发根农杆菌介导的菠菜毛状根遗传转化体系的建立

doi:10.11983/CBB18257

植物学报 ›› 2019, Vol. 54 ›› Issue (4): 515-521.DOI: 10.11983/CBB18257 cstr: 32102.14.CBB18257

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

徐悦¹,曹英萍²,王玉²,付春祥^2,^*(),戴绍军^1,^3,^*()

1 东北林业大学盐碱地生物资源环境研究中心, 东北盐碱植被恢复与重建教育部重点实验室, 哈尔滨 150040
2 中国科学院青岛生物能源与过程研究所, 中国科学院生物燃料重点实验室, 山东省能源生物遗传资源重点实验室, 山东省合成生物技术创新中心(筹), 青岛 266000
3 上海师范大学生命科学学院, 植物种质资源开发协同创新中心, 上海 200234;

收稿日期:2018-11-30 接受日期:2019-04-08 出版日期:2019-07-01 发布日期:2020-01-08
通讯作者: 付春祥,戴绍军
基金资助:
青岛市创业创新领军人才(16-8-3-15-zhc);中央高校基本科研业务费专项资金(2572017ET01);上海市科学技术委员会科研计划(17391900600)

Agrobacterium rhizogenes-mediated Transformation System of Spinacia oleracea

Yue Xu¹,Yingping Cao²,Yu Wang²,Chunxiang Fu^2,^*(),Shaojun Dai^1,^3,^*()

1 Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Harbin 150040, China
2 Shandong Technology Innovation Center of Synthetic Biology, Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266000, China
3 Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China

Received:2018-11-30 Accepted:2019-04-08 Online:2019-07-01 Published:2020-01-08
Contact: Chunxiang Fu,Shaojun Dai

摘要/Abstract

摘要： 发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。

关键词: 共转化, 毛状根, 植物再生体系, 菠菜

Abstract: The plant can produce hairy roots after infection by Agrobacterium rhizogenes harboring Ri plasmids. Spinach (Spinacia oleracea) is a common edible vegetable, and its hairy root system has yet to be reported. In this study, A. rhizogenes LBA9402 was screened to be suitable for hairy root induction of spinach. The highest induction efficiency, 16%, was reached when the spinach stem was infected by LBA9402. Hairy roots grew vigorously on SH solid medium without exogenous hormones. The hairy roots of spinach were white and had enormous root hairs. The regeneration plant obtained from the callus redifferentiated from spinach hair roots; the rate of regeneration was 8%. In addition, Ri T-DNA and Ti T-DNA carrying rolB genes and GFP gene, respectively, were co-transferred into cells of spinach and produced transgenic hairy roots. PCR analysis and fluorescence microscopy assays showed rolB gene and GFP gene in the hairy roots of spinach and expressed stably; the rate of co-transformation was 50%.

Key words: co-transformation, hairy root, plant regeneration system, Spinacia oleracea

徐悦,曹英萍,王玉,付春祥,戴绍军. 发根农杆菌介导的菠菜毛状根遗传转化体系的建立. 植物学报, 2019, 54(4): 515-521.

Yue Xu,Yingping Cao,Yu Wang,Chunxiang Fu,Shaojun Dai. Agrobacterium rhizogenes-mediated Transformation System of Spinacia oleracea. Chinese Bulletin of Botany, 2019, 54(4): 515-521.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18257

https://www.chinbullbotany.com/CN/Y2019/V54/I4/515

图/表 6

图1 pCAMBIA139IZ-eGFP (A)和Ri质粒(B)的T-DNA区示意图

Figure 1 Schematic map of T-DNAs of pCAMBIA139IZ-eGFP (A) and Ri plasmid (B)

图2 不同外植体和发根农杆菌菌种对菠菜毛状根诱导率的影响

Figure 2 Effects of different explants and Agrobacterium rhizogenes strains on the induction rate of spinach hairy roots

图3 菠菜毛状根诱导及其形态特点 (A) 发根农杆菌(LBA9402)侵染之后在1个位点产生多条毛状根; (B) 叶柄维管束部位产生的毛状根; (C) 菠菜叶脉部位产生的毛状根; (D) 茎的维管束部位产生的毛状根; (E) PCR分析毛状根中rolB (上)和VirG (下)基因(1: 2 kb DNA marker; 2-11: 不同毛状根再生植株; 12: LBA9402; 13: 非转化对照根; 14: 去离子水); (F) LBA9402诱导产生的毛状根; (G) 野生型根。(A)-(D), (F), (G) Bars=20 μm

