利用转基因技术培育抗病番茄新品系
摘要
番茄(Lycopersicon esculentum. Mill)是一种重要的经济作物,富含维生素C和多种微量元素,营养价值很高,在我国大面积栽培。它既可作为蔬菜,又可作为水果食用,还可加工成番茄酱、番茄汁等罐制品,出口创汇,具有很高的经济价值。番茄的产量和品质好坏将直接影响到农业和食品业的发展,因此,提高番茄产量,改善番茄品质对我省、我国的经济发展具有重要意义。然而,在番茄的生长过程中,病虫害问题依然较为突出。目前影响番茄的真菌病害主要是番茄早疫病、晚疫病、叶霉病、灰霉病、镰刀枯萎病、脐腐病等。这些病害常常发生于番茄的苗期至成株期的各个阶段,危害番茄的根、茎、叶、花、果等器官,严重影响了番茄的生长,破坏了番茄的品质,使国民经济遭受重大损失。目前防治作物真菌病害的主要方法还是喷洒化学农药,然而这一方法最终将导致病原菌抗药性增强,并且残留的农药又造成了环境污染,危害人类健康。
几丁质酶对细胞壁中含有几丁质的真菌有抑制作用。现已发现,很多动物、植物、微生物都可产生几丁质酶。随着对几丁质酶的深入研究,几丁质酶在植物抗病基因工程中的应用也被广泛开展起来。本研究的目的是利用从木霉中克隆的新型几丁质酶基因和从烟草中克隆的β-葡聚糖酶基因,通过基因工程方法,将这两种抗病基因导入番茄,培育出抗病的番茄新种质或新品系。该项目将首先探索番茄再生体系和相关技术,为番茄细胞工程和基因工程研究奠定理论基础和技术基础。研究结果将有助于解决当前严重的番茄病害问题,有利于提高番茄产量,改善番茄品质,促进我国农业以及食品行业的可持续发展。获得的转基因番茄具有较强的抗病能力,可显著减少化学农药的使用,减少番茄种植中的人力、物力、财力,降低生产成本。由于可减少化学农药对环境的污染和对人类健康的危害,研究结果将有利于环境保护。因此,该项研究将具有较好的理论意义和实际应用价值。
针对山东番茄种植情况,通过对几个主栽番茄品种的深入研究,利用转基因技术,设计实验方法和技术路线,完成了以下工作:
1.对山东几个主栽番茄品种进行组织培养,确定出2种子叶再生能力强的番茄品种
2.利用农杆菌介导法将外源抗病基因导入番茄子叶,筛选转化愈伤组织;
3.通过子叶再生获得转基因植株,并进行转基因植株的PCR检测和Southern杂交检测。
4.对获得的转基因植株进行抗病测验和筛选,得到高抗病的番茄新品系。
在完成上述工作的过程中,本文着重在以下三点进行了创新性研究:
1.研究了11个山东主栽番茄品种的组织培养和植株再生技术,鉴定出两个再生能力很强的品种。
2.将两个抗真菌病害的基因同时导入番茄,获得了增强抗病性的转基因番茄植株株系。
3.总结出一套番茄再生和转化的实验技术,为进一步开展番茄细胞工程和基因工程研究提供了基础。
细胞工程育种是一种重要的育种手段,是其它生物技术难以替代的,还应该继续开展。本研究针对11个番茄品种,或者说11个不同的基因型,进行了它们的组织培养和植株再生的研究,探索了高频再生方法系统,发现不同品种之间存在明显差异,并且从中筛选出具有高再生能力的2个品种,为进一步开展番茄细胞工程和基因工程研究提供了优良实验材料。
Tomato(Lycopersicon esculentum.Mill) is an important economic crop, rich in vitamin C and trace elements, with high nutritional value and large-scale cultivation in China. It can be used as vegetables, can be eaten as fruit, can also be processed into tomato paste, tomato juice cans products, export earnings, with high economic value. Tomato yield and quality will directly affect the development of agriculture and food industry, therefore, improving the yield of tomato and improving the quality of tomato have great significance to our province and China's economic development. However, the growth process in the tomato, pest and disease problems are still more prominent. The fungal diseases currently affecting tomato are mainly tomato early blight, late blight, leaf mold, gray mold, fusarium wilt, umbilical rot diseases, and so on. These diseases often occur in tomato at all stages from seedling to adult stage, harm tomato root, stem, leaf, flower, fruit and other organs, which seriously affect the growth of tomato and destroy the quality of tomato. The national economy thus suffer a major loss. The current main method of controlling plant fungal diseases is spraying chemical pesticides, but this approach will ultimately lead to enhanced resistance to pathogens and pesticide, and residues also cause environmental pollution and do harm to human health.
