发根农杆菌介导的野葛毛状根遗传转化体系

doi:10.11983/CBB24092

植物学报 ›› 2025, Vol. 60 ›› Issue (3): 425-434.DOI: 10.11983/CBB24092 cstr: 32102.14.CBB24092

发根农杆菌介导的野葛毛状根遗传转化体系

曾文丹¹, 严华兵¹^,^*(), 吴正丹², 尚小红¹, 曹升¹, 陆柳英¹, 肖亮¹, 施平丽¹, 程冬¹, 龙紫媛¹, 李婕宇¹

¹广西壮族自治区农业科学院经济作物研究所, 南宁 530007
²广西大学, 南宁 530004

收稿日期:2024-06-11 接受日期:2024-12-26 出版日期:2025-05-10 发布日期:2024-12-27
通讯作者: *严华兵, 男, 1979年5月生, 博士。现任广西农业科学院经济作物研究所所长, 兼任广西薯类创新团队首席专家、国家食药同源产业科技创新联盟葛根专委会主任委员, 荣获第八批广西“十百千”知识产权领军人才。其科研团队主要从事木薯、葛根等特色薯类植物资源收集、生物技术育种、良种繁育、高效栽培以及资源开发利用等研究工作。先后主持IAEA国际合作项目、国家重点研发计划子课题、国家自然科学基金、自治区主席科技资金等农业科研项目20余项。科研成果获得广西科技进步二等奖1项、广西科技进步三等奖2项。带领团队育成“桂木薯6号”等木薯新品种5个。以第一完成人获得国家发明专利10余项。以第一作者或通讯作者在Horticulture Research、DNA Research和植物生理学报等专业刊物上发表论文90余篇, 其中国际刊物20余篇。主编专著2部, 作为副主编合编著作2部。E-mail: h.b.yan@hotmail.com
基金资助:
广西自然科学基金(2023GXNSFBA026297);广西科技重大专项(桂科AA23023035);广西科技计划(桂科AB22080090);广西薯类创新团队(nycytxgxcxtd-2023-11);科技先锋队(桂农科盟202414)

Agrobacterium rhizogenes-mediated Transformation System of Pueraria lobata Hairy Roots

Zeng Wendan¹, Yan Huabing¹^,^*(), Wu Zhengdan², Shang Xiaohong¹, Cao Sheng¹, Lu Liuying¹, Xiao Liang¹, Shi Pingli¹, Cheng Dong¹, Long Ziyuan¹, Li Jieyu¹

¹Cash Crops Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
²Guangxi University, Nanning 530004, China

Received:2024-06-11 Accepted:2024-12-26 Online:2025-05-10 Published:2024-12-27
Contact: *E-mail: h.b.yan@hotmail.com

摘要/Abstract

摘要： 为建立高效的野葛(Pueraria lobata)毛状根遗传转化体系, 以野葛组培苗为外植体, 探讨不同因素对野葛毛状根遗传转化效率的影响。结果表明, 基因型是建立野葛毛状根高效遗传转化体系的主要限制因子; 发根农杆菌K599为最适宜的菌株; 以培养5-13代组培苗继代培养8天、第1-2节位刚展开的幼嫩叶片为最佳外植体材料, 预培养3天, 菌液侵染15分钟, 毛状根诱导率最高, 可达22.4%。野葛毛状根继代增殖的最佳培养基类型为固体培养基, 其毛状根鲜重是液体培养基中毛状根鲜重的75倍; PCR检测和荧光显微观察结果显示, GFP及rolB基因在野葛毛状根基因组中稳定表达, 共转化率为80%。研究初步建立了发根农杆菌介导的野葛毛状根遗传转化体系, 旨在为野葛基因功能研究奠定基础。

