根癌农杆菌介导的甜蛋白thaumatin基因导入烟草的遗传转化研究
|
摘要
甜蛋白Thaumatin是一种高甜度、低热量、安全无毒、甜味纯正的非糖类新型甜味剂，具有很高的经济价值。然而该蛋白的来源植物只产于西非Thaumatococcus danielli的果实中，该植物对生活环境要求苛刻，在世界各地的引种尚不成功，不能满足人们对甜蛋白Thaumatin的大量需求。研究人员试图用微生物表达甜蛋白，结果不如人意。因此，本试验构建了植物表达载体pBI_(121)-th，并将其转入根癌农杆菌LBA_(4404)中，然后通过农杆菌的介导将thaumatin基因导入烟草中，并对转基因植株进行了检测，借以初步探讨甜蛋白在植物中的转化和表达情况，为其今后在更多的植物上应用提供依据。实验结果如下：
1．甜蛋白thaumatin基因植物表达质粒pBI_(121)-th的构建与鉴定
利用DNA重组技术，将植物甜蛋白thaumatin基因克隆至植物表达载体pBI_(121)中，通过酶切、电泳，鉴定thaumatin基因已成功构建到植物表达质粒pBI_(121)中。
2．pBI_(121)-th质粒转化根癌农杆菌
重组质粒pBI_(121)-th通过直接转化法导入农杆菌LBA_(4404)中，卡那霉素(Km)平板筛选阳性克隆，有单菌落生长，证明重组质粒已转进农杆菌LBA_(4404)中。
3．植株再生体系的建立
采用无菌苗烟草叶片为外植体，以MS为基本培养基，筛选出MS+6-BA2.0mg／L+NAA0.3mg／L以诱导不定芽的分化，并于MS+NAA0.3mg／L的
PA 硕士学位论文
V沉了砂 “工队nk”
主根培养基上生根，获得完整再生植株。其叶片分化率达65石％，生根率达
95％。
4．转化体筛选及植株再生
将预培养2天的外植体与农杆菌菌液浸染5～6分钟后，共培养2～3天，
然后转化到含Km75m旮 的选择培养基上进行分化筛选，再转入K。为
75mg几、100mg／L的培养基上进行生根筛选，生根率为82．5％。转入外源基
因的转化体将会在这一系列筛选过程中发芽、生根，获得转基因植株。
5．转基因PCR检测
PCR扩增 nPt 11基因，证明 thaumatin基因己导入烟草中，转化率为
引．3％c
Sweet protein Thaumatin has great economic value because thaumatin is a no caloric sweetener that has many advantages such as high sweet intensity, low calories, no toxicity and so on. However, this protein can't meet people's dem--ands because of lacking of resources. Therefore, in this paper, the recombinant plant expression vector pBIi2i-th was constructed successfully and introduced into Agrobaterium tumefaciens LBA4404. Subsequently thaumatin gene was transformed to tobacco through Agrobaterium-mediated system. And transgenic plants were confirmed by a series of examinations. The results were as following:
1. Construction and identifcation of recombinant plant expression vector pBI!2i-th
By DNA recombination technology, sweet protein thaumatin gene was cloned into plant expression vector pBIi2i. Recombinant plasmid pBIi2i-th was constructed successfully by enzyme cutting and electrophoresis.
2. Introduction of pBI121-th plasmid into Agrobacterium tumefaciens . The pBI121-th was introduced into Agrobacterium LBAncn by direct transformation .By screening in the medium containing kanamycin , it was confirmed that the result was successful .
3. Regeneration system of plants.
The regeneration system of tobacco was established. The adventitious shoots were induced from leaf explants of tobacco based on MS basal medium supple--mented with 2.0mg/L 6-BA and 0.3mg/L NAA . Then the regenerated plants were rooted on MS medium containing 0.3mg/L NAA. And the frequency was about 65.6%, 95% respectively.
4. Transformation and selection of plants.
Leaf explants had been pre-cultured for 2 days, then immersed in Agroba--cterium suspension for 5~6minutes. The co-cultivation had been carried out for 2~3 days. After that, the transformants were obtained by transferring explants to selection medium containing 75mg/L kanamycin and rooting medium contain--ing 75mg/L, 100mg/L kanamycin .And its rooted frequency was 82.5%.
5. Detection of transgenic plants.
The PCR assay of kan-resistant plants showed that the target gene had been integrated into tobacco accompanying with npt II gene. The frequency of transf--ormation was 31.3%.