Chitinase has a confining role on fungi containing chitin on the cell wall. It has been found that many animals, plants and microbes can produce chitinase. With the deep study of chitinase, chitinase in plant disease resistance gene engineering has been widely carried out together. The purpose of this study is to use the new chitinase gene cloned from trichoderma andβ-glucanase gene cloned from tobacco, lead these two resistance genes into tomato by genetic engineering methods and cultivate new disease resistance germplasm or new line. The project will first explore the tomato regeneration system and related technologies and lay the theoretical and technical foundation for tomato cell engineering and gene engineering research. The results will help to resolve the serious tomato disease problems, help to improve tomato production, improve the quality of tomato and promote the sustainable development of agriculture and food industry. Transgenic tomato with a strong resistance to diseases, can significantly reduce the use of chemical pesticide, reduce the human, material and financial resources in tomato cultivation and reduce production costs. Since it can reduce the environmental pollution and harm to human health of chemical pesticides, the results will benefit the environmental protection. Therefore, the study will have a better theoretical and practical value.
Through the deep study of several main cultivated varieties of tomato according to tomato cultivation in Shandong, we make use of transgenic technology, design experimental methods and technical line and complete the following work:
1. Determine the 2 tomato varieties with strong leaf regeneration ability through tissue culturing for several main cultivated tomato varieties in Shandong;
2. Lead exogenous resistance genes into tomato leaf by the method of agrobacterium-mediated and screen transformed callus;
3. Acquire transgenic plants by leaf regeneration and conduct the transgenic plants PCR detection and Southern interbreeding detection;
4. Conduct resistant test, screen the transgenic plants and acquire new lines of tomato with high resistance to disease.
In the process of completing the above work, the paper mainly focuses on the following three innovative researches:
1. Study the tissue culture and plant regeneration technique of the 11 main cultivated tomato varieties in Shandong and identify two species with strong regenerative capacity.
2. Put the two antifungal genes into tomato at the same time and acquire the enhanced resistance in transgenic tomato lines.
3. Summarize a set of experimental tomato regeneration and transformation technology and provide a basis for the further research of tomato cell engineering and genetic engineering.
Cell engineering breeding is an important breeding method, which is difficult to replace by other biological technologies and should be continued to develop. We make a study of tissue culture and plant regeneration for 11 tomato varieties, or 11 different genotypes, explore the high-frequency regeneration methods system, find that significant differences exist between different species, and screen out 2 varieties of tomato of high regenerative capacity, which provides a good experimental material for further study of engineering and genetic engineering.
几丁质酶对细胞壁中含有几丁质的真菌有抑制作用。现已发现,很多动物、植物、微生物都可产生几丁质酶。随着对几丁质酶的深入研究,几丁质酶在植物抗病基因工程中的应用也被广泛开展起来。本研究的目的是利用从木霉中克隆的新型几丁质酶基因和从烟草中克隆的β-葡聚糖酶基因,通过基因工程方法,将这两种抗病基因导入番茄,培育出抗病的番茄新种质或新品系。该项目将首先探索番茄再生体系和相关技术,为番茄细胞工程和基因工程研究奠定理论基础和技术基础。研究结果将有助于解决当前严重的番茄病害问题,有利于提高番茄产量,改善番茄品质,促进我国农业以及食品行业的可持续发展。获得的转基因番茄具有较强的抗病能力,可显著减少化学农药的使用,减少番茄种植中的人力、物力、财力,降低生产成本。由于可减少化学农药对环境的污染和对人类健康的危害,研究结果将有利于环境保护。因此,该项研究将具有较好的理论意义和实际应用价值。
针对山东番茄种植情况,通过对几个主栽番茄品种的深入研究,利用转基因技术,设计实验方法和技术路线,完成了以下工作:
1.对山东几个主栽番茄品种进行组织培养,确定出2种子叶再生能力强的番茄品种
2.利用农杆菌介导法将外源抗病基因导入番茄子叶,筛选转化愈伤组织;
3.通过子叶再生获得转基因植株,并进行转基因植株的PCR检测和Southern杂交检测。
4.对获得的转基因植株进行抗病测验和筛选,得到高抗病的番茄新品系。
在完成上述工作的过程中,本文着重在以下三点进行了创新性研究:
1.研究了11个山东主栽番茄品种的组织培养和植株再生技术,鉴定出两个再生能力很强的品种。
2.将两个抗真菌病害的基因同时导入番茄,获得了增强抗病性的转基因番茄植株株系。
3.总结出一套番茄再生和转化的实验技术,为进一步开展番茄细胞工程和基因工程研究提供了基础。
细胞工程育种是一种重要的育种手段,是其它生物技术难以替代的,还应该继续开展。本研究针对11个番茄品种,或者说11个不同的基因型,进行了它们的组织培养和植株再生的研究,探索了高频再生方法系统,发现不同品种之间存在明显差异,并且从中筛选出具有高再生能力的2个品种,为进一步开展番茄细胞工程和基因工程研究提供了优良实验材料。
