根癌农杆菌介导的蒙古冰草稳定遗传转化体系建立

doi:10.11983/CBB24012

植物学报 ›› 2024, Vol. 59 ›› Issue (4): 600-612.DOI: 10.11983/CBB24012 cstr: 32102.14.CBB24012

根癌农杆菌介导的蒙古冰草稳定遗传转化体系建立

李宇琛¹^,², 赵海霞², 姜希萍², 黄馨田¹, 刘亚玲³, 吴振映², 赵彦¹^,^*(), 付春祥²^,^*()

¹内蒙古农业大学草原与资源环境学院/农业农村部饲草栽培、加工与高效利用重点实验室/草地资源教育部重点实验室, 呼和浩特 010018
²中国科学院青岛生物能源与过程研究所, 山东省能源研究院, 青岛新能源山东省实验室, 青岛 266101
³内蒙古草业技术创新中心有限公司, 呼和浩特 010052

收稿日期:2024-01-24 接受日期:2024-04-25 出版日期:2024-07-10 发布日期:2024-07-10
通讯作者: *E-mail: zhaoyannmg@163.com; fucx@qibebt.ac.cn
基金资助:
内蒙古自治区种业科技创新重大示范工程“揭榜挂帅”项目(2022JBGS0014);中央引导地方科技发展资金(2022ZY006);青岛新能源山东实验室“抓攻关”项目(QNESLKPP202302);国家林业和草原局重大应急科技“揭榜挂帅”项目(202201);呼和浩特市重大科技专项(2021-重-社-2)

Establishment of Agrobacterium-mediated Transformation System for Agropyron mongolicum

Yuchen Li¹^,², Haixia Zhao², Xiping Jiang², Xintian Huang¹, Yaling Liu³, Zhenying Wu², Yan Zhao¹^,^*(), Chunxiang Fu²^,^*()

¹Key Laboratory of Grassland Resources of Ministry of Education/Key Laboratory of Forage Cultivation, Processing and Efficient Utilization of Ministry of Agriculture and Rural Areas/College of Grassland, Resources and Environment of Inner Mongolia Agricultural University, Hohhot 010018, China
²Qingdao New Energy Shandong Laboratory, Shandong Energy Research Institute, Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences, Qingdao 266101, China
³Inner Mongolia Prataculture Technology Innovation Center Co., Ltd., Hohhot 010052, China

Received:2024-01-24 Accepted:2024-04-25 Online:2024-07-10 Published:2024-07-10
Contact: *E-mail: zhaoyannmg@163.com; fucx@qibebt.ac.cn

摘要/Abstract

摘要： 蒙古冰草(Agropyron mongolicum)亦称沙芦草, 为禾本科(Poaceae)小麦族(Triticeae)冰草属(Agropyron)多年生疏丛型牧草, 具有饲用价值高、抗寒耐旱以及耐盐、耐瘠薄、耐风沙等特性, 是改良天然草场的适宜草种与挖掘优良耐逆基因资源的重要材料。然而, 目前尚未建立蒙古冰草高效遗传转化体系, 制约了该物种的基因资源鉴定与遗传改良应用。以蒙农1号蒙古冰草种子为来源的高再生效率的胚性愈伤系#89为外植体, 建立了根癌农杆菌(Agrobacterium tumefaciens) EHA105介导的蒙古冰草稳定遗传转化体系, 转化效率达30%。此外, 针对多次继代后蒙古冰草愈伤系再生能力退化的难题, 通过在再生培养基中添加1 mg·L^-1 ABA或提高蔗糖浓度至45 g·L^-1, 成功将再生能力衰退的蒙古冰草愈伤系再生效率由5%分别提高至35%与42%。研究结果为后续蒙古冰草基因编辑体系建立、基因功能鉴定和新品种培育奠定了重要技术基础。

关键词: 蒙古冰草, 胚性愈伤组织, 根癌农杆菌, 遗传转化

Abstract: Agropyron mongolicum is a perennial shrub grass in the Triticeae tribe of Poaceae family. It has high forage quality and tolerance to cold, drought, salt and sandstorm. Therefore, it is an excellent grass species for mining stress tolerant genes and restoring deteriorated grasslands. However, a highly efficient transformation system has yet to be established in A. mongolicum. It restricts the identification of genetic resources and the application of genetic improvement of this species. Here, we report an embryogenic callus line (ECL) #89 that was induced from the seeds of A. mongolicum cultivar Mengnong No. 1. Our studies showed the ECL #89 had high regeneration and Agrobacterium-infection efficiencies. An Agrobacterium-mediated transformation system with a transformation efficiency up to 30% was established by infecting the ECL #89 with EHA105. Additionally, we found that subcultures could decrease severely the regeneration capacity of ECL #89, which could be partially restored by adding (1 mg·L^-1) ABA or high concentration (45 g·L^-1) sucrose in the regeneration medium, from 5% to 35% and 42%, respectively. Our work will facilitate the developing of genome editing system, gene function characterization, and molecular breeding in A. mongolicum.