关键词: 野葛, 发根农杆菌, 毛状根, 遗传转化

Abstract: INTRODUCTION:An efficient Agrobacterium rhizogenes-mediated transformation system for Pueraria lobata was established. RATIONALE: In this study, tissue-cultured plantlets of P. lobata were used as explants to investigate the effects of different genotypes, A. rhizogenes strains, explants, precultivation times, infection times, culture days, subculture times, and culture methods on the efficiency of hairy root genetic transformation in P. lobata. RESULTS: The results indicated that the induction rate of hairy root formation was the highest when the immature leaves of YG-19 were used as the explant material, reaching 10.2%. A. rhizogenes K599 was identified as the most suitable strain. The optimal explant material was immature leaves that had just unfolded from the first to second nodes of the 5^th to 13^th generation tissue culture plantlets subcultured for 8 days. After 3 days of pre-culture and 15 minutes of bacterial infection, the highest induction rate of hairy roots reached 22.4%. The optimal type of culture medium for the proliferation of hairy roots in P. lobatawas solid medium culture, and the fresh weight of hairy roots grown on solid medium was 75 times greater than that of hairy roots grown in liquid medium. PCR detection and fluorescence microscopy assays revealed that the expression of GFP and rolB genes in the hairy roots of P. lobata was stable, and the rate of cotransformation was 80%.CONCLUSION: Genotype, A. rhizogenes strain, and culture duration were the most critical factors for the efficient genetic transformation of hairy roots in P. lobata.

Key words: Pueraria lobata, Agrobacterium rhizogenes, hairy roots, genetic transformation

曾文丹, 严华兵, 吴正丹, 尚小红, 曹升, 陆柳英, 肖亮, 施平丽, 程冬, 龙紫媛, 李婕宇. 发根农杆菌介导的野葛毛状根遗传转化体系. 植物学报, 2025, 60(3): 425-434.

Zeng Wendan, Yan Huabing, Wu Zhengdan, Shang Xiaohong, Cao Sheng, Lu Liuying, Xiao Liang, Shi Pingli, Cheng Dong, Long Ziyuan, Li Jieyu. Agrobacterium rhizogenes-mediated Transformation System of Pueraria lobata Hairy Roots. Chinese Bulletin of Botany, 2025, 60(3): 425-434.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24092

https://www.chinbullbotany.com/CN/Y2025/V60/I3/425

图/表 12

图1 Ti质粒GFP载体信息

Figure 1 GFP vector information for the Ti plasmid

表1 引物名称及序列

Table 1 Primer names and sequences

Primer name	Primer sequence (5′−3′)
GFP	F: CAGTGCTTCAGCCGCTAC R: TTCTCGTTGGGGTCTTTG
rolB	F: AAGTGCTGAGGAACAATC R: CAAGTGAATGAACAAGGAAC
virG	F: CCTTGGGCGTCGTCATAC R: TCGTCCTCGGTCGTTTCC

表2 不同基因型及不同外植体对野葛毛状根诱导效率的影响

Table 2 Effects of different genotypes and different explants on the induction efficiency of Pueraria lobata hairy roots

Genotype	Hairy root frequency of different explants (%)
Genotype	Unfolding im- mature leaves	Mature leaves	Stems	Petioles	Shoot me- ristems
YG-18	0	0	0	0	0
YG-19	10.2±0.9	6.1±0.1	0	5.2±1.2	0
YG-50	0	0	0	0	0
YG-51	1.9±0.6	0	0	3.5±0.4	0
YG-81	2.1±0.3	1.1±0.4	0	1.9±0.1	0

图2 野葛毛状根诱导及继代培养 (A) 幼嫩叶片诱导产生的毛状根; (B) 叶柄诱导产生的毛状根; (C), (D) 继代培养的毛状根。Bars=1 cm

Figure 2 Induction and subculture of Pueraria lobata hairy roots (A) Hairy roots from immature leaf explants; (B) Hairy roots from petiole explants; (C), (D) Subcultured hairy roots. Bars=1 cm

表3 不同发根农杆菌对野葛毛状根诱导效率的影响

Table 3 Effects of different Agrobacterium rhizogenes strains on the induction efficiency in Pueraria lobata hairy roots

Agrobacterium rhizogenes	Rooting time (d)	Hairy root frequency (%)	Hairy root density
CK	-	0 c	0 c
K599	14	12.1±0.6 a	2.2±0.1 a
R1601	16	8.3±0.5 b	1.1±0.1 b
ATCC15834	-	0 c	0 c
Ar4	-	0 c	0 c

表4 不同部位幼嫩叶片对野葛毛状根诱导效率的影响

Table 4 Effects of immature leaves from different nodes on the induction efficiency of Pueraria lobata hairy roots