Tomato(Lycopersicon esculentum.Mill) is an important economic crop, rich in vitamin C and trace elements, with high nutritional value and large-scale cultivation in China. It can be used as vegetables, can be eaten as fruit, can also be processed into tomato paste, tomato juice cans products, export earnings, with high economic value. Tomato yield and quality will directly affect the development of agriculture and food industry, therefore, improving the yield of tomato and improving the quality of tomato have great significance to our province and China's economic development. However, the growth process in the tomato, pest and disease problems are still more prominent. The fungal diseases currently affecting tomato are mainly tomato early blight, late blight, leaf mold, gray mold, fusarium wilt, umbilical rot diseases, and so on. These diseases often occur in tomato at all stages from seedling to adult stage, harm tomato root, stem, leaf, flower, fruit and other organs, which seriously affect the growth of tomato and destroy the quality of tomato. The national economy thus suffer a major loss. The current main method of controlling plant fungal diseases is spraying chemical pesticides, but this approach will ultimately lead to enhanced resistance to pathogens and pesticide, and residues also cause environmental pollution and do harm to human health.
Chitinase has a confining role on fungi containing chitin on the cell wall. It has been found that many animals, plants and microbes can produce chitinase. With the deep study of chitinase, chitinase in plant disease resistance gene engineering has been widely carried out together. The purpose of this study is to use the new chitinase gene cloned from trichoderma andβ-glucanase gene cloned from tobacco, lead these two resistance genes into tomato by genetic engineering methods and cultivate new disease resistance germplasm or new line. The project will first explore the tomato regeneration system and related technologies and lay the theoretical and technical foundation for tomato cell engineering and gene engineering research. The results will help to resolve the serious tomato disease problems, help to improve tomato production, improve the quality of tomato and promote the sustainable development of agriculture and food industry. Transgenic tomato with a strong resistance to diseases, can significantly reduce the use of chemical pesticide, reduce the human, material and financial resources in tomato cultivation and reduce production costs. Since it can reduce the environmental pollution and harm to human health of chemical pesticides, the results will benefit the environmental protection. Therefore, the study will have a better theoretical and practical value.
Through the deep study of several main cultivated varieties of tomato according to tomato cultivation in Shandong, we make use of transgenic technology, design experimental methods and technical line and complete the following work:
1. Determine the 2 tomato varieties with strong leaf regeneration ability through tissue culturing for several main cultivated tomato varieties in Shandong;
2. Lead exogenous resistance genes into tomato leaf by the method of agrobacterium-mediated and screen transformed callus;
3. Acquire transgenic plants by leaf regeneration and conduct the transgenic plants PCR detection and Southern interbreeding detection;
4. Conduct resistant test, screen the transgenic plants and acquire new lines of tomato with high resistance to disease.
In the process of completing the above work, the paper mainly focuses on the following three innovative researches:
1. Study the tissue culture and plant regeneration technique of the 11 main cultivated tomato varieties in Shandong and identify two species with strong regenerative capacity.
2. Put the two antifungal genes into tomato at the same time and acquire the enhanced resistance in transgenic tomato lines.
3. Summarize a set of experimental tomato regeneration and transformation technology and provide a basis for the further research of tomato cell engineering and genetic engineering.
Cell engineering breeding is an important breeding method, which is difficult to replace by other biological technologies and should be continued to develop. We make a study of tissue culture and plant regeneration for 11 tomato varieties, or 11 different genotypes, explore the high-frequency regeneration methods system, find that significant differences exist between different species, and screen out 2 varieties of tomato of high regenerative capacity, which provides a good experimental material for further study of engineering and genetic engineering.
