铁棍山药微型块茎遗传转化体系的建立

doi:10.11983/CBB18118

植物学报 ›› 2019, Vol. 54 ›› Issue (1): 72-80.DOI: 10.11983/CBB18118

铁棍山药微型块茎遗传转化体系的建立

李俊华^1,^2,^3,^†,刘世宇^1,^†,李成龙¹,韩林林¹,董亚辉¹,张晓丽^1,^2,³,赵喜亭^1,^2,³,李明军^1,^2,^3,^*()

1 河南师范大学生命科学学院, 新乡 453007
2 河南省道地药材保育及利用工程技术研究中心, 新乡 453007
3 绿色药材生物技术河南省工程实验室, 新乡 453007

收稿日期:2018-05-15 接受日期:2018-09-13 出版日期:2019-01-01 发布日期:2019-07-31
通讯作者: 李俊华,刘世宇,李明军
基金资助:
国家自然科学基金(81274019);国家中医药管理局中医药行业科研专项子课题(201407005-08);河南省创新型科技人才队伍建设工程(C20130037)

Establishment of a Genetic Transformation System for Dioscorea opposita Using Microtuber

Junhua Li^1,^2,^3,^†,Shiyu Liu^1,^†,Chenglong Li¹,Linlin Han¹,Yahui Dong¹,Xiaoli Zhang^1,^2,³,Xiting Zhao^1,^2,³,Mingjun Li^1,^2,^3,^*()

1 College of Life Science, Henan Normal University, Xinxiang 453007, China
2 Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs of Henan Province, Xinxiang 453007, China
3 Engineering Laboratory of Green Medicinal Material Biotechnology of Henan Province, Xinxiang 453007, China

Received:2018-05-15 Accepted:2018-09-13 Online:2019-01-01 Published:2019-07-31
Contact: Junhua Li,Shiyu Liu,Mingjun Li

摘要/Abstract

摘要： 怀山药(Dioscorea opposita)遗传转化是对其进行基因功能分析和遗传改良的基础, 但目前国内外尚未见相关报道。以怀山药优良品种铁棍山药(D. opposita cv. ‘Tiegun’)的微型块茎为受体材料, 对影响遗传转化的因素进行优化, 建立了由根癌农杆菌介导的山药遗传转化体系。过表达质粒载体pCAMBIA1301-DoSERK2含GUS标记基因和潮霉素(Hyg)抗性筛选基因, 沉默质粒载体pART27-DoSERK2含卡那霉素(Kan)抗性筛选基因。根癌农杆菌抑制剂特美汀(Tim)的最佳浓度为500 mg·L ^-1; 再生芽和生根时, Hyg的最佳浓度分别为15和20 mg·L ^-1, Kan的最佳浓度分别为120和160 mg·L ^-1。对转化植株进行PCR和GUS组织化学检测, 结果显示外源基因已整合到铁棍山药转基因株系的基因组中并在细胞中表达。该研究建立了一套取材便利的铁棍山药遗传转化方法, 对其它品种山药的转化也具有参考价值。

关键词: 根癌农杆菌, 遗传转化, 微型块茎, 铁棍山药

Abstract: Genetic transformation system is the foundation for gene function study and genetic improvement of Dioscorea opposita. In this study, we established an Agrobacterium-mediated genetic transformation system using microtuber in D. opposita cv. ‘Tiegun’. The transformation was performed with the DoSERK2 gene overexpression plasmid pCAMBIA1301-DoSERK2 that harboring the GUS reporter gene and the hygromycin (Hyg) resistence gene as the selectable marker, and the gene-silencing vector pART27-DoSERK2 that containing kanamycin (Kan) resistence marker. The optimal concentration of timentin (Tim) for Agrobacterium counter selection was 500 mg·L ^-1. For shoot regeneration and rooting, the optimal concentrations of Hyg were 15 mg·L ^-1 and 20 mg·L ^-1, respectively, and the optimal concentration of Kan were 120 mg·L ^-1 and 160 mg·L ^-1, respectively. Results of PCR analysis and GUS histochemical staining indicated that the exogenous genes had been integrated into the genome of transgenic plants, and are expressed in the plants. Our study provides a genetic transformation system in D. opposita cv. ‘Tiegun’, which has the advantage that explants are easy available. This method has reference value for the genetic transformation of other varieties of D. opposita.

