毒蛋白cDNA的克隆及其转化番茄
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摘要
美洲商陆抗病毒蛋白(Pokeweed Antiviral Protein，PAP)是从美洲商陆(Phytolacca americana)叶片或种子分离出的一类碱性蛋白，分子量29-31KD。PAP是典型的单链核糖体失活蛋白，它可以催化水解原核和真核核糖体的大rRNA3’-端茎环结构(stem-loop)中的腺嘌呤残基，导致核糖体失活。PAP可以有效抑制若干种非相关病毒，包括植物病毒和动物病毒，利用PAP基因可以通过转单基因获得对多种病毒具有广谱抗性的转基因植株。成熟的PAP有262个氨基酸，其中靠C端的25个氨基酸为毒性区，PAP的C端缺失突变体PAP-c没有毒性区域，它的细胞毒性很低，但仍具有较强的抗病毒活性。
本研究采用PCR技术获得PAP和PAP-c的cDNA，同时利用番茄叶片特异表达启动子rbcS-3A，构建不同的植物表达载体，通过农杆菌介导的遗传转化体系将PAP和PAP-c基因转入番茄自交系TP002，获得转基因植株，是植物抗病毒基因工程研究领域一次新的探索；获得的转基因番茄植株对TMV和CMV病毒具有抗性，可以作为番茄育种的一个新的抗源材料。
主要研究结果如下：
1．采用PCR技术，分离并克隆到美洲商陆抗病毒蛋白cDNA(PAP)和C-端缺失突变体cDNA(PAP-c)。克隆到的PAP编码区为940bp，与Genebank中的序列同源率为99.7％；克隆到的PAP-c编码区为775bp，与相应的序列同源率为99.6％。
2．采用PCR技术，从番茄基因组中分离并克隆到rbcS-3A启动子。测序结果表明，克隆到的基因为1150bp(包括酶切位点)，与Genebank中的核苷酸序列同源率为99％，具有完整的TATA box和CAAT box，在增强子、沉默子和启动子功能区域与Genebank中的相应序列同源率为100％。
3．利用克隆到的基因和植物表达载体pBI121，成功构建了3个美洲商陆抗病毒蛋白的植物表达载体：pBPAP，pBPAP-c，pB-rbc-PAP-c。其中，pB-rbc-PAP-c为植物特异表达载体，启动子为番茄rbcS-3A启动子，在叶片中受光诱导表达，具有组织特异性。
4．通过农杆菌介导的遗传转化体系分别将2个植物表达载体pBPAP和pB-rbc-PAP-c导入番茄，经PCR检测和Southern检测，得到3株转基因番茄植株。RT-PCR检测结果表明，在CaMV 35S启动子的作用下，PAP基因在转基因番茄(9#植株)叶片和果实中均可以表达；在rbcS-3A启动子的作用下，PAP-c基因只在转基因番茄(6#植株)叶片中表达而不在果实中表达。人工接种病毒结果显示，与对照植株相比，5#和9#转基因植株对TMV具有抗性，6#转基因植株对TMV和CMV具有抗性。
Pokeweed antiviral protein(PAP), isolated from leaves and seeds of Phytolacca americana, are single-chain ribosome-inactive proteins(RIPs) with molecular mass of 29 to 31KD, which can inhibit translation by catalytically removing a specific adenine residue from a highly conserved, surface-exposed, stem-loop structure in the large rRNA of eukaryotic and prokaryotic ribosomes. PAP displays broad-spectrum antiviral activity against a number of different viruses, including plant and animal viruses. The property of PAP provides a way to produce multiple-virus-resistant transgenic plants by introducing and expressing a single gene. The C-terminal deletion mutant, PAP-c, which loses 25 amino acids near C-terminus comparing to the mature PAP of 262 amino acids, has lower cytotoxicity and shows resistance to viruses. These 25 amino acids near C-terminus is
necessary for toxicity but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity. Expression of PAP-c in transgenic plants will be safer than that of wild-type of PAP.
According to the published sequence of PAP, the cDNA encoding PAP and PAP-c were obtained by using PCR, respectively. The leaf specific expression promoter rbcS-3A, which confers light-inducible and organ-specific expression, was also amplified from tomato DMA by PCR. In order to transfer PAP or PAP-c gene into tomato, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 358 promoter and rbcS-3A promoter respectively. Agrobacterium tumefaciens containing plant transformation vectors was used to transform tomato self-line TP002 by the leaf disc method.
The results of this study are as follows:
1 The cDNA encoding PAP and C-terminal deletion mutant PAP-c were obtained by using PCR. The PAP cDNA has an open reading frame of 940bp, which is 99.7% homologous to the published sequence. The PAP-c cDNA has an open reading frame of 775bp, which is 99.6% homologous to the corresponding sequence of the published PAP cDNA.