引文
[1]番茄信息网.我国番茄青枯病及抗病育种研究进展[EB/OL]: http://www.chinatomato.net/news_view.asp?id=27,2007-12-19
[2]李钦存.日光温室番茄根腐病防治综述[J].陕西:西北园艺,2008,6:6-8
[3]福建工程学院图书馆.基因工程的发展现状及前景[EB/OL]: http://lib.fjut.edu.cn/news2/view.asp?id=9215,2009-2-19
[4]史开兵,李勇.作物基因工程进展和展望[J].湖南:湖南农业科学,2005, 3:10-13
[5] Powell A P. Delay of disease development in trans genic plant that express the tobacco mosaic virus coat protein genes [J]. Science, 1986, 232:738-743
[6]程英豪,王继伟,王英典等.由TMV54xlO3蛋白基因构建的转基因番茄及其对TMV的抗性[J].北京:北京师范大学学报(自然科学版)1999,35(1):93-96
[7]徐香玲,利用发根农杆菌介导二元载体向番茄导入TMV、CMV外壳蛋白的研究,植物研究,1994,14(4):416-423
[8] Nelson R.S., Dube P., Layton , J., Anderson, E.J. ,Kaniewska M., Proksch R.K. , Horsch R.B , RogersS.G., Fraley T.F., and Beachy R.N., 1988 , Virustolerance , plant growth , and field performance of trans2 genic tomato plants expressing coat protein from tomatomosaic virus[J] , Biotechnology, (6) : 403 - 409
[9] Loia P., Kim R., and Giovannoni J., 1992, Production ofthe sweet protein monellin in transgenic plants[J] ,Bio1Technology , 10 : 561 - 564
[10] Elad Y, Barak R,Chet I.Parasicism of Sclerotium rolfsii by Trichodermaharzianum [J]. Soil Biol Biochem, 1984,16:381-386
[11] Elad Y, Eak R, Chet I.The possible role of 1ectins in mycoparasitism [J]. Bacterial,1983.154:1431-1435
[12] Elad Y, Cbet I, Boyle P, et al. Parasitism of Trichodrma spp.on Rhizoctoniasolani andSclerotium rolfsii scanning electron microscopy and fiuorescence microscopy [J]. Phytopathnology, 1983, 73:85-88
[13] Sivasithamparam K, Ghisalbet EJ.Secondary metabo1ism in Trichoderm and Gliocladium [C].London:Taylor & Francis Ltd, 1998:139-191
[14] Lorito M.Hannan GE, Haves CK. et al, Chitinolytic enzymes produced by Trichodrerma harzianum; antifungal activity of purified endochitinase and chitobinase [J]. Phytopathology, 1993,83:302-307
[15] Sela-Buurlage MB, Ponstein AS et al. Only specific tobacco-chitinase and (-1,3-glucanase exhibit antinfungal activity [J].Plant Physiology,1993,101:857 - 863
[16]Vanden DJ, Jongdeu. KE, Mechersl S et al. Virus and fungal resistance:from laboratory to field [J]. Phil Trans R Soc Lond B,1993,342:271 -278
[17] Thomzik J.Z., Stenzel K., and Stocker R., 1997, Synthesisof a grapevine phytoalexin in transgenic tomato conditionsresistence against phytophthora infestans [J] , Physiol1 Mol1Plant Pathol1 , 51 (4) : 265 - 270
[18]仇润祥,扈延茂,王永胜,张晓海,刘中大.番茄1-氨基环内烷羧酸(ACC)合成酶反义的RNA-核酶嵌合DNA序列的构建[J]遗传, 1999,21(1):1-6
[19]叶志彪,李汉霞,刘勋甲,向长萍,郑世发,王春梅,余科秀.利用转基因技术育成耐贮番茄-华番一号[J],中国蔬菜, 1999,1:6-10
[20] Hightower R.C., Baden C., Penzes E., Lund P., and Dun2smuir P. Expression of antifreeze proteins intransgenic plants[J] , PMB , 1991,17 (5) : 1013 - 1021
[21]王跃驹,李刚强,刘德虎.利用转基因植物生产新型疫苗[J],高技术通讯,1997,12: 57-60
[22] Jongedijk E, Tiglaar H, Roekel J S C, et al. Syergistic activity of chitimases andβ-1,3-glucanases enhances fungal resistance in transgenic tomato plants [J]. Euthytica,1995,85(1-3):l73-l80