1．甜蛋白thaumatin基因植物表达质粒pBI_(121)-th的构建与鉴定
利用DNA重组技术，将植物甜蛋白thaumatin基因克隆至植物表达载体pBI_(121)中，通过酶切、电泳，鉴定thaumatin基因已成功构建到植物表达质粒pBI_(121)中。
2．pBI_(121)-th质粒转化根癌农杆菌
重组质粒pBI_(121)-th通过直接转化法导入农杆菌LBA_(4404)中，卡那霉素(Km)平板筛选阳性克隆，有单菌落生长，证明重组质粒已转进农杆菌LBA_(4404)中。
3．植株再生体系的建立
采用无菌苗烟草叶片为外植体，以MS为基本培养基，筛选出MS+6-BA2.0mg／L+NAA0.3mg／L以诱导不定芽的分化，并于MS+NAA0.3mg／L的
PA 硕士学位论文
V沉了砂 “工队nk”
主根培养基上生根，获得完整再生植株。其叶片分化率达65石％，生根率达
95％。
4．转化体筛选及植株再生
将预培养2天的外植体与农杆菌菌液浸染5～6分钟后，共培养2～3天，
然后转化到含Km75m旮 的选择培养基上进行分化筛选，再转入K。为
75mg几、100mg／L的培养基上进行生根筛选，生根率为82．5％。转入外源基
因的转化体将会在这一系列筛选过程中发芽、生根，获得转基因植株。
5．转基因PCR检测
PCR扩增 nPt 11基因，证明 thaumatin基因己导入烟草中，转化率为
引．3％c
Sweet protein Thaumatin has great economic value because thaumatin is a no caloric sweetener that has many advantages such as high sweet intensity, low calories, no toxicity and so on. However, this protein can't meet people's dem--ands because of lacking of resources. Therefore, in this paper, the recombinant plant expression vector pBIi2i-th was constructed successfully and introduced into Agrobaterium tumefaciens LBA4404. Subsequently thaumatin gene was transformed to tobacco through Agrobaterium-mediated system. And transgenic plants were confirmed by a series of examinations. The results were as following:
1. Construction and identifcation of recombinant plant expression vector pBI!2i-th
By DNA recombination technology, sweet protein thaumatin gene was cloned into plant expression vector pBIi2i. Recombinant plasmid pBIi2i-th was constructed successfully by enzyme cutting and electrophoresis.
2. Introduction of pBI121-th plasmid into Agrobacterium tumefaciens . The pBI121-th was introduced into Agrobacterium LBAncn by direct transformation .By screening in the medium containing kanamycin , it was confirmed that the result was successful .
3. Regeneration system of plants.
The regeneration system of tobacco was established. The adventitious shoots were induced from leaf explants of tobacco based on MS basal medium supple--mented with 2.0mg/L 6-BA and 0.3mg/L NAA . Then the regenerated plants were rooted on MS medium containing 0.3mg/L NAA. And the frequency was about 65.6%, 95% respectively.
4. Transformation and selection of plants.
Leaf explants had been pre-cultured for 2 days, then immersed in Agroba--cterium suspension for 5~6minutes. The co-cultivation had been carried out for 2~3 days. After that, the transformants were obtained by transferring explants to selection medium containing 75mg/L kanamycin and rooting medium contain--ing 75mg/L, 100mg/L kanamycin .And its rooted frequency was 82.5%.
5. Detection of transgenic plants.
The PCR assay of kan-resistant plants showed that the target gene had been integrated into tobacco accompanying with npt II gene. The frequency of transf--ormation was 31.3%.
引文
1.Horsch RB,Fry JE,Hoffmam NL,et al. A simple and general method for transferring genes into plant. Sc ience, 1985,227:1229-1231.
2.James C.Global review of commercialized transgenic crops. Ithaca NY: International Service for the Acquisition of Agro-Biotech Application, 1998.
3．成卓敏．抗病毒转基因小麦研究进展．生物技术通讯，1997，4：11-13．
4.Hilder VA, Gatehouse AMR, Sheerman SE, et al. A novel mechanism of insect-resistance engineering into tobacco. Nature, 1987,330:160-163.
5．刘秋云，贺竹梅，曹俊，等．利用农杆菌系统将超甜定基因导入烟草．遗传，1998，20(10)：24-27．
6．杨美珠，潘乃穟，陈章良．高效马铃薯遗传转化体系的建立及甜蛋白基因的导入．植物学报，1992,34(1)：31-36
7．陈章良．植物基因工程研究．北京大学出版社(第一版)，1993
8.Takamatsu N. Watanabe Y. Yanagi H, et al. Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector. FEBS lett, 1990,269:73-76.