Key words: Agrobacterium tumefaciens, genetic transformation, microtuber, Dioscorea opposita cv. ‘Tiegun’

李俊华,刘世宇,李成龙,韩林林,董亚辉,张晓丽,赵喜亭,李明军. 铁棍山药微型块茎遗传转化体系的建立. 植物学报, 2019, 54(1): 72-80.

Junhua Li,Shiyu Liu,Chenglong Li,Linlin Han,Yahui Dong,Xiaoli Zhang,Xiting Zhao,Mingjun Li. Establishment of a Genetic Transformation System for Dioscorea opposita Using Microtuber. Chinese Bulletin of Botany, 2019, 54(1): 72-80.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18118

https://www.chinbullbotany.com/CN/Y2019/V54/I1/72

图/表 8

图1 过表达载体和沉默载体结构示意图 (A) 过表达载体pCAMBIA1301-DoSERK2; (B) 沉默载体pART27-DoSERK2

Figure 1 Schematic maps of the overexpression vector and silencing vector used in this study (A) The overexpression vector pCAMBIA1301-DoSERK2; (B) The silencing vector pART27-DoSERK2

表1 铁棍山药遗传转化和植株再生培养基

Table 1 Medium used for genetic transformation and plant regeneration of Dioscorea opposita cv. ‘Tiegun’

Media	Composition
MS₀	MS major salts, MS minor salts and MS vitamins +30 g·L^-1 sucrose+6 g·L^-1 fungible agar, pH5.8- 6.2
MS₀ 1	MS₀+1 mg·L^-1 6-BA+1 mg?L^-1 IAA
MS₀ 2	MS₀+2 mg?L^-1PP₃₃₃+0.05 mg?L^-1 NAA
MS₀ 1-A	MS₀1+100 μmol?L^-1 Acetosyringone (AS)
MS₀ 1-T	MS₀1+500 mg?L^-1 Timentin (Tim)
MS₀1-TH	MS₀1+500 mg?L^-1 Tim+15 mg?L^-1Hyg
MS₀ 1-TK	MS₀ 1+500 mg?L^-1 Tim+120 mg?L^-1 Kan
MS₀ 2-H	MS₀ 2+20 mg?L^-1Hyg
MS₀ 2-K	MS₀2+160 mg?L^-1 Kan

表2 引物序列

Table 2 Sequences of primers

Primers	Sequences (5'-3')
DoSERK2-OE-F	ATGACGGCTTGGGTTTTC
DoSERK2-OE-R	TCACCTCGGACCAGATAGC
DoSERK2-RNAi-F	CAGATGATACAGAAAAGCACCG
DoSERK2-RNAi-R	TAACTTTCGGTAGAGCGGAC
DoActin-F	CTCATTGATCGGCATGGAAGC
DoActin-R	GGGGAACATAGTTGAACCACCAC
DoSERK2-qRT-F	TATCTGGACCAGTTCCATCC
DoSERK2-qRT-R	CTTCAGCAGGCACATCATAG

图2 不同浓度Tim对铁棍山药类原球茎(PLBs)增殖分化的影响 cv. ‘Tiegun’(A) 0 mg·L-1; (B) 100 mg·L-1; (C) 200 mg·L-1; (D) 300 mg·L-1; (E) 400 mg·L-1; (F) 500 mg·L-1。Bars=1 cm

Figure 2 Effects of different concentrations of Tim on the proliferation and differentiation of protocorm-like bodies (PLBs) in Dioscorea opposita (A) 0 mg·L-1; (B) 100 mg·L-1; (C) 200 mg·L-1; (D) 300 mg·L-1; (E) 400 mg·L-1; (F) 500 mg·L-1. Bars=1 cm

图3 筛选剂对铁棍山药微型块茎苗再生及再生苗生根的影响 (A)-(F) 不同浓度Hyg对微型块茎苗再生的影响, Hyg浓度分别为0、5、10、15、20和25 mg·L-1; (G)-(L) 不同浓度Kan对微型块茎苗再生的影响, Kan浓度分别为0、80、100、120、140和160 mg·L-1; (M) 不同浓度Hyg对再生苗生根的影响; (N) 不同浓度Kan对再生苗生根的影响。(A)-(L) Bars=0.5 cm; (M), (N) Bars=2 cm

Figure 3 Effects of antibiotic on microtuber regeneration and rooting of regenerated seedlings in Dioscorea opposita cv. ‘Tiegun’ (A)-(F) Effects of different concentrations of Hyg on microtuber regeneration, the concentrations of Hyg are 0, 5, 10, 15, 20, 25 mg·L-1, respectively; (G)-(L) Effects of different concentrations of Kan on microtuber regeneration, the concentrations of Kan are 0, 80, 100, 120, 140, 160 mg·L-1, respectively; (M) Effects of different concentrations of Hyg on rooting of regenerated plantlets; (N) Effects of different concentrations of Kan on rooting of regenerated plantlets. (A)-(L) Bars=0.5 cm; (M), (N) Bars=2 cm

