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花青素合成调节基因B1/C1转化东方百合‘索邦’的研究

崔祺,  杜运鹏,  魏,  迟,  高雪,  贾桂霞

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文章导航 > 北京林业大学学报  > 2015 >  37(11): 100-108
崔祺, 杜运鹏, 魏, 迟, 高雪, 贾桂霞. 花青素合成调节基因B1/C1转化东方百合‘索邦’的研究[J]. 北京林业大学学报, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
引用本文: 崔祺, 杜运鹏, 魏, 迟, 高雪, 贾桂霞. 花青素合成调节基因B1/C1转化东方百合‘索邦’的研究[J]. 北京林业大学学报, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
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CUI Qi, DU Yun-peng, WEI Chi, GAO Xue, JIA Gui-xia, .. Transformation of Lilium ‘Sorbonne’ (oriental hybrids) with anthocyanin regulatory gene B1/C1.[J]. Journal of Beijing Forestry University, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
Citation: CUI Qi, DU Yun-peng, WEI Chi, GAO Xue, JIA Gui-xia, .. Transformation of Lilium ‘Sorbonne’ (oriental hybrids) with anthocyanin regulatory gene B1/C1.[J]. Journal of Beijing Forestry University, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
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花青素合成调节基因B1/C1转化东方百合‘索邦’的研究

doi: 10.13332/j.1000-1522.20140414
  • 1.

    1 花卉种质创新与分子育种北京市重点实验室,国家花卉工程技术研究中心,北京林业大学园林学院

  • 2.

    2 北京市农业生物技术研究中心,北京市农林科学院

基金项目: 

“863”国家高技术研究发展计划项目(2013AA102706)。

详细信息
    作者简介:

    崔祺,博士生。主要研究方向: 园林植物资源与育种。Email: cuiqivivi@163.com 地址:100083北京市海淀区清华东路35号北京林业大学园林学院。
    责任作者: 贾桂霞,教授,博士生导师。主要研究方向: 园林植物资源与育种。Email: gxjia@bjfu.edu.cn 地址: 同上。

    崔祺,博士生。主要研究方向: 园林植物资源与育种。Email: cuiqivivi@163.com 地址:100083北京市海淀区清华东路35号北京林业大学园林学院。
    责任作者: 贾桂霞,教授,博士生导师。主要研究方向: 园林植物资源与育种。Email: gxjia@bjfu.edu.cn 地址: 同上。

Transformation of Lilium ‘Sorbonne’ (oriental hybrids) with anthocyanin regulatory gene B1/C1.

  • 1.

    1 Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, College of Landscape Architecture, Beijing Forestry University, Beijing, 100083, P. R. China;

  • 2.

    2 Beijing Research Center for Agricultural Biotechnology, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, P. R. China.

  • 摘要: 百合是全球著名的切花商品花卉,较低的遗传转化效率限制了百合转基因育种的发展,建立高效、稳定的遗传转化体系对于培育转基因新品种至关重要。外源花青素合成调节基因的表达可使花青素在植物细胞内积累, 使植物体外观上表现出色彩的变化,易于观察, 因此可作为报告基因用于植物转基因研究,快速报告细胞、组织、器官或植株是否被转化。本文以东方百合‘索邦’无菌苗鳞片为受体材料,利用农杆菌介导法将花青素合成调节基因B1/C1导入‘索邦’中。通过草甘膦敏感性试验,确定了草甘膦筛选的质量浓度为2.1 mg/L,对农杆菌介导的遗传转化体系进行了优化,研究了侵染液及共培养基成分对抗性芽诱导率的影响,以MS培养基为基本培养基,设置了7种类型改良MS培养基。结果表明:当MS培养基中去除大量元素时,抗性芽获得率相对较高,可达(18.31±1.71)%。在此条件下,将培养基中30 g/L的蔗糖替换成70 g/L的麦芽糖,可使抗性苗诱导率增至(22.27±3.48)%。热激和超声波结合使用能够明显提高抗性苗的获得率,其中以42 ℃热激1.5 min、120 W超声波超声20 s抗性苗获得率最高,可达(26.80±2.24)%。抗性植株经PCR和Southern检测,获得了1株单拷贝转基因植株,初步证明B1/C1基因已整合到‘索邦’基因组DNA中,并且转基因植株叶片、叶柄、鳞茎的花青素含量均高于非转基因植株,说明花青素合成调节基因B1/C1在转基因百合中获得了表达。
    Abstract: The Lilium is the one of world’s leading cut flowers and commodity flowers, but low transformation efficiencies make it difficult to reproduce genetically modified cultivars, which limits the development of breeding. Thus, the establishment of a rapid, efficient and reproducible transformation protocol for Lilium is very important. Expression of the metabolic regulation gene causes the accumulation of anthocyanins in plant cells, resulting in varied colors of plant organs easy to observe. As a visual reporter, the metabolic regulation gene makes the transformed plants easily distinguishable even only by their color appearance at the early stage. In this study, the Agrobacterium-mediated genetic transformation system of Lilium ‘Sorbonne’ (oriental hybrids) was optimized by using the bulblet scales as receptor materials and the anthocyanin regulatory gene B1/C1 as the target gene. The resistance of plantlets from ‘Sorbonne’ to glyphosate was studied for culture selection, and the optimum concentration was 2.1 mg/L. Seven different types of modified Murashige and Skoog (MS) media in both inoculation and co-cultivation media were evaluated, and a dramatic increase in the rate of resistant plantlets 18.31±1.71% was observed when MS medium was used after removing macro elements. Under this condition, using the inoculation and co-cultivation media containing 6% maltose (instead of 3% sucrose) could improve the rate of resistant plantlets to 22.27±3.48%. A 42 ℃ heat shock treatment for 1.5 min followed by the ultrasonic power 120 W for 20 s brought the highest rate of resistant plantlets 26.80±2.24%. Some resistant plantlets transformed with B1/C1 by Agrobacterium-mediation were obtained after the preliminary screening. PCR screening and Southern blot results identified one single copy positive Gly-resistant plantlet. Analysis of PCR, Southern blot and anthocyanin content showed that exogenous genes were well integrated in the genome of Lilium ‘Sorbonne’ (oriental hybrids) and expressed in leaves, petiole and bulb.
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  • 收稿日期:  2014-11-14
  • 刊出日期:  2015-11-30

