农杆菌介导的转Bt cry1Iα基因抗虫花椰菜的研究
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摘要
花椰菜(Brassica oleracea Var.botrytis)是十字花科芸薹属中重要的蔬菜作物,不仅在我国的蔬菜生产中占有重要地位,在世界范围内也广泛种植。但是近年来,花椰菜的虫害十分严重,其中以鳞翅目的菜青虫(Pieris rapae)和小菜蛾(Plutella xylostella)为主。在我国,小菜蛾对花椰菜的危害呈明显上升趋势。常用的化学防治措施不仅污染环境,且害虫容易产生抗药性,因此,选育和应用抗虫品种,既经济有效又安全环保,是防治害虫最理想的方法。
Bt cry1Ia杀虫晶体蛋白基因是由本课题组从苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)的野生菌株Btc008中克隆得到的一种具有很高杀虫活性的杀虫晶体蛋白(Insecticidal crystal proteins,ICPs)。它是一类特殊的cry1类基因,与生产中常用的cry1A类基因相似性较低。Cry1Ia蛋白有较强的杀虫活性和相对较广的杀虫谱,对亚洲玉米螟、小菜蛾等有很强的杀虫活性,其杀虫效果与CrylAb和CrylAc相当,但与CrylAb和CrylAc无交互抗性。
本研究以具柄子叶和下胚轴为外植体,通过根癌农杆菌介导法将苏云金芽胞杆菌杀虫蛋白基因Cry1Ia导人花椰菜中,并优化了转化体系,研究了不同因素对花椰菜转化效率的影响。试验结果表明:花椰菜的基因型与外植体对抗性芽的再生有显著影响,本试验中瑞士雪球的具柄子叶和下胚轴是比较理想的外植体;不同激素对花椰菜芽再生的影响较大,本试验中添加2mg/L的6-BA和0.05mg/L的NAA时不定芽的再生频率较高;生根培养基中添加0.1mg/L的IAA时,生根效果最好;另外,用农杆菌EHA105介导的转化,转化效率要高于LBA4404;在侵染液及共培养基中添加乙酰丁香酮,能提高花椰菜的转化频率;在培养基中添加银离子并没有促进芽的再生;使用10mg/L的卡那霉素筛选阳性植株,可有效筛选出转基因植株;使用300mg/L的羧苄青霉素能完全抑制住农杆菌的污染;同时对农杆菌的侵染时间进行了优化,结果表明以下胚轴8 min、具柄子叶15min为最佳。
转化后的外植体在含有卡那霉素的培养基上诱导不定芽和诱导生根,共获得了42株抗性植株,经PCR检测,其中的18株呈阳性。进一步通过RT-PCR和Western blot检测,共有14株均呈阳性反应,检测结果初步说明cry1Ia基因已整合到花椰菜的基因组中并得到有效地表达。转基因植株用小菜蛾幼虫进行饲虫试验,结果表明昆虫幼虫的校正死亡率高达99.96%,同时,存活幼虫的生长发育也受到了明显抑制。
Cauliflower (Brassica oleracea Var.botrytis), one of the most important vegeTab. crops, is planted all over the world. Nowadays, it is also a main vegeTab. in China. But insect-pests have caused severe damage. Among them green worm (Pieris rapae L.) and diamondback moth (Plutella xylostella L.) are the primary pests. In China,diamondback moth is one of the most destructive pests and its damage has risen rapidly especially for cruciferous vegeTab.s. Because the pests are easy to induce resistance to insecticide, it is the best way to obtain the cultivars of insect-resistance in pest control.
Bt cry1Ia gene has been cloned from Bacillus thuringiensis strains Btc008 and has no homology with cry1A-type which is commonly used in pest control. Cry1Ia protein have relatively broad insecticidal controlling spectrum, strong insecticidal activity for corn borer, diamondback moth. Insecticidal activity of Cry1Ia protein is similar to Cry1Ab and Cry1AC, but didn’t have cross-resistance with both of them.