2 The rbcS-3A promoter sequence was amplified from tomato genome DNA by using PCR. Sequencing results showed, the 1150bp fragment obtained in this study has 99% homology to the rbcS-3A promoter sequence published in GeneBank and contains intact TATA box and
CAAT box. Its enhancer region, silencer region and promoter function region are 100% homologous to those of published rbcS-3A.
3 Based on pBI121, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 35S promoter and rbcS-3A promoter respectively. The pBPAP has PAP gene expressed from CaMV 35S promoter and pBPAP-c has PAP-c gene with the same promoter. The pB-rbc-PAP-c has PAP-c gene modulated by rbcS-3A promoter from tomato.
4 By Agrobacterium tumefaciens mediated transformation, PAP and PAP-c gene were induced into tomato self-line(TP002) through two different plant expression vectors, pBPAP and pB-rbc-PAP-c, respectively. Three regenerated tomato plants were proved to be transgenic plants by PCR and Southern blotting. The results of RT-PCR for transgenic
plants showed, PAP gene was expressed in both leaves and fruits of No.9 transgenic plant under CaMV 35S promoter, PAP-c gene was only expressed in the leaves not in the fruits of No.6 transgenic plant under rbcS-3A promoter. To test the resistance to virus of transgenic tomato plants, they were mechanically inoculated with TMV and CMV respectively. The results indicated , comparing to the non-transformed control plants, No.5 and No.9 plants displayed resistance to TMV, No.6 plant displayed resistance to TMV and CMV.
本研究采用PCR技术获得PAP和PAP-c的cDNA，同时利用番茄叶片特异表达启动子rbcS-3A，构建不同的植物表达载体，通过农杆菌介导的遗传转化体系将PAP和PAP-c基因转入番茄自交系TP002，获得转基因植株，是植物抗病毒基因工程研究领域一次新的探索；获得的转基因番茄植株对TMV和CMV病毒具有抗性，可以作为番茄育种的一个新的抗源材料。
主要研究结果如下：
1．采用PCR技术，分离并克隆到美洲商陆抗病毒蛋白cDNA(PAP)和C-端缺失突变体cDNA(PAP-c)。克隆到的PAP编码区为940bp，与Genebank中的序列同源率为99.7％；克隆到的PAP-c编码区为775bp，与相应的序列同源率为99.6％。
2．采用PCR技术，从番茄基因组中分离并克隆到rbcS-3A启动子。测序结果表明，克隆到的基因为1150bp(包括酶切位点)，与Genebank中的核苷酸序列同源率为99％，具有完整的TATA box和CAAT box，在增强子、沉默子和启动子功能区域与Genebank中的相应序列同源率为100％。
3．利用克隆到的基因和植物表达载体pBI121，成功构建了3个美洲商陆抗病毒蛋白的植物表达载体：pBPAP，pBPAP-c，pB-rbc-PAP-c。其中，pB-rbc-PAP-c为植物特异表达载体，启动子为番茄rbcS-3A启动子，在叶片中受光诱导表达，具有组织特异性。
4．通过农杆菌介导的遗传转化体系分别将2个植物表达载体pBPAP和pB-rbc-PAP-c导入番茄，经PCR检测和Southern检测，得到3株转基因番茄植株。RT-PCR检测结果表明，在CaMV 35S启动子的作用下，PAP基因在转基因番茄(9#植株)叶片和果实中均可以表达；在rbcS-3A启动子的作用下，PAP-c基因只在转基因番茄(6#植株)叶片中表达而不在果实中表达。人工接种病毒结果显示，与对照植株相比，5#和9#转基因植株对TMV具有抗性，6#转基因植株对TMV和CMV具有抗性。
Pokeweed antiviral protein(PAP), isolated from leaves and seeds of Phytolacca americana, are single-chain ribosome-inactive proteins(RIPs) with molecular mass of 29 to 31KD, which can inhibit translation by catalytically removing a specific adenine residue from a highly conserved, surface-exposed, stem-loop structure in the large rRNA of eukaryotic and prokaryotic ribosomes. PAP displays broad-spectrum antiviral activity against a number of different viruses, including plant and animal viruses. The property of PAP provides a way to produce multiple-virus-resistant transgenic plants by introducing and expressing a single gene. The C-terminal deletion mutant, PAP-c, which loses 25 amino acids near C-terminus comparing to the mature PAP of 262 amino acids, has lower cytotoxicity and shows resistance to viruses. These 25 amino acids near C-terminus is
necessary for toxicity but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity. Expression of PAP-c in transgenic plants will be safer than that of wild-type of PAP.
