发根农杆菌介导的芜菁高效遗传转化体系建立

doi:10.16420/j.issn.0513-353x.2025-0140

园艺学报 ›› 2025, Vol. 52 ›› Issue (5): 1389-1398.doi: 10.16420/j.issn.0513-353x.2025-0140

• 新技术与新方法 • 上一篇

发根农杆菌介导的芜菁高效遗传转化体系建立

马曦¹^,^*, 张金睿¹^,^*, 庄红梅², 陈巍³, 张蜜¹, 袁晓琪¹, 易小芳¹, 王聪聪¹, 王海云¹, 王燕¹^,^**()

¹ 南京农业大学园艺学院，作物遗传与种质创新利用全国重点实验室，农业农村部华东地区园艺作物生物学与种质创制重点实验室，南京 211800
² 新疆维吾尔自治区农业科学院果蔬研究所（国家果树种质资源轮台果树资源圃），新疆蔬菜工程技术研究中心，乌鲁木齐 830091
³ 江苏农林职业技术学院，江苏现代园艺工程技术中心，江苏镇江 212400

收稿日期:2025-02-22 修回日期:2025-04-28 出版日期:2025-05-23 发布日期:2025-05-21
通讯作者:
**E-mail：wangyanhs@njau.edu.cn
作者简介:
*共同第一作者
基金资助:
国家自然科学基金项目(31960600); 新疆维吾尔自治区重点研发计划项目(2022B02058); 新疆维吾尔自治区自然科学基金面上项目(2022D01A268); 新疆农业科学院青年科技骨干创新能力培养项目(xjnkq-2019003)

Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium rhizogenes in Turnip

MA Xi¹, ZHANG Jinrui¹, ZHUANG Hongmei², CHEN Wei³, ZHANG Mi¹, YUAN Xiaoqi¹, YI Xiaofang¹, WANG Congcong¹, WANG Haiyun¹, WANG Yan¹^,^**()

¹ State Key Laboratory of Crop Genetics and Germplasm Enhancement and Utilization，Key Laboratory of Horticultural Crop Biology and Genetic Improvement（East China），College of Horticulture，Nanjing Agricultural University，Nanjing 211800，China
² Xinjiang Vegetable Engineering Technology Research Center，Institute of Fruit and Vegetable Research，Xinjiang Uyghur Autonomous Region Academy of Agricultural Sciences（Luntai Fruit Tree Resource Garden of the National Fruit Tree Germplasm Resources），Urumqi 830091，China
³ Jiangsu Engineering and Technology Center for Modern Horticulture，Jiangsu Polytechnic College of Agriculture and Forestry，Zhenjiang，Jiangsu 212400，China

Received:2025-02-22 Revised:2025-04-28 Published:2025-05-23 Online:2025-05-21

摘要/Abstract

摘要：

芜菁（Brassica rapa var. rapa L.）存在再生困难、转化效率低等问题。以新疆柯坪白皮芜菁为试材，对外植体类型、菌液侵染浓度、侵染与共培养时间及乙酰丁香酮（AS）浓度等进行筛选，建立发根农杆菌介导的芜菁毛状根转化体系。以6 d苗龄的芜菁无根苗为外植体，采用添加200 μmol · L^-1 AS，菌液浓度OD₆₀₀值为0.4时侵染12 min，共培养36 h后再转入含有400 mg · L^-1羧苄青霉素（Carb）的MS抑菌培养基的转化效率最高，并将可视化标记RUBY应用于转基因阳性毛状根高效筛选。基于该体系结合生长发育调节因子（developmental regulators，DRs）成功获得稳定的阳性转基因芜菁植株，阳性转化效率可达10.61%，从种子萌发、芽再生到生根整个过程可以在3个月内完成。

关键词: 芜菁, 发根农杆菌, 毛状根, RUBY, 发育调节因子

Abstract:

Turnip（Brassica rapa var. rapa L.）has problems such as poor regeneration and low transformation efficiency. The influenced factors including explants type，bacterial infection concentration，infection time，co-cultivation time and acetosyringone（AS）were firstly screened to establish the genetic transformed hairy roots system by Agrobacterium rhizogenes with a white skin turnip variety from Xinjiang Keping. The results showed that 6 d rootless seedlings were selected as explant and the suspension added 200 μmol · L^-1 AS with the bacterial concentration OD₆₀₀ of 0.4 was infected for 12 min. After 36 h co-cultivation，hairy roots were exhibited the highest transformation efficiency in MS bacteriostatic medium with 400 mg · L^-1 carbenicillin，as well as the visual marker RUBY was applied to efficiently screening for transgenic positive hairy roots. Furthermore，based on this system combined with developmental regulators，the stable positive transgenic plants were successfully obtained with a transformation efficiency of 10.61%.The whole process from seed germination，shoot regeneration to rooting could be completed within 3 months.

Key words: turnip, Agrobacterium rhizogenes, hairy root, RUBY, developmental regulator

马曦, 张金睿, 庄红梅, 陈巍, 张蜜, 袁晓琪, 易小芳, 王聪聪, 王海云, 王燕. 发根农杆菌介导的芜菁高效遗传转化体系建立[J]. 园艺学报, 2025, 52(5): 1389-1398.

MA Xi, ZHANG Jinrui, ZHUANG Hongmei, CHEN Wei, ZHANG Mi, YUAN Xiaoqi, YI Xiaofang, WANG Congcong, WANG Haiyun, WANG Yan. Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium rhizogenes in Turnip[J]. Acta Horticulturae Sinica, 2025, 52(5): 1389-1398.

图/表 11

图1 不同外植体类型

Fig. 1 Different types of explants

表1 外植体类型对毛状根诱导的影响

Table 1 The effects of explant types on hairy root induction

外植体类型 Explant type	诱导率/% Inductivity	平均毛状根数 Average number of hairy roots	阳性率/% Positive rate
带柄子叶 Cotyledon with petiole	89.66 ± 4.78 a	12.83 ± 1.49 a	2.15 ± 1.60 b
下胚轴 Hypocotyl	16.67 ± 9.43 b	0.20 ± 0.14 c	8.33 ± 5.89 ab
生长点 Growing point	35.00 ± 14.72 b	3.77 ± 2.29 b	3.98 ± 1.50 ab
无根苗 Rootless seedling	92.06 ± 4.88 a	12.98 ± 0.80 a	10.64 ± 1.82 a

图2 侵染30 d后不同外植体毛状根诱导情况（A ~ D）及阳性情况（E ~ H）

Fig. 2 Induction of hairy roots in different explants 30 d after infection（A-D）and positive results（E-H）

表2 发根农杆菌菌液浓度对毛状根诱导的影响

Table 2 The effect of OD600 value on hairy root induction

OD₆₀₀值 OD₆₀₀ value	诱导率/% Inductivity	平均毛状根数 Average number of hairy roots	溢菌数 Number of Agrobacterium overflow
0.2	81.03 ± 6.52 b	1.79 ± 1.09 b	少 Little
0.3	85.96 ± 4.97 ab	2.74 ± 0.71 b	无 None
0.4	92.06 ± 4.88 ab	12.98 ± 0.80 a	无 None
0.5 0.6	96.61 ± 2.42 a 63.33 ± 8.50 c	15.08 ± 0.86 a 3.97 ± 0.65 b	较少 Less 多 Much

表3 侵染时间对毛状根诱导的影响

Table 3 The effect of infection time on hairy root induction

侵染时间/min Infection time	诱导率/% Inductivity	平均毛状根数 Average number of hairy roots	阳性率/% Positive rate	毛状根长势 Hairy root growing condition
4	90.00 ± 4.89 a	4.25 ± 0.37 b	6.67 ± 7.37 a	细长，稀疏 Slender and sparse
8	95.00 ± 7.44 a	4.47 ± 0.81 b	1.49 ± 1.47 b	细长，稀疏 Slender and sparse
12	92.06 ± 4.88 a	12.98 ± 0.80 a	10.64 ± 1.82 a	粗长，茂密 Thick，long，and dense
16	93.33 ± 6.24 a	10.98 ± 2.81 a	1.37 ± 1.92 b	细长，茂密 Slender and dense