Key words: Agropyron mongolicum, embryogenic callus, Agrobacterium tumefaciens, genetic transformation

李宇琛, 赵海霞, 姜希萍, 黄馨田, 刘亚玲, 吴振映, 赵彦, 付春祥. 根癌农杆菌介导的蒙古冰草稳定遗传转化体系建立. 植物学报, 2024, 59(4): 600-612.

Yuchen Li, Haixia Zhao, Xiping Jiang, Xintian Huang, Yaling Liu, Zhenying Wu, Yan Zhao, Chunxiang Fu. Establishment of Agrobacterium-mediated Transformation System for Agropyron mongolicum. Chinese Bulletin of Botany, 2024, 59(4): 600-612.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24012

https://www.chinbullbotany.com/CN/Y2024/V59/I4/600

图/表 7

图1 pANIC6B空载体示意图(参考Mann et al., 2012) LB: T-DNA区段左边界; OsAct1: 水稻Act1基因的启动子与内含子; hph: 潮霉素抗性筛选标记基因; 35S T: 35S终止子; PvUbi1: 柳枝稷Ubi1基因的启动子与内含子; GUSplus: 葡萄糖苷酸酶报告基因; NOS T: 根癌农杆菌胭脂碱合酶基因终止子; ZmUbi1: 玉米Ubi1基因的启动子与内含子; Gateway cassette: attR1-CmR/ccdb-attR2; OCS T: 章鱼碱合酶终止子; RB: T-DNA区段右边界

Figure 1 Schematic diagram of pANIC6B empty vector (refer to Mann et al., 2012) LB: Left border of T-DNA; OsAct1: Rice Act1 promoter and intron; hph: Screening marker gene for hygromycin resistance; 35S T: 35S terminator; PvUbi1: Switchgrass Ubi1 promoter and intron; GUSplus: β-glucuronidase plus reporter gene; NOS T: Agrobacterium tumefaciens carmine synthase gene terminator; ZmUbi1: Maize Ubi1 promoter and intron; Gateway cassette: attR1-CmR/ccdb-attR2; OCS T: Octopine synthase terminator; RB: Right border of T-DNA

图2 蒙农1号种子来源的愈伤系类型 (A)-(D) 不同愈伤系外观特征(上部图为愈伤组织在M5培养基中的生长情况, 下部图为代表性愈伤组织的显微放大情况。Bars=1 cm); (E) 胚性愈伤系(ECL)在总愈伤系中的比例(单因素方差分析, Duncan’s检验, P<0.05)。

Figure 2 Phenotypes of callus lines induced from Mengnong No.1 seeds (A)-(D) Phenotypes of different callus lines (the up photos are calli grown on M5 medium, the down photos are calli under the microscope. Bars=1 cm); (E) Percentage of embryogenic callus lines (ECL) in total callus lines (One-way analysis of variance, Duncan’s test, P<0.05).

图3 蒙农1号种子胚性愈伤系(ECL)的再生情况 (A) 不同基因型的#88和#89胚性愈伤系再生情况(bars=1 cm); (B) 大于30%再生效率的胚性愈伤系占全部胚性愈伤系的比例。不同小写字母表示各处理间差异显著(单因素方差分析, Duncan’s检验, P<0.05)。

Figure 3 Regeneration analyses of embryogenic callus lines (ECL) induced from Mengnong No.1 seeds (A) Regeneration of embryogenic callus lines #88 and #89 with different genotypes (bars=1 cm); (B) Percentage of embryogenic callus lines with more than 30% regeneration efficiency in total embryogenic callus lines. Different lowercase letters indicate significant differences among three experimental groups (One-way analysis of variance, Duncan’s test, P<0.05).