花青素合成调节基因B1/C1转化东方百合‘索邦’的研究

doi: 10.13332/j.1000-1522.20140414
  • 1. 1 花卉种质创新与分子育种北京市重点实验室,国家花卉工程技术研究中心,北京林业大学园林学院
  • 2. 2 北京市农业生物技术研究中心,北京市农林科学院
    • 基金项目:

    “863”国家高技术研究发展计划项目(2013AA102706)。

    作者简介:

    崔祺,博士生。主要研究方向: 园林植物资源与育种。Email: cuiqivivi@163.com 地址:100083北京市海淀区清华东路35号北京林业大学园林学院。
    责任作者: 贾桂霞,教授,博士生导师。主要研究方向: 园林植物资源与育种。Email: gxjia@bjfu.edu.cn 地址: 同上。

    崔祺,博士生。主要研究方向: 园林植物资源与育种。Email: cuiqivivi@163.com 地址:100083北京市海淀区清华东路35号北京林业大学园林学院。
    责任作者: 贾桂霞,教授,博士生导师。主要研究方向: 园林植物资源与育种。Email: gxjia@bjfu.edu.cn 地址: 同上。

  • 收稿日期: 2014-11-14
  • 刊出日期: 2015-11-30

摘要: 百合是全球著名的切花商品花卉,较低的遗传转化效率限制了百合转基因育种的发展,建立高效、稳定的遗传转化体系对于培育转基因新品种至关重要。外源花青素合成调节基因的表达可使花青素在植物细胞内积累, 使植物体外观上表现出色彩的变化,易于观察, 因此可作为报告基因用于植物转基因研究,快速报告细胞、组织、器官或植株是否被转化。本文以东方百合‘索邦’无菌苗鳞片为受体材料,利用农杆菌介导法将花青素合成调节基因B1/C1导入‘索邦’中。通过草甘膦敏感性试验,确定了草甘膦筛选的质量浓度为2.1 mg/L,对农杆菌介导的遗传转化体系进行了优化,研究了侵染液及共培养基成分对抗性芽诱导率的影响,以MS培养基为基本培养基,设置了7种类型改良MS培养基。结果表明:当MS培养基中去除大量元素时,抗性芽获得率相对较高,可达(18.31±1.71)%。在此条件下,将培养基中30 g/L的蔗糖替换成70 g/L的麦芽糖,可使抗性苗诱导率增至(22.27±3.48)%。热激和超声波结合使用能够明显提高抗性苗的获得率,其中以42 ℃热激1.5 min、120 W超声波超声20 s抗性苗获得率最高,可达(26.80±2.24)%。抗性植株经PCR和Southern检测,获得了1株单拷贝转基因植株,初步证明B1/C1基因已整合到‘索邦’基因组DNA中,并且转基因植株叶片、叶柄、鳞茎的花青素含量均高于非转基因植株,说明花青素合成调节基因B1/C1在转基因百合中获得了表达。

English Abstract

崔祺, 杜运鹏, 魏, 迟, 高雪, 贾桂霞. 花青素合成调节基因B1/C1转化东方百合‘索邦’的研究[J]. 北京林业大学学报, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
引用本文: 崔祺, 杜运鹏, 魏, 迟, 高雪, 贾桂霞. 花青素合成调节基因B1/C1转化东方百合‘索邦’的研究[J]. 北京林业大学学报, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
shu
CUI Qi, DU Yun-peng, WEI Chi, GAO Xue, JIA Gui-xia, .. Transformation of Lilium ‘Sorbonne’ (oriental hybrids) with anthocyanin regulatory gene B1/C1.[J]. Journal of Beijing Forestry University, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
Citation: CUI Qi, DU Yun-peng, WEI Chi, GAO Xue, JIA Gui-xia, .. Transformation of Lilium ‘Sorbonne’ (oriental hybrids) with anthocyanin regulatory gene B1/C1.[J]. Journal of Beijing Forestry University, 2015, 37(11): 100-108. doi: 10.13332/j.1000-1522.20140414
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