Figure 3 The induction and morphological characteristics of spinach hairy roots (A) After Agrobacterium rhizogenes LBA9402 infection, multiple hairy roots were produced at one site; (B) The induction of hairy roots around the vascular bundle of the petiole; (C) The induction of hairy roots in veins; (D) The induction of hairy roots around the vascular bundle of stem; (E) Genomic PCR analysis of rolB (up) and virG (down) genes in hairy roots (Lane 1: 2 kb DNA marker; Lane 2-11: Different hairy roots plantlets; Lane 12: LBA9402; Lane 13: Untransformed roots; Lane 14: Mili Q water); (F) The hairy root induced by LBA9402; (G) The wild type root. (A)-(D), (F), (G) Bars=20 μm

图4 在液体和固体培养基上培养的菠菜毛状根的形态特征 (A) 接种于固体培养基0天的毛状根; (B), (F) 接种于固体培养基14天的毛状根; (C) 接种于液体培养基0天的毛状根; (D), (G) 接种于液体培养基14天的毛状根; (E) 毛状根鲜重在2种培养基中的增长倍数。(A)-(D) Bars=8 cm; (F), (G) Bars=20 μm

Figure 4 The morphological characterization of spinach hairy roots grown on solid medium and in liquid medium (A) Hairy roots grown on solid medium in 0 day after inoculation; (B), (F) Hairy roots grown on solid medium in 14 days after inoculation; (C) Hairy roots grown in liquid medium in 0 day after inoculation; (D), (G) Hairy roots grown in liquid medium in 14 days after inoculation; (E) Increase times of spinach hairy roots fresh weight on solid medium and in liquid medium, respectively. (A)-(D) Bars=8 cm; (F), (G) Bars=20 μm

图5 菠菜毛状根再生体系 (A) 毛状根在愈伤诱导培养基上产生愈伤组织; (B) 毛状根诱导的愈伤组织在分化培养基中生长并产生不定芽; (C) 毛状根经由愈伤组织诱导分化产生再生植株; (D) 野生型种子萌发的植株; (E) PCR检测rolB (上)和VirG (下)基因(1: 2 kb DNA marker; 2-4: 不同毛状根再生株系; 5: LBA9402; 6: 野生型; 7: 去离子水)。(A)-(D) Bars=4 cm

Figure 5 Regeneration system of spinach hairy roots (A) Calli produced from hairy roots on callus induction medium; (B) Calli induced from hairy roots and produced adventitious buds on the regeneration medium; (C) The plantlets regenerated from the calli produced from hairy roots; (D) The wild type plantlets; (E) Genomic PCR analysis of rolB (up) and virG (down) genes in the plantlets regenerated from the calli produced from hairy roots (Lane 1: 2 kb DNA marker; Lane 2-4: Different hairy roots plantlets regenerated from the calli produced; Lane 5: LBA9402; Lane 6: The wild type plantlets; Lane 7: Mili Q water). (A)-(D) Bars=4 cm

图6 菠菜毛状根rolB基因和GFP基因的PCR检测和荧光观察 (A) 在自然光下的转基因毛状根; (B) 在蓝色激发光下观察的转基因毛状根; (C) PCR检测转rolB (上)、VirG (中)和GFP (下)基因(1: 2 kb DNA marker; 2-11: 不同转基因毛状根株系; 12: LBA9402; 13: 野生型根; 14: 去离子水)。(A), (B) Bars=20 μm

Figure 6 PCR analysis and GFP green fluorescence assay of the rolB gene and GFP gene in spinach transgenic hairy roots (A) Transgenic hairy root under the natural light; (B) Transgenic hairy root under the blue exciting light; (C) Genomic PCR analysis of rolB (up) and virG (middle) and GFP (down) genes in transgenic hairy roots (Lane 1: 2 kb DNA marker; Lane 2-11: Different of transgenic hairy roots lines; Lane 12: LBA9402 (GFP); Lane 13: The wild type root; Lane 14: Milli Q water). (A), (B) Bars=20 μm

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发根农杆菌介导的菠菜毛状根遗传转化体系的建立

Agrobacterium rhizogenes-mediated Transformation System of Spinacia oleracea

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