The cotyledon petioles and hypocotyls of cauliflower(Brassica oleracea Var.botrytis))were transformed with Agrobacterium tumefaciens harboring CaMV35S-1Ia-NOS expression vector containing CaMV35S promoter,nptⅡmarker gene and the insecticidal protein gene Bt cry1Ia gene. In this study the effects of influencing factors on genetic transformation frequency of cauliflower were investigated. The results showed: Both the hypocotyls and cotyledon of RuiXue were perfect explants for transformation; The hormone was an important factor on the regeneration of adventitious buds, we could obtain high regeneration frequency by supplementing 6-BA 2 mg/L and NAA 0.05 mg/L on the MS medium; Adding 0.lmg/L IAA to MS medium had good effect on rooting period and root quality of regenerate shoots; Infectious activity of Agrobacterium tumefaciens EHA105 was superior to LBA4404; Acetosyringone could promote the regeneration of adventitious buds in the transformation; It was no obvious difference to add silver nitrate on transformation frequency of cauliflower; The result showed that 10mg/L kanamycin would completely inhibit green shoot regeneration from explants of cauliflower, so it was available to selecting transgenic plants; The carbenicillin concentration was decided to use with 300mg/L; The experiment detected that the optimized infection time was 8 min for cotyledon and 15 min for hypocotyls.
Through successive selection in the period of shoot and root induction under kanamycin selecting pressure,42 kanamycin resistant regenerated plants were obtained,18 of them were positive resulted by PCR detection which demostrated that cry1Ia gene was integrated into the genome. Moreover, RT-PCR and Western blot analysis demonstrated that cry1Ia gene was expressed in RNA and protein levels in 14 plants. The transformation plants were given insect feeding test with larvae of diamondback moth.The results showed the mortality of insect larvae was accounted for 99.6%,and the growth of the survival larvae were seriously inhibited.
Bt cry1Ia杀虫晶体蛋白基因是由本课题组从苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)的野生菌株Btc008中克隆得到的一种具有很高杀虫活性的杀虫晶体蛋白(Insecticidal crystal proteins,ICPs)。它是一类特殊的cry1类基因,与生产中常用的cry1A类基因相似性较低。Cry1Ia蛋白有较强的杀虫活性和相对较广的杀虫谱,对亚洲玉米螟、小菜蛾等有很强的杀虫活性,其杀虫效果与CrylAb和CrylAc相当,但与CrylAb和CrylAc无交互抗性。
本研究以具柄子叶和下胚轴为外植体,通过根癌农杆菌介导法将苏云金芽胞杆菌杀虫蛋白基因Cry1Ia导人花椰菜中,并优化了转化体系,研究了不同因素对花椰菜转化效率的影响。试验结果表明:花椰菜的基因型与外植体对抗性芽的再生有显著影响,本试验中瑞士雪球的具柄子叶和下胚轴是比较理想的外植体;不同激素对花椰菜芽再生的影响较大,本试验中添加2mg/L的6-BA和0.05mg/L的NAA时不定芽的再生频率较高;生根培养基中添加0.1mg/L的IAA时,生根效果最好;另外,用农杆菌EHA105介导的转化,转化效率要高于LBA4404;在侵染液及共培养基中添加乙酰丁香酮,能提高花椰菜的转化频率;在培养基中添加银离子并没有促进芽的再生;使用10mg/L的卡那霉素筛选阳性植株,可有效筛选出转基因植株;使用300mg/L的羧苄青霉素能完全抑制住农杆菌的污染;同时对农杆菌的侵染时间进行了优化,结果表明以下胚轴8 min、具柄子叶15min为最佳。
转化后的外植体在含有卡那霉素的培养基上诱导不定芽和诱导生根,共获得了42株抗性植株,经PCR检测,其中的18株呈阳性。进一步通过RT-PCR和Western blot检测,共有14株均呈阳性反应,检测结果初步说明cry1Ia基因已整合到花椰菜的基因组中并得到有效地表达。转基因植株用小菜蛾幼虫进行饲虫试验,结果表明昆虫幼虫的校正死亡率高达99.96%,同时,存活幼虫的生长发育也受到了明显抑制。
Cauliflower (Brassica oleracea Var.botrytis), one of the most important vegeTab. crops, is planted all over the world. Nowadays, it is also a main vegeTab. in China. But insect-pests have caused severe damage. Among them green worm (Pieris rapae L.) and diamondback moth (Plutella xylostella L.) are the primary pests. In China,diamondback moth is one of the most destructive pests and its damage has risen rapidly especially for cruciferous vegeTab.s. Because the pests are easy to induce resistance to insecticide, it is the best way to obtain the cultivars of insect-resistance in pest control.
