基因枪介导的老芒麦遗传转化体系的建立

doi:10.11983/CBB20174

植物学报 ›› 2021, Vol. 56 ›› Issue (1): 62-70.DOI: 10.11983/CBB20174

基因枪介导的老芒麦遗传转化体系的建立

杜鹏飞¹^,²^,³, 王玉², 曹英萍², 杨松², 孙志超², 毛德才³^,⁴, 鄢家俊³^,⁴, 李达旭³^,⁴, 孙美贞⁵, 付春祥²^,^*(), 白史且³^,⁴^,^*()

¹西南民族大学, 成都 610041
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, 青岛 266101
³四川省草原科学研究院, 成都 611731
⁴国家林草局青藏高原高寒草地生态修复工程技术研究中心, 成都 611731
⁵青岛市中心血站, 青岛 266071

收稿日期:2020-11-02 接受日期:2021-01-05 出版日期:2021-01-01 发布日期:2021-01-15
通讯作者: 付春祥,白史且
作者简介:610852681@qq.com
*E-mail: fucx@qibebt.ac.cn;
基金资助:
首批万人计划四川天府杰出科学家项目(2018);四川省重点研发项目(2019YFN0170);西南民族大学研究生创新型科研项目(CX2019SZ91);山东省农业良种工程(2019LZGC010)

Establishment of Biolistic Mediated Transformation System for Elymus sibiricus

Pengfei Du¹^,²^,³, Yu Wang², Yingping Cao², Song Yang², Zhichao Sun², Decai Mao³^,⁴, Jiajun Yan³^,⁴, Daxu Li³^,⁴, Meizhen Sun⁵, Chunxiang Fu²^,^*(), Shiqie Bai³^,⁴^,^*()

¹Southwest University for Nationalities, Chengdu 610041, China
²Shandong 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 266101, China
³Sichuan Grassland Science Research Institute, Chengdu 611731, China
⁴Engineering Research Center for Ecological Restoration of Alpine Grassland on the Qinghai-Tibet Plateau, National Forestry and Grassland Administration, Chengdu 611731, China
⁵Qingdao Blood Center, Qingdao 266071, China

Received:2020-11-02 Accepted:2021-01-05 Online:2021-01-01 Published:2021-01-15
Contact: Chunxiang Fu,Shiqie Bai

摘要/Abstract

摘要： 川草2号老芒麦(Elymus sibiricus)是青藏高原地区治理荒漠化和建设高产人工草地的主要栽培草种。用川草2号老芒麦5种外植体诱导愈伤组织, 经分化测试, 仅幼穗愈伤组织能分化再生。以当代培养25天和35天的结构致密坚硬的幼穗愈伤组织为受体, 分别进行农杆菌侵染和基因枪转化, 结果只有基因枪能转化成功。在基因枪转化过程中, 采用高渗培养和滤纸干燥2种方式预处理愈伤组织, 结果表明滤纸干燥处理比高渗处理转化效率高。当代诱导25天的幼穗愈伤组织, 滤纸干燥处理2小时转化效率最高, 达40%。该研究成功获得了基因枪转化的以川草2号老芒麦幼穗愈伤为受体的阳性愈伤组织。

关键词: 川草2号老芒麦, 幼穗, 农杆菌侵染, 基因枪转化, 愈伤组织处理

Abstract: Elymus sibiricus cv. ‘Chuancao No.2’ is the main cultivated grass species for desertification control and construction of high-yield and high-quality pasture in northwest Sichuan Plateau. In this study, we tested five explants of E. sibiricus cv. ‘Chuancao No.2’ for callus induction, and found that only inflorescence calli were able to differentiate and regenerate. The calli of inflorescence with dense and hard structure cultured for 25 d and 35 d were used for Agrobacterium and biolistic mediated transformation respectively. The results showed that only biolistic-mediated transformation could produce positive transgenic calli of ‘Chuancao No.2’. In the process of biolistic-mediated transformation, the calli was pretreated in two ways: hyperosmotic culture and filter paper drying. The results revealed that the transformation efficiency of filter paper drying was higher than that of hyperosmotic treatment. For the inflorescence callus after 25 d induction, the transformation efficiency under the condition of 2 h drying of filter paper was highest which reached about 40%. In short, we applied the biolistic technology in ‘Chuancao No.2’ for the first time and successfully obtained the positive transgenic inflorescence calli. This work will lead to establishment of the robust transformation system for E. sibiricus in future.