According to the published sequence of PAP, the cDNA encoding PAP and PAP-c were obtained by using PCR, respectively. The leaf specific expression promoter rbcS-3A, which confers light-inducible and organ-specific expression, was also amplified from tomato DMA by PCR. In order to transfer PAP or PAP-c gene into tomato, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 358 promoter and rbcS-3A promoter respectively. Agrobacterium tumefaciens containing plant transformation vectors was used to transform tomato self-line TP002 by the leaf disc method.
The results of this study are as follows:
1 The cDNA encoding PAP and C-terminal deletion mutant PAP-c were obtained by using PCR. The PAP cDNA has an open reading frame of 940bp, which is 99.7% homologous to the published sequence. The PAP-c cDNA has an open reading frame of 775bp, which is 99.6% homologous to the corresponding sequence of the published PAP cDNA.
2 The rbcS-3A promoter sequence was amplified from tomato genome DNA by using PCR. Sequencing results showed, the 1150bp fragment obtained in this study has 99% homology to the rbcS-3A promoter sequence published in GeneBank and contains intact TATA box and
CAAT box. Its enhancer region, silencer region and promoter function region are 100% homologous to those of published rbcS-3A.
3 Based on pBI121, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 35S promoter and rbcS-3A promoter respectively. The pBPAP has PAP gene expressed from CaMV 35S promoter and pBPAP-c has PAP-c gene with the same promoter. The pB-rbc-PAP-c has PAP-c gene modulated by rbcS-3A promoter from tomato.
4 By Agrobacterium tumefaciens mediated transformation, PAP and PAP-c gene were induced into tomato self-line(TP002) through two different plant expression vectors, pBPAP and pB-rbc-PAP-c, respectively. Three regenerated tomato plants were proved to be transgenic plants by PCR and Southern blotting. The results of RT-PCR for transgenic
plants showed, PAP gene was expressed in both leaves and fruits of No.9 transgenic plant under CaMV 35S promoter, PAP-c gene was only expressed in the leaves not in the fruits of No.6 transgenic plant under rbcS-3A promoter. To test the resistance to virus of transgenic tomato plants, they were mechanically inoculated with TMV and CMV respectively. The results indicated , comparing to the non-transformed control plants, No.5 and No.9 plants displayed resistance to TMV, No.6 plant displayed resistance to TMV and CMV.
引文
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80．李继红，1997，几丁质酶和β-1,3-葡聚糖酶基因的克隆及转化番茄的研究，华南热带农业大学博士研究生学位论文
81．李一琨，王金发，1998，高等植物启动子研究进展，植物学通报，15(增刊)：1-6
82．刘后利，1993，作物育种研究进展(第一集)，北京：农业出版社
83．刘迎芳，王春霞，赵进东等，1999，美洲商陆中新发现的一种抗真菌蛋白基因的克隆和表达，植物学报，41(10)：1036-1040
84．刘志毅等，1995，基因表达的转录调控的研究进展，生命科学，7(2)：22-26