Bt cry1Ia gene has been cloned from Bacillus thuringiensis strains Btc008 and has no homology with cry1A-type which is commonly used in pest control. Cry1Ia protein have relatively broad insecticidal controlling spectrum, strong insecticidal activity for corn borer, diamondback moth. Insecticidal activity of Cry1Ia protein is similar to Cry1Ab and Cry1AC, but didn’t have cross-resistance with both of them.
The cotyledon petioles and hypocotyls of cauliflower(Brassica oleracea Var.botrytis))were transformed with Agrobacterium tumefaciens harboring CaMV35S-1Ia-NOS expression vector containing CaMV35S promoter,nptⅡmarker gene and the insecticidal protein gene Bt cry1Ia gene. In this study the effects of influencing factors on genetic transformation frequency of cauliflower were investigated. The results showed: Both the hypocotyls and cotyledon of RuiXue were perfect explants for transformation; The hormone was an important factor on the regeneration of adventitious buds, we could obtain high regeneration frequency by supplementing 6-BA 2 mg/L and NAA 0.05 mg/L on the MS medium; Adding 0.lmg/L IAA to MS medium had good effect on rooting period and root quality of regenerate shoots; Infectious activity of Agrobacterium tumefaciens EHA105 was superior to LBA4404; Acetosyringone could promote the regeneration of adventitious buds in the transformation; It was no obvious difference to add silver nitrate on transformation frequency of cauliflower; The result showed that 10mg/L kanamycin would completely inhibit green shoot regeneration from explants of cauliflower, so it was available to selecting transgenic plants; The carbenicillin concentration was decided to use with 300mg/L; The experiment detected that the optimized infection time was 8 min for cotyledon and 15 min for hypocotyls.
Through successive selection in the period of shoot and root induction under kanamycin selecting pressure,42 kanamycin resistant regenerated plants were obtained,18 of them were positive resulted by PCR detection which demostrated that cry1Ia gene was integrated into the genome. Moreover, RT-PCR and Western blot analysis demonstrated that cry1Ia gene was expressed in RNA and protein levels in 14 plants. The transformation plants were given insect feeding test with larvae of diamondback moth.The results showed the mortality of insect larvae was accounted for 99.6%,and the growth of the survival larvae were seriously inhibited.
引文
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Tabashnik B E, Carrière Y. 2006. Bt transgenic crops do not have favorable effects on resistant insects. Journal of Insect Science. 4(4): 1～3
Tabashnik B E, Cushing N L, Finson N, et al. 1990. Field development of resistance to Bacillus thuringiensis in diamondback moth(Lepidoptera:Plutellidae) . Journal of Economic Entomology. 83(5): 1671～1676
Vernader D, Wang K, el a1. 1988. Glycine betainc allows enhanced induction of the agribacterium tumefaciens vir genes by aeetosyringone at low Ph. Bacteriol. 170(12): 5822～5829
Xiang Y, Wong W R, Ma M C, et al. 2000. Agrobacterium-mediated transformation of Brassica campestrisssp Parachinensis with synthetic Bacillus thuringgiensis cry1Ab and cry1Ac genes. Plant Cell Report. 19(2): 201～256
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Zheng S J, HenKen B, Maggd R, et al. 2005. Two different bacillus thuringiensis toxin genes confer resistance to beet armyworm in transgenic Bt-shallots. Transgenic Research. 14(3): 261～272
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