Key words: Elymus sibiricus cv. ‘Chuancao No.2’, inflorescence, Agrobacterium infection, biolistic mediated transformation, callus treatment

杜鹏飞, 王玉, 曹英萍, 杨松, 孙志超, 毛德才, 鄢家俊, 李达旭, 孙美贞, 付春祥, 白史且. 基因枪介导的老芒麦遗传转化体系的建立. 植物学报, 2021, 56(1): 62-70.

Pengfei Du, Yu Wang, Yingping Cao, Song Yang, Zhichao Sun, Decai Mao, Jiajun Yan, Daxu Li, Meizhen Sun, Chunxiang Fu, Shiqie Bai. Establishment of Biolistic Mediated Transformation System for Elymus sibiricus. Chinese Bulletin of Botany, 2021, 56(1): 62-70.

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图/表 8

图1 PANIC6A (A)、PANIC6D (B)和PANIC6E (C) T-DNA区段 LB: T-DNA区段左边界; OsAct1: 水稻启动子; hph/bar: 筛选标记基因; PvUbi1: 柳枝稷启动子; pporRFP/GUSplus: 红色荧光/葡萄糖苷酸酶报告基因; ZmUbi1: 玉米启动子; Cmr: 氯霉素抗性基因; ccdB: 目的基因; RB: T-DNA区段右边界

Figure 1 T-DNA segment of PANIC6A (A), PANIC6D (B) and PANIC6E (C) LB: Left boundary of T-DNA segment; OsAct1: Rice promoter; hph/bar: Screening marker genes; PvUbi1: Switchgrass promoter; pporRFP/GUSplus: Red fluorescence/glucuronidase reporter gene; ZmUbi1: Maize promoter; Cmr: Chloramphenicol resistant gene; ccdB: Target gene; RB: Right boundary of T-DNA segment

表1 4种农杆菌侵染方案

Table 1 Four Agrobacterium infection methods

Infect schemes	Infection methods
Infect schemes	Predrying treatment (10 min)	Vacuum treatment (10 min)	Ultrasonic treatment (5 min)	Vacuum treatment (10 min)
1	-	+	+	+
2	-	+	+	-
3	-	-	+	+
4	+	+	+	+

图2 不同外植体诱导出愈伤后约30天的显微图 (A)-(E) 分别表示用种子(成熟胚)、根、下胚轴、茎和幼穗诱导出的愈伤。Bars=50 μm

Figure 2 Micrographs of calli from different explants (30 d) (A)-(E) Callus induced by seeds (mature embryo), roots, hypocotyl, stem and inflorescence, respectively. Bars=50 μm

图3 川草2号老芒麦不同外植体愈伤诱导所用时间及诱导率误差线为标准误(SE), 样本容量为60。不同小写字母表示差异显著(P<0.05)。

Figure 3 Callus induction time and induction rate of different explants in Elymus sibiricus cv. ‘Chuancao No.2’ The error bars indicate standard error (SE), the sample size was 60. Different lowercase letters indicate significant differences (P<0.05).

图4 川草2号老芒麦组培再生体系建立及幼穗愈伤分化效率随继代时间的变化 (A) 幼穗接种于愈伤诱导培养基; (B) 幼穗诱导35天后的愈伤; (C) 挑选图(B)中结构致密坚硬的愈伤置于继代培养基15天; (D) 图(C)愈伤置于分化培养基约35天; (E) 图(D)中红色框内愈伤分化显微图; (F) 图(D)中分化苗置于生根培养基约30天; (G) 种子接种于愈伤诱导培养基; (H) 种子诱导35天后的愈伤; (I) 挑选图(H)中结构致密坚硬的愈伤置于继代培养基15天; (J) 图(I)愈伤置于分化培养基约35天; (K) 图(J)中红色框内愈伤分化显微图; (L) 不同再生时间下的幼穗愈伤分化效率(误差线为标准误, 样本容量为30, 不同小写字母表示差异显著(P<0.05))。(D), (F), (G), (J) Bars=2 cm; (A)-(C), (E), (H), (I), (K) Bars=1 cm