图4 添加ABA和高浓度蔗糖对退化的#89胚性愈伤系再生能力的影响 (A) 再生能力退化的#89胚性愈伤系在MSBK培养基上的分化情况; (B) 再生能力退化的#89胚性愈伤系在添加1 mg·L-1 ABA的MSBKA培养基上的分化情况; (C) 再生能力退化的#89胚性愈伤系在含(45 g·L-1)蔗糖的MSBKS45培养基上的分化情况; (D) 在ABA处理的MSBKA培养基上, 再生能力退化的#89胚性愈伤系的再生效率; (E) 在30 g·L-1和45 g·L-1蔗糖处理的MSBK45培养基上, 再生能力退化的#89胚性愈伤系的再生效率。不同小写字母表示各处理间差异显著(单因素方差分析, Duncan’s检验, P<0.05)。(A), (B), (C)中右图为对应左图的显微镜下观察。Bars=1 cm

Figure 4 Effects of ABA and high concentration sucrose on the regeneration capacity of deteriorated embryogenic callus line #89 (A) Differentiation of deteriorated embryogenic callus line #89 grown in MSBK medium; (B) Differentiation of deteriorated embryogenic callus line #89 grown in MSBKA medium supplemented with 1 mg·L-1 ABA; (C) Differentiation of deteriorated embryogenic callus line #89 grown in MSBKS45 medium supplemented with 45 g·L-1 sucrose; (D) Regeneration efficiency of deteriorated embryogenic callus line #89 grown in MSBKA medium with ABA treatment; (E) Regeneration efficiency of deteriorated embryogenic callus line #89 grown in MSBKS45 medium with 30 g·L-1 and 45 g·L-1 sucrose. Different lowercase letters indicate significant differences between treatments (One-way analysis of variance, Duncan’s test, P<0.05). The callus depicted in (A), (B), and (C) on the right side represents the microscopic observation of the corresponding structures on the left side. Bars=1 cm

图5 蒙农1号种子胚性愈伤系(ECL)的侵染效率 (A) GUS染色分析农杆菌EHA105对不同基因型胚性愈伤系的侵染效果; (B) 侵染效率在40%以上的胚性愈伤系占总胚性愈伤系的比例。不同小写字母表示各处理间差异显著(单因素方差分析, Duncan’s检验, P<0.05)。

Figure 5 Infection analyses of embryogenic callus lines (ECL) induced from Mengnong No.1 seeds (A) The infection efficiency of EHA105 on embryogenic callus lines through GUS staining; (B) The percentage of embryogenic callus lines with high infection efficiency (>40%) in total embryogenic callus lines. Different lowercase letters indicated significant differences among three experimental groups (One-way analysis of variance, Duncan’s test, P<0.05).

图6 农杆菌介导的蒙农1号遗传转化体系的建立 (A) 生长在M2培养基上的#89愈伤系; (B) 愈伤组织与农杆菌共培养; (C) 共培养后的GUS染色分析; (D) 共培养后的愈伤组织生长在M2H30筛选培养基上; (E) 抗性愈伤组织的GUS染色分析; (F) 抗性愈伤组织在MSBKH2筛选培养基上的分化情况; (G) 生根后的抗性植株移栽至土中; (H) 抗性植株叶片的GUS染色。Bars=1 cm

Figure 6 Establishment of the Agrobacterium-mediated transformation in Mengnong No.1 (A) #89 embryogenic callus line induced from Mengnong No.1 seeds grown on M2 medium; (B) Co-cultivation of #89 calli with Agrobacterium strain EHA105; (C) Agrobacterium infection analysis of #89 embryogenic calli by GUS staining; (D) Selection of hygromycin resistant calli through M2H30 medium; (E) GUS staining of hygromycin resistant callus; (F) Differentiation of the hygromycin resistant calli on MSBKH2 medium; (G) Growth of the hygromycin resistant plant in soil; (H) GUS staining of leaves from the hygromycin resistant plants. Bars=1 cm

图7 蒙农1号蒙古冰草转基因植株的潮霉素抗性基因hph的PCR分析 M: 2000 bp DNA分子量标准; 1-19: PCR所用的DNA模板(1: pANIC6B空载体质粒; 2: ddH2O; 3: #89号胚性愈伤系未经遗传转化的愈伤组织再生植株; 4-19: #89号胚性愈伤组织遗传转化获得的抗性植株。hph的PCR产物大小为375 bp)。

Figure 7 PCR analysis of hph in Mengnong No.1 Agropyron mongolicum transgenic plants M: 2000 bp DNA marker; 1-19: PCR template (1: pANIC6B empty vector; 2: ddH2O; 3: Non-transgenic plant regenerated from embryogenic callus line #89; 4-19: Transgenic plants regenerated from embryogenic callus #89. The sizes of PCR products are 375 bp for hph).

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根癌农杆菌介导的蒙古冰草稳定遗传转化体系建立

Establishment of Agrobacterium-mediated Transformation System for Agropyron mongolicum

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