新疆甜瓜组培体系的优化及抗病转基因研究
摘要
近几年来在新疆甜瓜的生产中，真菌病害已成为影响新疆甜瓜产量和品质的重要因素。作者试图通过植物基因工程方法，利用根癌农杆菌将抗真菌蛋白的几丁质酶基因和β-1,3-葡聚糖酶基因导入新疆甜瓜中，以提高其抗真菌病害的能力。
选用新疆优良甜瓜品种：红蜜脆、皇后、黄醉仙、8601，用未成熟子叶切块作为外植体，经过组织培养获得再生植株。以MS为基本培养基，取3日龄子叶块外植体，选用MS+6-BA0-2.5mg/L诱导丛生芽,最高诱导率可达89%。其中杂交种的芽诱导率高于本地种。由于基因型的差异，不同品种甜瓜诱导不定芽的最适激素浓度不同，而且在整个生长过程中，6-BA的浓度逐渐降低。在生根培养时，用1/2MS+0.5mg/L IBA或 NAA诱导生根率较高。
在建立甜瓜高效再生体系的基础上，用根癌农杆菌介导法将几丁质酶基因和β-1,3-葡聚糖酶基因导入新疆甜瓜中，经卡那霉素的抗性筛选，获得转化的再生植株。用PCR反应、点杂交及SDS-PAGE等手段，进一步研究了再生植株的基因整合与表达情况。经PCR扩增反应和琼脂糖电泳检测，在转化植株中扩增出了特异片段，证明外源基因已整合到甜瓜基因组中。点杂交检测转化植株有阳性反应。SDS-PAGE分析表明，转化植株比对照在35.5KD处有一条明显的蛋白质谱带，其分子量与几丁质酶基因表达产物分子量相符。可初步确定该基因在转化植株内得到正常表达。
Fungal disease has been a serious threat to production and trait of Xinjiang muskmelon in recent years. We attempt to transfer Xinjiang muskmelon via Agrobacterium tumefaciens carrying chitinase and β -1,3-glucanase in order to enhance the abilities of anti-fungal.
In order to establish the transgenic plant system, the cotyledons of muskmelon as explants, regenerated plantlets were used in culture. Shoots regeneration were induced on basic medium MS medium supplemented with 0-2.5mg/L 6-BA.The highest frequency shoot regeneration observed is 89%. The induction frequency of shoot of melon varieties is depended on different concentration of hormones in the medium. To achieve the high rate of rooting, the concentration of hormone 6-BA was gradually decreased during the whole growth period. The 1/2 MS medium with 0.5mg/L IBA and NAA was used for this propose.
On the basis of regeneration protocol of Xinjiang muskmelon, two genes were transfected into muskmelon plant and selected of kanamycin in the medium. After kanamycin resistant plants were obtained, transgenic plants were determined by PCR and confirmed by Southern dot blotting. The expression of chitinase was observed by using SDS-PAGE analysis.
选用新疆优良甜瓜品种：红蜜脆、皇后、黄醉仙、8601，用未成熟子叶切块作为外植体，经过组织培养获得再生植株。以MS为基本培养基，取3日龄子叶块外植体，选用MS+6-BA0-2.5mg/L诱导丛生芽,最高诱导率可达89%。其中杂交种的芽诱导率高于本地种。由于基因型的差异，不同品种甜瓜诱导不定芽的最适激素浓度不同，而且在整个生长过程中，6-BA的浓度逐渐降低。在生根培养时，用1/2MS+0.5mg/L IBA或 NAA诱导生根率较高。
在建立甜瓜高效再生体系的基础上，用根癌农杆菌介导法将几丁质酶基因和β-1,3-葡聚糖酶基因导入新疆甜瓜中，经卡那霉素的抗性筛选，获得转化的再生植株。用PCR反应、点杂交及SDS-PAGE等手段，进一步研究了再生植株的基因整合与表达情况。经PCR扩增反应和琼脂糖电泳检测，在转化植株中扩增出了特异片段，证明外源基因已整合到甜瓜基因组中。点杂交检测转化植株有阳性反应。SDS-PAGE分析表明，转化植株比对照在35.5KD处有一条明显的蛋白质谱带，其分子量与几丁质酶基因表达产物分子量相符。可初步确定该基因在转化植株内得到正常表达。
Fungal disease has been a serious threat to production and trait of Xinjiang muskmelon in recent years. We attempt to transfer Xinjiang muskmelon via Agrobacterium tumefaciens carrying chitinase and β -1,3-glucanase in order to enhance the abilities of anti-fungal.