9.Goy PA, Duesing JH. Frompots to pots: Genetically modified plants on trial. Bio Technology, 1995,13:454-458.
10.Hooykaas GMS, Hooykaas PJJ, Schilperoort PA. Expression of Ti plasmid genes in monocotyledonous plants infected with Agrobacterium tumefaciens. Nature, 1984, 311:763-764.
11．陶余敏．农杆菌转化单子叶植物的可能性及问题．植物生理学通讯，1992，28(6)：402-406．
12.Rainri DM, Botino P, Gordon MP, et al. Agrobacterium-mediated transformation of rice (Oryza sativa L.). Bio/Technology, 1990,8:33-38.
13.Graves ACF, Goldman SL. The transformation of Zea mays seedlings with Agrobacterium tumefaciens: detection of T-DNA specific enzyme activities. Plant Molecular Biology, 1986,7:43-50.
14.Chen M, Fry JE, Pang SI, et al.Genetic transformation of wheat mediated by Agrobac terium tumefaciens. Plant Physiology, 1997,115:971-980.
15.Cohen A, Meredith CP. Agrobacterium-mediated transformation of Lilium. Acta Horticulturae, 1992325:611-618.
16.Hernalsteens JP, Toong LT, Schell J, et al. An Agrobacterium transformed cell culture from the monocot Asparagus officinalis. EMBO J, 1984,3:3 039-3401.
17.Graves ACF, Goldman SL, Agrobacterium tumefaciens-mediated transformation of the monocot genus Gladiolus: detection of expression of T-DNA encoded genes. J Bacteriol, 1987, 169:1745-1746.
18．傅荣昭，孙勇如，贾士荣．植物遗传转化技术手册，中国科学技术出版社，1994．
19．施建科，叶蕴华，田桂玲．有甜味的蛋白质．化学通报， 1998，(8)：21-25
20．范长胜，陈永青，李爽等．超甜蛋白基因工程及开发研究进展．工业微生物，1999，29(1)：29-33
21.Faus I. Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl Microbiol B iotechnol, 2000, 53(2):145-51
22．崔洪志，李敏，郭三堆．植物甜蛋白的研究与进展．生物技术通报，1997(2)：10-13
23.Rokert Dansby. Sweet science: Over expressing of monellin in yeast. Nature Biotechnology, 1997, 15(5): 419-420
24.Lee J.H, et al. Expression of Synthetic Thaumatin Genes in Yeast. Biochcmistry. 1998, 27:5101-5107
25．毛建民．甜味蛋白及其基因工程．生物学通报，1998，33(7)：24-25
26.Ogata C, et al. Crystal structure of a sweet tasting protein, thaumatin Ⅱ, at 1.65 A resolution. J.Mol. Biol, 1992,228:893-908
27.Beynon R, Cusack M. Thaumatin not proteolytic, Nature (London), 1990, 344:498
28.Dave Rs, Mitra RK. A low temperature induced apoplastic protein isolated from Arachis hypogaea. Phytochemistny, 1998,49(8):2207-13
29.Hiilovaara-Teijo M, et al. Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity. Plant physiol, 1999,121 (2): 665-74
30.Kuwabara C, et al. Abscisic acid-induced secretary protein in suspension-cultured cells of winter wheat. Plant Cell Physiol, 1999,40(2): 184-91
31.Grenier J, et al. Some thaumatin-like hydrolyse pohymeric beta-1,3-glucans. Plant J, 1999,19(4): 473-80
32．洛科·菲尔曼．THAUMATIN：一种强力甜味蛋白．生命的化学，1994，14(5)：39-42
33.Schiffman SS et al. Selective inhibition of sweetness by the sodium salt of +/-2-(4-methoxyphenoxy) propanoic acid. Chem Senses, 1999, 24(4): 439-47
34.Schiffman SS, Sattely-Miller EA, et al. Effect of temperature, pH, and ions on sweet taste. Physiol Behav, 2000, 68(4): 469-81
35.Moreno A, Soriano-Garcia M.Crystal-grouth kinetics of protein single crystals along capillary tubes in the gel-acupuncture technique. Acta Crystallogar D Biol Crystallogr, 1999,55:577-80.
36.Lorber B,etaI.Crystallization within agarose gel in microgravity improves the quality of thaumatin Crystals. Acta Crystallogr D Biol crystallogr, 1999,55:1491-1494
37.Moralejo FJ,et al.Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage. Appl Enviorn Microbiol, 1999,65(3): 1168-74.
38.Murashige T, Skoog F. Revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiology Plant, 1962,15:473-497.
