根癌农杆菌介导万寿菊遗传转化体系的建立

doi:10.11983/CBB22141

植物学报 ›› 2023, Vol. 58 ›› Issue (5): 760-769.DOI: 10.11983/CBB22141

根癌农杆菌介导万寿菊遗传转化体系的建立

余晓敏¹, 王亚琴¹, 刘雨菡¹, 易庆平², 程文翰², 朱钰¹, 段枫¹, 张莉雪¹, 何燕红¹^,²^,^*()

1.华中农业大学园艺林学学院, 果蔬园艺作物种质创新与利用全国重点实验室, 武汉 430070
2.荆楚理工学院, 特色花卉生物育种湖北省工程研究中心, 荆门 448000

收稿日期:2022-07-02 接受日期:2022-12-02 出版日期:2023-09-01 发布日期:2023-09-21
通讯作者: *E-mail: hyh2010@mail.hzau.edu.cn
基金资助:
国家自然科学基金(32172616);特色花卉生物育种湖北省工程研究中心开放课题(2022ZD007)

Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System of Marigold (Tagetes erecta)

Yu Xiaomin¹, Wang Yaqin¹, Liu Yuhan¹, Yi Qingping², Cheng Wenhan², Zhu Yu¹, Duan Feng¹, Zhang Lixue¹, He Yanhong¹^,²^,^*()

1. National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China
2. Hubei Engineering Research Center for Specialty Flowers Biological Breeding, Jingchu University of Technology, Jingmen 448000, China

Received:2022-07-02 Accepted:2022-12-02 Online:2023-09-01 Published:2023-09-21
Contact: *E-mail: hyh2010@mail.hzau.edu.cn

摘要/Abstract

摘要： 以万寿菊(Tagetes erecta)里程碑·黄色的小叶为外植体, 采用农杆菌介导法探究抗生素浓度、菌株类型、菌液浓度、侵染时间、共培养时间、乙酰丁香酮浓度和抗褐化剂种类对万寿菊遗传转化效率的影响。结果表明, 头孢霉素(Cef)和硫酸卡那霉素(Kan)的适宜浓度分别为100 mg·L^-1和10 mg·L^-1; EHA105菌株的稳定转化效率最高; 菌液浓度OD₆₀₀=0.1、侵染5分钟及共培养1天为最佳侵染条件。此外, 在侵染过程中添加100 µmol·L^-1乙酰丁香酮(AS)和筛选培养基中添加0.2 g·L^-1聚乙烯吡咯烷酮(PVP)均能提高出芽率。经GUS染色、PCR检测以及Southern blot检测证明转化成功, 转化效率达到4%左右。研究结果为万寿菊基因功能研究和转基因育种奠定了基础。

关键词: 根癌农杆菌, 遗传转化, 小叶, 万寿菊

Abstract: In this study, we used the leaflets of marigold (Tagetes erecta) Milestone Yellow as explants to investigate the major factors impacting the transformation efficiency of Agrobacterium-mediated method. The factors included antibiotic concentration, strain type, bacterial concentration, infection time, co-culture time, acetosyringone concentration and anti-browning agent type. We found that the suitable concentrations of cefotaxim sodium salt and kanamycin sulfate were 100 mg·L^-1 and 10 mg·L^-1, respectively. We also found that the strain EHA105 had the highest transformation efficiency up to 4%. The best infection conditions for EHA105 were at bacterial concentration of 0.1 for OD₆₀₀, infected for 5 minutes, and co-cultured for 1 day. In addition, the bud germination rate could be improved by both applying 100 μmol·L^-1acetosyringone during the infection process and adding 0.2 g·L^-1polyvinyl pyrrolidone in the screening medium. This study laid a foundation for marigold gene function research and transgenic breeding.

Key words: Agrobacterium tumefaciens, genetic transformation, leaflet, marigold

余晓敏, 王亚琴, 刘雨菡, 易庆平, 程文翰, 朱钰, 段枫, 张莉雪, 何燕红. 根癌农杆菌介导万寿菊遗传转化体系的建立. 植物学报, 2023, 58(5): 760-769.