In order to establish the transgenic plant system, the cotyledons of muskmelon as explants, regenerated plantlets were used in culture. Shoots regeneration were induced on basic medium MS medium supplemented with 0-2.5mg/L 6-BA.The highest frequency shoot regeneration observed is 89%. The induction frequency of shoot of melon varieties is depended on different concentration of hormones in the medium. To achieve the high rate of rooting, the concentration of hormone 6-BA was gradually decreased during the whole growth period. The 1/2 MS medium with 0.5mg/L IBA and NAA was used for this propose.
On the basis of regeneration protocol of Xinjiang muskmelon, two genes were transfected into muskmelon plant and selected of kanamycin in the medium. After kanamycin resistant plants were obtained, transgenic plants were determined by PCR and confirmed by Southern dot blotting. The expression of chitinase was observed by using SDS-PAGE analysis.
引文
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2. Broglie K,Chet I,Holliday M et al. Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani.Science,1991.254:1194-1197
3. Logemann J,Jach G, Tommerup H.Expression of a barley ribosome-inactivating protein leads to increasedfungal protection in transgenic tobacco plant. Bio/Techonlogy, 1992.10:305
4. Leah R. Tommerup H.Svendsen Ib et al.Biochemical and molecular haracterization of three barley seed proteins with antifungal properties.J Biol Chem,1991.266:1564-1570
5. .Neuhaus J-M,Ahl-Goy P.Hinz U et al.High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cerospora nicotianae infection. Plant Mol Biol,1991.16:141-151
6. Woloshuk C P,Meulenhoff J S, Sela-Buurlage M et al.Pathogen-induced proteins with inhibitory activity toward Phytophthora infestaus.Plant Cell,1991.3:619-628
7. Meeley R B，Jobal GS，Briggs SP. A biochemical phenotypes for a resistance gene of maize.Plant Cell,1992.4:71
8. De Wit PJGM.Molecular characterization of gene-for-gene system in plant fungus interaction and the application of avirulence genes in control of plant pathogenu.Ann Rev Phytopathol,1992.30:391-418
9. van Kan JAL， van den Ackerveken GF J M，de Wit P M.Cloning and characterization of Cdna of avirulence gene avr9 of the fungal pathogen Cladosporium fulvum,causal agent of tomato leaf mold,Mol Plant-Microb Inter,1991.4:52-59
10. Dixon RA,Day PM,Lamb CJ.Phytoalexins:Enzymology and molecular biology.Adv Enzymol,1993.55:1
11. Vanetter HD,Matthews P S.Phytoalexin detoxification.Importance for pathogenicity and practical implications.Ann Rev Phytopathol,1989.27:143-164
12. Hain R,Bieseleer B,Kindl H.Ecppression of astilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol.Pl Mol Biol,1990,15:325
13.
14. Hahn MG，BucheH P，Cervone F.Roles of cell wall constituents in plant-pathogen interactions.In Kosuge T,Nester EY(eds),Plant-Microbe Interactions:Molccular and Genetic Perspectives,1989,3:131
15. Toubart P,Desiderio A,Salvi G et al.Cloning and characterization of the gene encoding the endopolyglacturonase inhibiting protein of Phaseolus vulgaris L.Plant,1992.2:367-373
16. Bayer S M.New plant caffeoyl-coA-3-O-methyl transferase gene.Derwent Biotechnology Abstract.1993.12(4):9302188
17. Mauch F.Mauch-ManiB,Boller T.Antifungal hydrolases in pea tissue II.Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase.Plant Physiol,1988.88:936-942
18. Norelli-JL, Bolar-JP, Harman-GE, Transgenic apple plants expressing chitinases from Trichoderma have increased resistance to scab (Venturia inaequalis). Acta-Horticulturae. 2000, (Vol.2), 617-618
19. Punja-ZK; Raharjo-SHT, Response of transgenic cucumber and carrot plants expressing different chitinase enzymes to inoculation with fungal pathogens. Plant-Disease. 1996, 80: 9, 999-1005;
20. Jongedijk-E, Tigelaar-H, Roekel-SC-van,et al.Synergistic activity of chitinases and beta-1,3-glucanases enhances fungal resistance in transgenic tomato plants. Eucarpia Genetic Manipulation in Plant Breeding section meeting, 1995, 85: 173-180
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