39．卢圣栋．现代分子生物学试验技术．高等教育出版社(北京)，1993．
40．顾红雅，瞿礼嘉，明小天等．植物基因与分子操作，北京大学出版社(第一版)，1995．
41．王关林，方宏均主编．植物基因工程原理与技术．科学出版社，1998．
42.Babic V, Datla RS, Scole GJ. Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata. Plant Cell Reports, 1998,17:183-188.
43．余迪求，邓庆丽，沈亚楠等．金鱼草基因转化和转基因植株的再生．热带亚热带植物学报，1996，4(4)：196-199．
44．周冀明，卫志明，许志宏等．根癌农杆菌介导转化诸葛菜子叶获得转基因植株．生物工程学报，1996，12(1)：34-39．
45.John RZ, Patricia Z. Transfer of T-DNA from Agrobacterium to the plantcell. Plant Physiology, 1995, 107: 1041-1047.
46．李学宝，秦明辉，施荣华等．芥菜型油菜抗虫转基因植株及其后代株系的研究．生物工程学报，1999，15(4)：482-488．
47．李学宝，郑世学，董五辈等．甘蓝型油菜抗虫转基因植株及其抗性分析．遗传学报，1999，26(3)：262-268．
48.Mukhopadhyay A, Arumugan N, Nandakumar PBA, et al. Agrobacterium-mediated transformation of oilseed Brassicacampestris: Transformation frequency is strongly influenced by the mode of shoot regeneration. Plant Cell Reports, 1992, 11: 506-513.
49．蓝海燕，王长海，张丽华等．导入β—1，3-葡聚糖酶及几丁质酶基因的转基因可育油菜及其抗菌核病的研究．生物工程学报，2000，16(2)：142-145．
50．傅荣昭，刘敏，梁红健等．通过根癌农杆菌介导法获得菊花转基因植株．植物生理学报，1998，24(1)：72-76．
2.James C.Global review of commercialized transgenic crops. Ithaca NY: International Service for the Acquisition of Agro-Biotech Application, 1998.
3．成卓敏．抗病毒转基因小麦研究进展．生物技术通讯，1997，4：11-13．
4.Hilder VA, Gatehouse AMR, Sheerman SE, et al. A novel mechanism of insect-resistance engineering into tobacco. Nature, 1987,330:160-163.
5．刘秋云，贺竹梅，曹俊，等．利用农杆菌系统将超甜定基因导入烟草．遗传，1998，20(10)：24-27．
6．杨美珠，潘乃穟，陈章良．高效马铃薯遗传转化体系的建立及甜蛋白基因的导入．植物学报，1992,34(1)：31-36
7．陈章良．植物基因工程研究．北京大学出版社(第一版)，1993
8.Takamatsu N. Watanabe Y. Yanagi H, et al. Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector. FEBS lett, 1990,269:73-76.
9.Goy PA, Duesing JH. Frompots to pots: Genetically modified plants on trial. Bio Technology, 1995,13:454-458.
10.Hooykaas GMS, Hooykaas PJJ, Schilperoort PA. Expression of Ti plasmid genes in monocotyledonous plants infected with Agrobacterium tumefaciens. Nature, 1984, 311:763-764.
11．陶余敏．农杆菌转化单子叶植物的可能性及问题．植物生理学通讯，1992，28(6)：402-406．
12.Rainri DM, Botino P, Gordon MP, et al. Agrobacterium-mediated transformation of rice (Oryza sativa L.). Bio/Technology, 1990,8:33-38.
13.Graves ACF, Goldman SL. The transformation of Zea mays seedlings with Agrobacterium tumefaciens: detection of T-DNA specific enzyme activities. Plant Molecular Biology, 1986,7:43-50.
14.Chen M, Fry JE, Pang SI, et al.Genetic transformation of wheat mediated by Agrobac terium tumefaciens. Plant Physiology, 1997,115:971-980.
15.Cohen A, Meredith CP. Agrobacterium-mediated transformation of Lilium. Acta Horticulturae, 1992325:611-618.
16.Hernalsteens JP, Toong LT, Schell J, et al. An Agrobacterium transformed cell culture from the monocot Asparagus officinalis. EMBO J, 1984,3:3 039-3401.
17.Graves ACF, Goldman SL, Agrobacterium tumefaciens-mediated transformation of the monocot genus Gladiolus: detection of expression of T-DNA encoded genes. J Bacteriol, 1987, 169:1745-1746.