85．鲁德全，1995，中国商陆属植物的校订，武汉植物学研究，13(1)：27-29
86．P．B．沃斯，S．G．布利克斯特主编，刘秉华等译，1993，植物育种的现代基础，北京：农业出版社
87．单丽波，贾旭，2000，核糖体失活蛋白及其在植物抗真菌病基因工程中的应用，生物工程进展，20(6)：74-78
88．施曼玲，薛宝娣，1999，转CMV-CP基因番茄植株的抗病性表现，浙江农业科学，6：276-279
89，杨荣昌，1995，表达黄瓜花叶病毒外壳蛋白的转基因番茄及其对CMV的抗性，江苏农业学报，11(1)：40-44
90．王得元，何晓明，王鸣，2002，蔬菜生物技术概论，北京：中国农业出版社
91．王关林，方宏筠，1998，植物基因工程原理与技术，北京：科学出版社
92．王海廷，王鸣，李长年，1988，番茄育种，上海：上海科学技术出版社
93．王金生，1995，植物抗病性分子机制，植物病理学报，25(4)：289-295
94．王军，1994，植物诱导抗病性的研究进展·，华南农业大学学报，15(4)：121-126
95．王莉江，安成才，陈章良，2000，核糖体灭活蛋白在植物中的应用，生物技术通报，2：1-4
96．奚正德，1995，核糖体失活蛋白及相关毒素的生物学活性研究进展，生命科学，7(3)：18-21
97．夏兰芹等，2000，外源基因在转基因植物中的表达与稳定性，生物技术通报，3：8-12
98．余诞年，吴定华，陈竹君，1999，番茄遗传学，长沙：湖南科学技术出版社
99．俞德超 1993，抗病毒基因工程的研究进展及评估 植物生物学通讯 29(2)：141-147
100．张德水，陈受宜，1997，植物抗病性的分子生物学研究进展，植物病理学报，27(2)：97-103
101．张海燕，刘桂珍，陈正华，1999，商陆抗病毒蛋白cDNA的克隆、测序及其植物表达载体的构建，植物学报41(2)：226-228
102．张增艳，孔凡晶，辛志勇，2000，植物抗病基因克隆的进展，生物技术通报，2：8-11
103．曾士迈，张树榛，1998，植物抗病育种的流行学研究，北京：科学出版社
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65．Borojevise S．编著，郑彦平 主译，1992，作物育种原理与方法，北京：农村读物出版社
66．程玉忠，1991，植物基因工程进展，遗传，13(4)：45-48
67．董汉松，1995，植物诱导抗病性，北京：科学出版社
68．董金皋，黄梧芳，1994，植物的形态结构与抗病性，植物病理学报，25(1)：1-3
69．樊金献，薛洁，董建寅，1995，药用有毒植物商陆与美洲商陆，植物杂志，5：21
70．樊龙江，周雪平，2001，转基因作物安全性争论与事实，北京：中国农业出版社
71．方中达编著，1998，植病研究方法(第三版)，北京：中国农业出版社
72．傅荣昭，孙勇如，贾士荣，1994，植物遗传转化技术手册，北京：中国科学技术出版社
73．顾红雅等，1995，植物基因与分子操作，北京：北京大学出版社
74．黄绍兴，阎隆飞，1995，植物发育过程中特异性基因的表达，31(3)：161-166
75，贾士荣，曹冬孙，1992，转基因植物，植物学通报，9(2)：3-15
76．贾士荣，邓燕华，1993，农业生物技术进展与展望，合肥：中国科学技术大学出版社
77．蒋国勇，1994，天花粉蛋白转化番茄的研究，植物学报，41(3)：334-336
78．J.萨姆布鲁克，E.F.弗里奇等，1996，分子克隆实验指南(金冬雁等译)，北京：科学出版社
79．康俊根，1997，植物抗病毒基因工程研究进展，生物技术，7(2)：4-7
80．李继红，1997，几丁质酶和β-1,3-葡聚糖酶基因的克隆及转化番茄的研究，华南热带农业大学博士研究生学位论文
81．李一琨，王金发，1998，高等植物启动子研究进展，植物学通报，15(增刊)：1-6
82．刘后利，1993，作物育种研究进展(第一集)，北京：农业出版社
83．刘迎芳，王春霞，赵进东等，1999，美洲商陆中新发现的一种抗真菌蛋白基因的克隆和表达，植物学报，41(10)：1036-1040
84．刘志毅等，1995，基因表达的转录调控的研究进展，生命科学，7(2)：22-26
85．鲁德全，1995，中国商陆属植物的校订，武汉植物学研究，13(1)：27-29
86．P．B．沃斯，S．G．布利克斯特主编，刘秉华等译，1993，植物育种的现代基础，北京：农业出版社
87．单丽波，贾旭，2000，核糖体失活蛋白及其在植物抗真菌病基因工程中的应用，生物工程进展，20(6)：74-78
88．施曼玲，薛宝娣，1999，转CMV-CP基因番茄植株的抗病性表现，浙江农业科学，6：276-279
89，杨荣昌，1995，表达黄瓜花叶病毒外壳蛋白的转基因番茄及其对CMV的抗性，江苏农业学报，11(1)：40-44
90．王得元，何晓明，王鸣，2002，蔬菜生物技术概论，北京：中国农业出版社
91．王关林，方宏筠，1998，植物基因工程原理与技术，北京：科学出版社
92．王海廷，王鸣，李长年，1988，番茄育种，上海：上海科学技术出版社
93．王金生，1995，植物抗病性分子机制，植物病理学报，25(4)：289-295
94．王军，1994，植物诱导抗病性的研究进展·，华南农业大学学报，15(4)：121-126
95．王莉江，安成才，陈章良，2000，核糖体灭活蛋白在植物中的应用，生物技术通报，2：1-4
96．奚正德，1995，核糖体失活蛋白及相关毒素的生物学活性研究进展，生命科学，7(3)：18-21
97．夏兰芹等，2000，外源基因在转基因植物中的表达与稳定性，生物技术通报，3：8-12
98．余诞年，吴定华，陈竹君，1999，番茄遗传学，长沙：湖南科学技术出版社
99．俞德超 1993，抗病毒基因工程的研究进展及评估 植物生物学通讯 29(2)：141-147
100．张德水，陈受宜，1997，植物抗病性的分子生物学研究进展，植物病理学报，27(2)：97-103
101．张海燕，刘桂珍，陈正华，1999，商陆抗病毒蛋白cDNA的克隆、测序及其植物表达载体的构建，植物学报41(2)：226-228
102．张增艳，孔凡晶，辛志勇，2000，植物抗病基因克隆的进展，生物技术通报，2：8-11
103．曾士迈，张树榛，1998，植物抗病育种的流行学研究，北京：科学出版社