Yu Xiaomin, Wang Yaqin, Liu Yuhan, Yi Qingping, Cheng Wenhan, Zhu Yu, Duan Feng, Zhang Lixue, He Yanhong. Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System of Marigold (Tagetes erecta). Chinese Bulletin of Botany, 2023, 58(5): 760-769.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22141

https://www.chinbullbotany.com/CN/Y2023/V58/I5/760

图/表 11

表1 万寿菊里程碑·黄色遗传转化培养基

Table 1 Medium used for genetic transformation of marigold Milestone Yellow

Culture medium types	Formula
MS	4.405 g·L^-1 MS+30 g·L^-1 sucrose+8 g·L^-1 agar
Regeneration medium	4.405 g·L^-1 MS+40 g·L^-1 sucrose+8 g·L^-1 agar+0.2 mg·L^-1 TDZ+0.5 mg·L^-1 IBA
Co-culture medium	4.405 g·L^-1 MS+40 g·L^-1 sucrose+8 g·L^-1 agar+0.2 mg·L^-1 TDZ+0.5 mg·L^-1 IBA+0.5 g·L^-1 MES+ 100 μmol·L^-1 AS
Screening medium	4.405 g·L^-1 MS+40 g·L^-1 sucrose+8 g·L^-1 agar+0.2 mg·L^-1 TDZ+0.5 mg·L^-1 IBA+0.5 g·L^-1 MES+10 mg·L^-1 Kan+100 mg·L^-1 Cef
Elongation medium/ rooting medium	4.405 g·L^-1 MS+30 g·L^-1 sucrose+8 g·L^-1 agar+0.5 g·L^-1 MES+10 mg·L^-1 Kan+100 mg·L^-1 Cef

表2 不同浓度Kan对万寿菊小叶再生的影响

Table 2 Effect of Kan concentration on leaflet regeneration of marigold

Kan concentration (mg·L^-1)	Callus induction rate (%)	Regeneration rate (%)
0	100.00±0.00 a	72.50±5.00 a
2	100.00±0.00 a	67.50±12.58 a
4	100.00±0.00 a	50.00±14.14 b
6	97.50±5.00 a	15.00±10.00 c
8	95.00±5.77 ab	7.50±5.00 d
10	92.50±5.00 b	0.00±0.00 e
15	65.00±5.77 c	0.00±0.00 e
20	37.50±12.58 d	0.00±0.00 e
30	27.50±5.00 d	0.00±0.00 e
40	15.00±5.77 e	0.00±0.00 e
50	0.00±0.00 f	0.00±0.00 e

表3 不同浓度Cef对万寿菊小叶再生和菌落生长的影响

Table 3 Effect of Cef concentration on leaflet regeneration of marigold and bacteria growth

Cef concentration (mg·L^-1)	Callus induction rate (%)	Regeneration rate (%)	Growth of bacteria
0	100.00±0.00 a	60.00±8.16 a	Grow well
100	97.50±5.00 a	47.50±12.58 ab	Completely inhibited
200	97.50±5.00 a	40.00±8.16 abc	Completely inhibited
300	65.00±5.77 b	35.00±12.91 bc	Completely inhibited
400	37.50±12.58 c	37.50±17.08 bc	Completely inhibited
500	27.50±5.00 c	27.50±12.58 c	Completely inhibited

图1 菌株类型对万寿菊遗传转化效率的影响 (A) 3种菌株的GUS基因瞬时表达和抗性愈伤组织示意图(bars=1 cm); (B) 菌株类型对GUS基因瞬时表达效率及抗性愈伤组织诱导效率的影响(相同颜色柱形图上不同小写字母表示在0.05水平差异显著)

Figure 1 Effects of strain types on genetic transformation efficiency of marigold (A) Schematic diagram of transient expression of GUS gene and resistant callus in three strains (bars=1 cm); (B) Effect of strain types on transient expression efficiency of GUS gene and induction rate of resistant calli (different lowercase letters on the same color histogram indicate significant differences at 0.05 level)

表4 不同侵染条件对万寿菊转化效率的影响

Table 4 Effects of different infection conditions on transformation efficiency of marigold

No.	OD₆₀₀	Infection time (d)	Co-culture time (d)	Transient expression rate of GUS gene (%)	Callus induction rate (%)	Resistance bud rate (%)	PCR positive rate (%)
1	0.1	5	1	100	90.67±2.31 abcd	45.33	4.00
2	0.1	10	2	100	91.85±7.83 abcd	27.55	0.00
3	0.1	20	3	100	77.11±6.99 e	20.25	0.00
4	0.1	30	4	100	86.20±11.95 cde	0.00	0.00
5	0.5	5	2	100	96.67±2.88 abc	28.33	0.00
6	0.5	10	1	100	86.67±0.00 bcde	22.22	3.33
7	0.5	20	4	100	93.62±6.45 abc	3.16	0.00
8	0.5	30	3	100	96.30±3.21 abc	0.93	0.00
9	1	5	3	100	97.10±2.51 ab	23.53	0.00
10	1	10	4	100	91.61±4.93 abcd	13.51	0.00
11	1	20	1	100	81.25±6.25 de	28.57	0.00
12	1	30	2	100	86.03±1.27 cde	9.00	0.00
13	2	5	4	100	95.45±7.87 abc	17.65	1.96
14	2	10	3	100	100.00±0.00 a	5.71	0.00
15	2	20	2	100	86.18±3.66 cde	12.64	0.00
16	2	30	1	100	86.11±4.34 cde	0.00	0.00