Figure 4 The process diagram of tissue culture and regeneration system and the plot of callus differentiation efficiency of Elymus sibiricus cv. ‘Chuancao No.2’ with subculture time (A) Inflorescence of ‘Chuancao No.2’ placed in callus induction medium; (B) Callus induced by inflorescence of ‘Chuancao No.2’ for 35 d; (C) The callus with dense and hard structure in figure (B) was selected and placed in medium for 15 d; (D) Callus in figure (C) cultured in differentiation medium for 35 d; (E) The micrograph of callus in red frame in figure (D); (F) Differentiated seedlings in figure (D) placed in rooting medium for 30 d; (G) Seeds placed in callus induction medium; (H) Callus induced by seeds for 35 d; (I) The callus with dense and hard structure in figure (H) was selected and placed in subculture medium for 15 d; (J) Callus in figure (I) cultured in differentiation medium for 35 d; (K) The micrograph of callus in red frame in figure (J); (L) The differentiation efficiency of callus induced by inflorescence at different time periods (The error line is the standard error, and the sample size is 30; Different lowercase letters indicate significant differences at P<0.05). Bars in (D), (F), (G), (J)=2 cm; Bars in (A)-(C), (E), (H), (I), (K)=1 cm

图5 根癌农杆菌侵染川草2号老芒麦幼穗和幼穗愈伤 (A), (B) 农杆菌侵染幼穗愈伤GUS染色前后图片; (C), (D) 农杆菌侵染幼穗愈伤RFP荧光下明场和暗场图片; (E), (F) 农杆菌直接侵染幼穗后GUS染色前后图片; (G), (H) 农杆菌直接侵染幼穗后RFP下明场和暗场图片。Bars=50 μm

Figure 5 Callus of inflorescence and inflorescence infected by Agrobacterium tumefaciens in Elymus sibiricus cv. ‘Chuancao No.2’ (A), (B) Callus induced by inflorescence before and after GUS staining after Agrobacterium infection; (C), (D) The bright and dark field images of RFP fluorescence after Agrobacterium infection of callus induced by inflorescence; (E), (F) The images before and after GUS staining directly after Agrobacterium infection with inflorescence; (G), (H) The bright and dark field images of RFP fluorescence after Agrobacterium infection of inflorescence, respectively. Bars=50 μm

图6 不同诱导时间和预处理方式下的幼穗愈伤基因枪轰击后的荧光显微图 (A), (B) 25天幼穗愈伤(未进行轰击); (C), (D) 25天幼穗愈伤常规高渗处理; (E), (F) 25天幼穗愈伤滤纸干燥处理2小时; (G), (H) 35天的幼穗愈伤滤纸干燥处理2小时。箭头所示为阳性转化愈伤。Bars=20 μm

Figure 6 Fluorescence micrographs of inflorescence callus after bombardment with different induction time and pretreatment methods (A), (B) Callus of inflorescence on 25 d (no bombardment); (C), (D) Conventional hyperosmotic treatment of inflorescence callus at 25 d; (E), (F) Inflorescence callus filter paper dried for 2 h on 25 d; (G), (H) Inflorescence callus filter paper dried for 2 h on 35 d. Arrows indicate the positive transgenic calli. Bars =20 μm

图7 基因枪轰击后荧光愈伤百分比随持续观测时间的变化趋势误差线为标准误差, 样本容量为30。25天幼穗愈伤滤纸干燥2小时荧光愈伤比率均显著高于其它2种处理方式(* P<0.05, ** P<0.01, *** P<0.001)。

Figure 7 The trend of percentage of fluorescent callus after gene gun bombardment with continuous observation time The error line is the standard error, and the sample size is 30. The fluorescence callus ratio of the 25-day inflorescence callus after 2 h drying on filter paper was significantly higher than that of the other two treatments (* P<0.05, ** P<0.01, *** P<0.001).

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基因枪介导的老芒麦遗传转化体系的建立

Establishment of Biolistic Mediated Transformation System for Elymus sibiricus

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