RNAi技术培育抗ToMV加工番茄研究
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摘要
本研究是利用RNAi技术培育加工番茄抗病毒新材料。(1)通过农杆菌介导法将抗ToMVRNAi载体转化烟草和加工番茄,在实验过程中优化加工番茄的转化条件,提高加工番茄的转化效率,为培育新的抗病毒种子资源构建平台。(2)对得到的转基因烟草和加工番茄植株通过攻毒试验验证对ToMV有抗性,从而得到抗病毒的转基因加工番茄植株。
试验中首先以子叶为外植体,在MS基本培养基中添加不同浓度的卡那霉素(Kan10、30、50mg/L),检测子叶对卡那霉素的抗性,发现子叶外植体的分化被不同程度地抑制。然后诱导培养基卡那霉素的选择选取了梯度变化,0-10d时10mg/L；10-20d时30mg/L；20d以后50mg/L。芽诱导培养基分别添加玉米素(ZT)0.5mg/L和1.0mg/L;6-BA2.0mg/L不同的细胞分裂素,研究分裂素的种类和浓度对芽分化的影响。将侵染后的外植体分别放在23-24℃、26-28℃温度条件下,其他条件相同,研究温度对抗性芽分化的影响。最后对获得的抗卡那霉素抗性植株进行PCR检测,发现ToMV的干扰片段已转入加工番茄品种黑格尔87-5。最后对转基因植株通过攻毒试验RT-PCR检测RNAi效果。
通过试验建立加工番茄子叶为外植体的高效遗传转化体系：确定了当预培养时间4d,共培养时间3d,,侵染时间20min,诱导培养基ZT (0.5mg/L), IAA (0.1mg/L),卡那霉素0-10d时10mg/L;10-20d时30mg/L；20d以后50mg/L放在23-24℃分化的温度条件下转化率最高。攻毒试验表明：转基因加工番茄和烟草对ToMV有很好的干扰效果。不同的干扰片段对病毒的基因的降解效果也不同,出现了免疫型和对病毒敏感的不同效果。
上述研究发现遗传转化条件的优化,可以提高加工番茄的遗传转化率；基因工程为培育新的抗病毒品种开辟了新途径。试验验证了RNAi技术对培育加工番茄抗病毒新材料的可行性。
In this research, RNAi technology was used to repress the homology target gene expression of Tomato mosaic virus by degrading mRNA of target gene aiming to resist ToMV. Agrobacterium-mediated transformation has been used to introduce the RNAi vector into processing tomatoes.The conditions for regeneration and transformation of processing tomato mediated by Agrobacterium were studied with the aim of optimizing the transformation protocol.(2)Transgenic plants were tested in order to make sure that they were resistant to ToMV.
During the experimmt cotyledons were taken as explants,first We added different concentrations of Kanamycin into MS basic medium in order to test whether they were resistant to Kanamycin.We Found that the differentiation of cotyledon explants were inhibited in different degrees.And then different concentrations of kanamycin was added into induction medium. The first ten days we added 10mg/L kanamycin into induction medium,The second ten days we added 30mg/L,and from then on we added 50mg/L. We added different concentrations and types of cytokinins into bud induction medium.ZT0.5 mg/L,1.0 mg/L or 6-BA2.0 mg/1 was added into bud induction medium in order to choose the best concentration and type of cytokinins for bud induction.After infection,we studied inflution of temperature on bud induction.explants were placed in 23～24℃and 26～28℃temperature, other things were equal.
PCR amplication showed that fragments of ToMV was integrated into genome of transgenic tomato plants. The result showed transgenic tomato plants are resistant to ToMV when infected with ToMV
Through the experimmt we got such result:(1) After 4d pre-culture,3d co-culture, and 12weeks of selective culture with 10～30～50 mg/L kanamycin, in the first ten days concentrations of kanamycin in bud induction medium were 10mg/L, in the second ten days concentrations of kanamycin in bud induction medium were 30mg/L, and from then on concentrations of kanamycin were 50mg/L. 0.5mg/LZT, 0.1mg/LIAA was added into bud induction medium, explants were placed under 23～24℃condition,the best transformation efficiencies could be obtained.(2) Transgenic plants are resistant to ToMV.Different fragments of ToMV were selected to contruct RNAi vector.The result showed fragments of ToMV has influences on resistance to ToMV
Through the experiment we found that the best transformation efficiencies could be obtained by improving conditions.Genetic engineering technology open up new ways to plant antiviral breeding. It is possible to cultivate processing tomato resistant to ToMV using RNAi.