图3 不同侵染时间下30 d时毛状根生长情况

Fig. 3 Growth of hairy roots at 30 d under different infection time

表4 共培养时间对毛状根诱导的影响

Table 4 The effect of co-cultivation time on hairy root induction

共培养时间/h Co-cultivation time	诱导率/% Inductivity	平均毛状根数 Average number of hairy roots	阳性率/% Positive rate	侵染后发根时间/d Rooting time after infection
12	96.61 ± 2.50 a	10.58 ± 0.89 b	1.44 ± 0.68 b	9 ~ 10
24	96.67 ± 4.71 a	8.43 ± 1.05 c	3.56 ± 1.71 b	7 ~ 8
36	92.06 ± 4.88 a	12.98 ± 0.80 a	10.64 ± 1.82 a	7 ~ 8
48	94.92 ± 4.08 a	8.25 ± 0.76 c	3.08 ± 0.27 b	4 ~ 5

图4 不同共培养时间的毛状根诱导（30 d时）情况（A ~ D）及荧光情况（E ~ H）

Fig. 4 Induction of hairy roots at 30 d with different co-cultivation time（A-D）and fluorescence conditions（E-H）

表5 乙酰丁香酮（AS）浓度对毛状根诱导的影响

Table 5 Effect of AS concentration on hairy root induction

AS/（μmol · L^-1）	诱导率/% Inductivity	平均毛状根数 Average number of hairy roots	阳性率/% Positive rate
0	100.00 ± 0 a	9.15 ± 1.57 b	1.64 ± 0.48 c
100	100.00 ± 0 a	9.05 ± 0.78 b	8.84 ± 1.50 a
200	92.06 ± 4.88 b	12.98 ± 0.80 a	10.64 ± 1.82 a
300	95.00 ± 4.08 ab	9.90 ± 0.74 b	5.39 ± 0.82 b
400	98.33 ± 2.36 a	13.33 ± 0.66 a	1.88 ± 0.90 c
500	100.00 ± 0 a	13.33 ± 0.91 a	1.65 ± 0.40 c

图5 基于RUBY报告基因筛选阳性毛状根

Fig. 5 Screening of positive hairy roots based on the RUBY reporter gene

图6 ZmWus2-ipt-AtPLT5-RUBY系统简单高效遗传转化体系的建立 A：6 d苗龄无根苗；B：外植体共培养后转移至MS + 400 mg · L-1羧苄青霉素（Carb）抑菌培养基；C：外植体培养35 d；D ~ G：再生芽生根培养1、10、35、50 d芽生长状态；H：再生芽生根培养50 d根系生长状态；I ~ J：再生芽生根培养65 d驯化3 ~ 5 d并移栽至营养钵；K：转基因芜菁植株在营养钵中生长40 d状态

Fig. 6 The establishment of a simple and efficient genetic transformation system with ZmWus2-ipt-AtPLT5-RUBY A：Rootless seedlings for growing 6 d；B：The explants were transferred to MS bacteriostatic medium containing 400 mg · L-1 Carbenicillin（Carb）after co-cultivation；C：The explants on MS medium containing 400 mg · L-1 Carb for 35 d；D-G：The emerged shoots cut off from the infected seedling were transferred to the roots induction medium for 1，10，35，50 d；H：Root growth status of the shoots for 50 d；I-J：The shoots were cultured for 65 d and transplanted into pot after acclimatization for 3-5 d；K：Transgenic turnip plants were grown in the pot for 40 d

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发根农杆菌介导的芜菁高效遗传转化体系建立

Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium rhizogenes in Turnip

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发根农杆菌介导的芜菁高效遗传转化体系建立

Establishment of an Efficient Genetic Transformation System Mediated by Agrobacterium rhizogenes in Turnip

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