表3 不同浓度筛选剂对铁棍山药微型块茎切片再生的影响

Table 3 Effects of antibiotic concentrations on regeneration of microtuber slices in Dioscorea opposita cv. ‘Tiegun’

Concentration of Hyg (mg·L^-1)	Regeneration (%)	Concentration of Kan (mg·L^-1)	Regeneration (%)	Growth of seedlings
0	60.00±2.00	0	65.56±2.52	Strong growth vigor
5	55.56±1.15	80	48.89±1.53	Well
10	33.33±2.00	100	34.44±1.53	Slow, albefaction in some seedlings
15	25.56±0.58	120	8.89±3.06	Slow, albefaction in many seedlings
20	2.22±1.15	140	1.11±0.58	Growth stopped, albefaction seriously
25	0	160	0	All dead

表4 不同浓度筛选剂对铁棍山药再生苗生根的影响

Table 4 Effects of antibiotic concentrations on rooting of regenerated seedlings in Dioscorea opposita cv. ‘Tiegun’

Concentration of Hyg (mg·L^-1)	Rooting (%)	Concentration of Kan (mg·L^-1)	Rooting (%)	Growth of roots
0	100	0	100	Strong and flourishing
5	100	120	100	Well
15	3±2.00	140	1±1.20	Seldom rooting
20	0	160	0	Hardly rooting
25	0	180	0	Hardly rooting, albefaction

图4 铁棍山药微型块茎切片的遗传转化、植株再生及转基因植株的分子鉴定 (A) 农杆菌侵染后的切片; (B) 共培养3天后的切片; (C) 再生苗培养基上培养40天的愈伤组织; (D) 再生苗培养基上培养70天的愈伤组织; (E) 抗性再生苗转入生根培养基; (F) 生根培养基上生长30天的再生苗; (G) 转基因植株基因组DNA的PCR鉴定(M: DNA分子量标记; P: 分别以质粒pCAMBIA1301-DoSERK2 (左)和pART27-DoSERK2 (右)为模板; WT: 以野生型植株的基因组DNA为模板; OE: 以过表达转基因株系基因组DNA为模板; RNAi-1-6: 以沉默载体转基因株系1-6的基因组DNA为模板); (H) 过表达转化叶片及叶柄中的GUS表达情况, 野生型植株(WT)和转基因植株(OE)分别经GUS染色液染色; (I) 内源DoSERK2表达水平的RT-qPCR检测(WT: 以野生型植株的cDNA为模板; RNAi-1-6: 以沉默载体转基因株系1-6 cDNA为模板)。(A)-(F), (H) Bars=1 cm

Figure 4 Genetic transformation and regeneration of Dioscorea opposita cv. ‘Tiegun’ microtuber slices and molecular verification of trans- genic plants (A) Microtuber slices infected by Agrobacterium; (B) Microtuber slices after co-cultivation with Agrobacterium for 3 days; (C) Tissues cultured on regeneration medium for 40 days; (D) Tissues cultured on regeneration medium for 70 days; (E) Antibiotic-resistant regenerated seedlings that had been transferred to rooting medium; (F) Regenerated seedlings that had been cultured on rooting medium for 30 days; (G) PCR assay of genome DNA from transgenic plants (M: DNA marker; P: Plasmid pCAMBIA1301-DoSERK2 (left panel) and pART27-DoSERK2 (right panel) as template; WT: Genome DNA of wild-type plants as template; OE: Genome DNA from overexpression transgenic plants as template; RNAi-1-6: Genome DNA from line 1-6 of transgenic plants of the silencing vector as template); (H) GUS activity assay in the leaf of an overexpression transformant, nodal or leaf segments of a wild-type plant (WT) or transgenic plant (OE) were stained with GUS staining solution; (I) RT-qPCR assay of the endogenous expression levels of DoSERK2 (WT: cDNA of wild-type plants as template; RNAi-1-6: cDNA from line 1-6 of transgenic plants of the silencing vector as template). (A)-(F), (H) Bars=1 cm

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铁棍山药微型块茎遗传转化体系的建立

Establishment of a Genetic Transformation System for Dioscorea opposita Using Microtuber

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