试验中首先以子叶为外植体,在MS基本培养基中添加不同浓度的卡那霉素(Kan10、30、50mg/L),检测子叶对卡那霉素的抗性,发现子叶外植体的分化被不同程度地抑制。然后诱导培养基卡那霉素的选择选取了梯度变化,0-10d时10mg/L；10-20d时30mg/L；20d以后50mg/L。芽诱导培养基分别添加玉米素(ZT)0.5mg/L和1.0mg/L;6-BA2.0mg/L不同的细胞分裂素,研究分裂素的种类和浓度对芽分化的影响。将侵染后的外植体分别放在23-24℃、26-28℃温度条件下,其他条件相同,研究温度对抗性芽分化的影响。最后对获得的抗卡那霉素抗性植株进行PCR检测,发现ToMV的干扰片段已转入加工番茄品种黑格尔87-5。最后对转基因植株通过攻毒试验RT-PCR检测RNAi效果。
通过试验建立加工番茄子叶为外植体的高效遗传转化体系：确定了当预培养时间4d,共培养时间3d,,侵染时间20min,诱导培养基ZT (0.5mg/L), IAA (0.1mg/L),卡那霉素0-10d时10mg/L;10-20d时30mg/L；20d以后50mg/L放在23-24℃分化的温度条件下转化率最高。攻毒试验表明：转基因加工番茄和烟草对ToMV有很好的干扰效果。不同的干扰片段对病毒的基因的降解效果也不同,出现了免疫型和对病毒敏感的不同效果。
上述研究发现遗传转化条件的优化,可以提高加工番茄的遗传转化率；基因工程为培育新的抗病毒品种开辟了新途径。试验验证了RNAi技术对培育加工番茄抗病毒新材料的可行性。
In this research, RNAi technology was used to repress the homology target gene expression of Tomato mosaic virus by degrading mRNA of target gene aiming to resist ToMV. Agrobacterium-mediated transformation has been used to introduce the RNAi vector into processing tomatoes.The conditions for regeneration and transformation of processing tomato mediated by Agrobacterium were studied with the aim of optimizing the transformation protocol.(2)Transgenic plants were tested in order to make sure that they were resistant to ToMV.
During the experimmt cotyledons were taken as explants,first We added different concentrations of Kanamycin into MS basic medium in order to test whether they were resistant to Kanamycin.We Found that the differentiation of cotyledon explants were inhibited in different degrees.And then different concentrations of kanamycin was added into induction medium. The first ten days we added 10mg/L kanamycin into induction medium,The second ten days we added 30mg/L,and from then on we added 50mg/L. We added different concentrations and types of cytokinins into bud induction medium.ZT0.5 mg/L,1.0 mg/L or 6-BA2.0 mg/1 was added into bud induction medium in order to choose the best concentration and type of cytokinins for bud induction.After infection,we studied inflution of temperature on bud induction.explants were placed in 23～24℃and 26～28℃temperature, other things were equal.
PCR amplication showed that fragments of ToMV was integrated into genome of transgenic tomato plants. The result showed transgenic tomato plants are resistant to ToMV when infected with ToMV
Through the experimmt we got such result:(1) After 4d pre-culture,3d co-culture, and 12weeks of selective culture with 10～30～50 mg/L kanamycin, in the first ten days concentrations of kanamycin in bud induction medium were 10mg/L, in the second ten days concentrations of kanamycin in bud induction medium were 30mg/L, and from then on concentrations of kanamycin were 50mg/L. 0.5mg/LZT, 0.1mg/LIAA was added into bud induction medium, explants were placed under 23～24℃condition,the best transformation efficiencies could be obtained.(2) Transgenic plants are resistant to ToMV.Different fragments of ToMV were selected to contruct RNAi vector.The result showed fragments of ToMV has influences on resistance to ToMV
Through the experiment we found that the best transformation efficiencies could be obtained by improving conditions.Genetic engineering technology open up new ways to plant antiviral breeding. It is possible to cultivate processing tomato resistant to ToMV using RNAi.
引文
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[2]李生, 加工番茄病虫害防治技术植物保护.农村科技[J]2010(1)
[3]齐士发,石书兵,俞钧山新疆加工番茄产业现状及障碍因子对策.经济作物[J]2007,(1)
[4]Meshi T, Ohno T, Okada Y (1982) Nucleotide sequence and its character of cistron coding for the 30 K protein of tobacco mosaic virus (OM strain). J Biochem 91:1441-1444
[9]Gabriel DW, Rolfe BG. Working models of specific recognitionin plant-microbe interaction Annu Rev Phytopathology,1990,28:365-391
[10]姜国勇,杨仁崔,番茄花叶病毒移动蛋白和番茄抗病基因J.微生物学报2005 45(2)301-304
[11]马庆虎,宋艳茹,多聚乳半糖醛酸酶反义基因在转基番茄中的表达J.生物多样性1995 3：(1)21-25.
[12]李晓东,刘敬梅, 陈杭,甜蛋白基因对番茄的遗传转化J.。西北植物学报.2004,24(5)：808-811
[13]于源华,何秀霞,郭中满,乙肝表面抗原小蛋白基因导入番茄的遗传转化体系的建立东北师大学报J.2003.35：(4)72-75
[14]郭兴启,李菡,张杰道,竺晓平表达马铃薯,病毒外壳蛋白基因的转基因烟草抗病机制.应用与环境生物学报[J]2003.9(4)：372-376
[15]李盼,梁慧媛.外壳蛋白介导的病毒抗性.生物学通报[J]61-62
[16]项瑜,杨兰英,周雪平等.表达马铃薯Y病毒外壳蛋白的转基因烟草的抗病性研究[J].病毒学报,1995,11(2)：158-162
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[18]徐香玲,李兴华,刘伟华,李集临,王毅.Ri质粒介导大豆花叶病毒外壳蛋白基因转化大豆的研究[J].大豆科学,1996,(04)
[19]朱俊华,朱常香,温孚江,宋云枝.正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究[J].植物病理学报,2004,(02)
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