18．傅荣昭，孙勇如，贾士荣．植物遗传转化技术手册，中国科学技术出版社，1994．
19．施建科，叶蕴华，田桂玲．有甜味的蛋白质．化学通报， 1998，(8)：21-25
20．范长胜，陈永青，李爽等．超甜蛋白基因工程及开发研究进展．工业微生物，1999，29(1)：29-33
21.Faus I. Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl Microbiol B iotechnol, 2000, 53(2):145-51
22．崔洪志，李敏，郭三堆．植物甜蛋白的研究与进展．生物技术通报，1997(2)：10-13
23.Rokert Dansby. Sweet science: Over expressing of monellin in yeast. Nature Biotechnology, 1997, 15(5): 419-420
24.Lee J.H, et al. Expression of Synthetic Thaumatin Genes in Yeast. Biochcmistry. 1998, 27:5101-5107
25．毛建民．甜味蛋白及其基因工程．生物学通报，1998，33(7)：24-25
26.Ogata C, et al. Crystal structure of a sweet tasting protein, thaumatin Ⅱ, at 1.65 A resolution. J.Mol. Biol, 1992,228:893-908
27.Beynon R, Cusack M. Thaumatin not proteolytic, Nature (London), 1990, 344:498
28.Dave Rs, Mitra RK. A low temperature induced apoplastic protein isolated from Arachis hypogaea. Phytochemistny, 1998,49(8):2207-13
29.Hiilovaara-Teijo M, et al. Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity. Plant physiol, 1999,121 (2): 665-74
30.Kuwabara C, et al. Abscisic acid-induced secretary protein in suspension-cultured cells of winter wheat. Plant Cell Physiol, 1999,40(2): 184-91
31.Grenier J, et al. Some thaumatin-like hydrolyse pohymeric beta-1,3-glucans. Plant J, 1999,19(4): 473-80
32．洛科·菲尔曼．THAUMATIN：一种强力甜味蛋白．生命的化学，1994，14(5)：39-42
33.Schiffman SS et al. Selective inhibition of sweetness by the sodium salt of +/-2-(4-methoxyphenoxy) propanoic acid. Chem Senses, 1999, 24(4): 439-47
34.Schiffman SS, Sattely-Miller EA, et al. Effect of temperature, pH, and ions on sweet taste. Physiol Behav, 2000, 68(4): 469-81
35.Moreno A, Soriano-Garcia M.Crystal-grouth kinetics of protein single crystals along capillary tubes in the gel-acupuncture technique. Acta Crystallogar D Biol Crystallogr, 1999,55:577-80.
36.Lorber B,etaI.Crystallization within agarose gel in microgravity improves the quality of thaumatin Crystals. Acta Crystallogr D Biol crystallogr, 1999,55:1491-1494
37.Moralejo FJ,et al.Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage. Appl Enviorn Microbiol, 1999,65(3): 1168-74.
38.Murashige T, Skoog F. Revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiology Plant, 1962,15:473-497.
39．卢圣栋．现代分子生物学试验技术．高等教育出版社(北京)，1993．
40．顾红雅，瞿礼嘉，明小天等．植物基因与分子操作，北京大学出版社(第一版)，1995．
41．王关林，方宏均主编．植物基因工程原理与技术．科学出版社，1998．
42.Babic V, Datla RS, Scole GJ. Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata. Plant Cell Reports, 1998,17:183-188.
43．余迪求，邓庆丽，沈亚楠等．金鱼草基因转化和转基因植株的再生．热带亚热带植物学报，1996，4(4)：196-199．
44．周冀明，卫志明，许志宏等．根癌农杆菌介导转化诸葛菜子叶获得转基因植株．生物工程学报，1996，12(1)：34-39．
45.John RZ, Patricia Z. Transfer of T-DNA from Agrobacterium to the plantcell. Plant Physiology, 1995, 107: 1041-1047.
46．李学宝，秦明辉，施荣华等．芥菜型油菜抗虫转基因植株及其后代株系的研究．生物工程学报，1999，15(4)：482-488．
47．李学宝，郑世学，董五辈等．甘蓝型油菜抗虫转基因植株及其抗性分析．遗传学报，1999，26(3)：262-268．
48.Mukhopadhyay A, Arumugan N, Nandakumar PBA, et al. Agrobacterium-mediated transformation of oilseed Brassicacampestris: Transformation frequency is strongly influenced by the mode of shoot regeneration. Plant Cell Reports, 1992, 11: 506-513.
49．蓝海燕，王长海，张丽华等．导入β—1，3-葡聚糖酶及几丁质酶基因的转基因可育油菜及其抗菌核病的研究．生物工程学报，2000，16(2)：142-145．
50．傅荣昭，刘敏，梁红健等．通过根癌农杆菌介导法获得菊花转基因植株．植物生理学报，1998，24(1)：72-76．