[23]陈玉辉,许向阳,李景富.转基因技术在番茄育种上的应用[J].分子植物育种,2004,2(1):133-138
[2]李钦存.日光温室番茄根腐病防治综述[J].陕西:西北园艺,2008,6:6-8
[3]福建工程学院图书馆.基因工程的发展现状及前景[EB/OL]: http://lib.fjut.edu.cn/news2/view.asp?id=9215,2009-2-19
[4]史开兵,李勇.作物基因工程进展和展望[J].湖南:湖南农业科学,2005, 3:10-13
[5] Powell A P. Delay of disease development in trans genic plant that express the tobacco mosaic virus coat protein genes [J]. Science, 1986, 232:738-743
[6]程英豪,王继伟,王英典等.由TMV54xlO3蛋白基因构建的转基因番茄及其对TMV的抗性[J].北京:北京师范大学学报(自然科学版)1999,35(1):93-96
[7]徐香玲,利用发根农杆菌介导二元载体向番茄导入TMV、CMV外壳蛋白的研究,植物研究,1994,14(4):416-423
[8] Nelson R.S., Dube P., Layton , J., Anderson, E.J. ,Kaniewska M., Proksch R.K. , Horsch R.B , RogersS.G., Fraley T.F., and Beachy R.N., 1988 , Virustolerance , plant growth , and field performance of trans2 genic tomato plants expressing coat protein from tomatomosaic virus[J] , Biotechnology, (6) : 403 - 409
[9] Loia P., Kim R., and Giovannoni J., 1992, Production ofthe sweet protein monellin in transgenic plants[J] ,Bio1Technology , 10 : 561 - 564
[10] Elad Y, Barak R,Chet I.Parasicism of Sclerotium rolfsii by Trichodermaharzianum [J]. Soil Biol Biochem, 1984,16:381-386
[11] Elad Y, Eak R, Chet I.The possible role of 1ectins in mycoparasitism [J]. Bacterial,1983.154:1431-1435
[12] Elad Y, Cbet I, Boyle P, et al. Parasitism of Trichodrma spp.on Rhizoctoniasolani andSclerotium rolfsii scanning electron microscopy and fiuorescence microscopy [J]. Phytopathnology, 1983, 73:85-88
[13] Sivasithamparam K, Ghisalbet EJ.Secondary metabo1ism in Trichoderm and Gliocladium [C].London:Taylor & Francis Ltd, 1998:139-191
[14] Lorito M.Hannan GE, Haves CK. et al, Chitinolytic enzymes produced by Trichodrerma harzianum; antifungal activity of purified endochitinase and chitobinase [J]. Phytopathology, 1993,83:302-307
[15] Sela-Buurlage MB, Ponstein AS et al. Only specific tobacco-chitinase and (-1,3-glucanase exhibit antinfungal activity [J].Plant Physiology,1993,101:857 - 863
[16]Vanden DJ, Jongdeu. KE, Mechersl S et al. Virus and fungal resistance:from laboratory to field [J]. Phil Trans R Soc Lond B,1993,342:271 -278
[17] Thomzik J.Z., Stenzel K., and Stocker R., 1997, Synthesisof a grapevine phytoalexin in transgenic tomato conditionsresistence against phytophthora infestans [J] , Physiol1 Mol1Plant Pathol1 , 51 (4) : 265 - 270
[18]仇润祥,扈延茂,王永胜,张晓海,刘中大.番茄1-氨基环内烷羧酸(ACC)合成酶反义的RNA-核酶嵌合DNA序列的构建[J]遗传, 1999,21(1):1-6
[19]叶志彪,李汉霞,刘勋甲,向长萍,郑世发,王春梅,余科秀.利用转基因技术育成耐贮番茄-华番一号[J],中国蔬菜, 1999,1:6-10
[20] Hightower R.C., Baden C., Penzes E., Lund P., and Dun2smuir P. Expression of antifreeze proteins intransgenic plants[J] , PMB , 1991,17 (5) : 1013 - 1021
[21]王跃驹,李刚强,刘德虎.利用转基因植物生产新型疫苗[J],高技术通讯,1997,12: 57-60
[22] Jongedijk E, Tiglaar H, Roekel J S C, et al. Syergistic activity of chitimases andβ-1,3-glucanases enhances fungal resistance in transgenic tomato plants [J]. Euthytica,1995,85(1-3):l73-l80
[23]陈玉辉,许向阳,李景富.转基因技术在番茄育种上的应用[J].分子植物育种,2004,2(1):133-138