表5 不同侵染条件下PCR阳性率的极差分析

Table 5 Range analysis of PCR positive rate under different infection conditions

Factor	OD₆₀₀	Infection time (d)	Co-culture time (d)
K₁	4.00	5.96	7.33
K₂	3.33	3.33	0.00
K₃	0.00	0.00	0.00
K₄	1.96	0.00	1.96
K₁	1.00	1.49	1.83
K₂	0.83	0.83	0.00
K₃	0.00	0.00	0.00
K₄	0.49	0.00	0.49
R	1.00	1.49	1.83

图2 乙酰丁香酮(AS)对万寿菊转化效率的影响相同颜色柱形图上不同小写字母表示在0.05水平差异显著。

Figure 2 Effect of acetosyringone (AS) on transformation efficiency of marigold Different lowercase letters on the same color histogram indicate significant differences at 0.05 level.

表6 柠檬酸(CA)和聚乙烯吡咯烷酮(PVP)对万寿菊转化效率的影响

Table 6 Effects of citric acid (CA) and polyvinyl pyrrolidone (PVP) on transformation efficiency of marigold

Anti-browning agent and concentration	No. of resistant buds	Resistance buds rate (%)	No. of positive seedlings	PCR positive rate (%)	Description of budding state
CK	4	4.00	0	0.00	Grow well
0.2 g·L^-1CA	6	6.45	0	0.00	Hard texture, more clustered buds, basically no main stem
0.4 g·L^-1CA	9	9.00	0	0.00	Hard texture, more clustered buds, basically no main stem
0.6 g·L^-1CA	12	12.00	0	0.00	Hard texture, more clustered buds, basically no main stem
0.2 g·L^-1PVP	12	12.00	4	4.00	Grow well
0.4 g·L^-1PVP	7	7.00	0	0.00	Grow well
0.6 g·L^-1PVP	7	7.37	1	1.11	Grow well

图3 万寿菊遗传转化流程图 (A) 万寿菊复叶; (B) 小叶培养; (C) 小叶诱导形成愈伤组织; (D) 瞬时转化叶片GUS染色; (E) 抗性芽; (F) 抗性苗; (G) 抗性苗生根培养; (H) 阳性苗; (I) 阳性苗移栽。(A)-(D), (G)-(I) Bars=1 cm; (E), (F) Bars=0.5 cm

Figure 3 Genetic transformation process diagram of marigold (A) Marigold compound leaves; (B) Leaflet culture; (C) Leaflet-induced calli; (D) GUS staining of transiently transformed leaves; (E) Resistance buds; (F) Resistant seedlings; (G) Rooting culture of resistant seedlings; (H) Positive seedlings; (I) Transplantation of positive seedlings. (A)-(D), (G)-(I) Bars=1 cm; (E), (F) Bars=0.5 cm

图4 万寿菊阳性植株不同器官的GUS染色效果 ars=2 mm

Figure 4 GUS staining effect on different organs of marigold positive plants Bars=2 mm

图5 万寿菊抗性苗PCR检测及Southern blot验证 (A) 抗性苗PCR扩增检测(M: 2000 DNA分子量标准; 1: ddH2O; 2: 质粒; 3, 4: 阴性对照, 即未侵染的叶片DNA; 5-15: 抗性苗DNA); (B) 阳性苗Southern blot检测(用HindIII酶切, 以NPT-II为探针。M: DNA分子量标准; 1: 阴性对照, 即未侵染的叶片DNA; 2: 阳性对照, 即质粒; 3-5: 阳性苗DNA)。

Figure 5 PCR amplification detection and Southern blot results of marigold resistant seedlings (A) PCR amplification detection of resistant seedlings (M: 2000 DNA marker; 1: ddH2O; 2: Plasmid; 3, 4: Negative control, DNA of uninfected leaf; 5-15: DNA of resistant seedlings); (B) Southern blot results of positive seedlings (the NPT-II probe was digested with HindIII. M: DNA marker; 1: Negative control, DNA of uninfected leaf; 2: Positive control, plasmid; 3-5: DNA of positive seedlings).

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根癌农杆菌介导万寿菊遗传转化体系的建立

Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System of Marigold (Tagetes erecta)

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