Different nodes	Rooting time (d)	Hairy root frequency (%)	Hairy root density
Unfully expanded immature leaves	16	2.3±0.2 bc	1.0±0 b
Immature leaves from 1^st to 2^nd nodes	13	18.1±1.2 a	1.9±0.4 a
Immature leaves from 3^rd to 4^th nodes	14	10.7±0.3 b	1.7±0.2 a
Immature leaves from 5^th to 6^th nodes	16	5.5±0.5 c	1.2±0.2 b

表5 不同预培养时间对野葛毛状根诱导效率的影响

Table 5 Effects of preculture time on the induction efficiency of Pueraria lobata hairy roots

Precultivation time (d)	Rooting time (d)	Hairy root frequency (%)	Hairy root density
0	16	2.4±0.1 c	1.1±0.4 b
1	13	8.1±1.5 b	1.2±0.1 b
2	13	18.9±2.1 a	2.0±0.0 a
3	10	21.3±1.7 a	1.8±0.4 a
4	14	11.1±0.9 b	1.0±0.2 b

表6 不同侵染时间对野葛毛状根诱导效率的影响

Table 6 Effects of infection time on the induction efficiency of Pueraria lobata hairy roots

Infection time (min)	Rooting time (d)	Hairy root frequency (%)	Hairy root density
15	10	20.9±2.7 a	1.7±0.6 a
20	11	19.7±3.3 a	1.8±0.2 a
25	13	14.3±2.1 a	1.0±0 b
30	16	9.1±1.4 b	1.0±0 b

表7 不同培养天数对野葛毛状根诱导效率的影响

Table 7 Effects of culture days on the induction efficiency of Pueraria lobata hairy roots

Culture days (d)	Rooting time (d)	Hairy root frequency (%)	Hairy root density
5	11	14.7±4.5 bc	2.0±0 a
8	10	22.4±3.7 a	1.9±0.5 a
11	11	16.4±3.5 b	1.6±0.3 ab
14	13	12.0±2.5 c	1.0±0 b
17	14	6.1±3.1 d	1.0±0 b
20	-	0 e	-
23	-	0 e	-
26	-	0 e	-
29	-	0 e	-

表8 不同继代次数对野葛毛状根诱导效率的影响

Table 8 Effects of subculture time on the induction efficien- cy of Pueraria lobata hairy roots

Subculture times (t)	Rooting time (d)	Hairy root frequency (%)	Hairy root density
1	9	2.2±0.8 d	1.0±0 b
3	10	11.1±2.3 b	1.0±0 b
5	9	18.9±1.7 a	1.9±0.2 a
7	10	16.7±2.6 a	2.1±0.1 a
9	10	17.0±1.1 a	2.0±0 a
11	11	15.6±3.7 a	1.8±0.2 a
13	11	14.1±2.1 ab	1.8±0.4 a
15	12	11.2±0.4 b	1.4±0.2 ab
17	13	6.7±2.2 c	1.1±0.3 b
19	13	4.3±1.9 cd	1.0±0 b
21	-	0 e	-
23	-	0 e	-
25	-	0 e	-

图3 野葛毛状根的继代增殖 (A) 不同培养方式下野葛毛状根的鲜重; (B) 接种于固体培养基20天的毛状根; (C) 接种于液体培养基20天的毛状根。Bars=1 cm

Figure 3 Subculture proliferation of Pueraria lobata hairy roots (A) Fresh weight of P. lobata hairy roots under different cultivation methods; (B) Morphological characterization of P. lobata hairy roots grown on solid media 20 d after inoculation; (C) Morphological characterization of P. lobata hairy roots grown in liquid media 20 d after inoculation. Bars=1 cm

图4 野葛转基因毛状根GFP荧光观察及PCR检测 (A) 毛状根GFP荧光观察(bar=1 cm); (B) PCR检测(M: DL2000 DNA marker; N: 超纯水; V: K599; P: 阳性对照; 1-20: 不同转基因毛状根株系)

Figure 4 Green fluorescence assay and PCR analysis of the GFP in Pueraria lobata transgenic hairy roots (A) GFP green fluorescence assay of hairy roots (bar=1 cm); (B) PCR detection (M: DL2000 DNA marker; N: ddH2O; V: K599; P: Positive control; 1-20: Different transgenic hairy root lines)

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发根农杆菌介导的野葛毛状根遗传转化体系

Agrobacterium rhizogenes-mediated Transformation System of Pueraria lobata Hairy Roots

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