农杆菌介导大麦无筛选标记转基因植株的获得

doi:10.3864/j.issn.0578-1752.2020.18.002

摘要/Abstract

摘要：

【目的】目前,国内外大麦遗传转化主要利用Golden Promise品种,基因依赖性严重,尤其是大麦的转化效率较低,并且获得安全型转基因大麦植株对其进一步产业化非常重要。建立高效、无筛选标记大麦遗传转化体系,拓展大麦遗传转化的受体基因型,为大麦基因功能解析和大麦转基因育种及商业化种植提供技术保障。【方法】以优良大麦品种Vlamingh为受体,取开花授粉后14 d左右的幼胚为转化材料,通过对培养基成分及培养步骤优化,建立农杆菌介导的高效遗传转化体系,并利用该体系将Bar和GUS在不同T-DNA区段的双T-DNA表达载体pWMB123转化大麦,获得候选转基因植株,然后利用PCR、Bar试纸条、组织化学染色和Southern blot等检测方法,在T₁代转基因植株中成功获得无筛选标记大麦转基因植株。【结果】在愈伤组织分化阶段,发现培养基中添加1.0 mg·L^-1 KT、0.5 mg·L^-1 6-BA和0.05 mg·L^-1 NAA明显促进愈伤组织分化。在转基因植株生根阶段,发现采用添加1.0 mg·L^-1的IBA的SM1（无其他生长素）的生根效果最佳,培养基中添加2.5 mg·L^-1 CuSO₄显著降低了大麦转基因植株白化现象。共转化了138个幼胚,最终获得14株大麦转基因植株,转化效率10.14%。PCR、Bar试纸条、GUS染色等检测证实,T₀代转基因植株中均含有Bar,而仅有10株含有GUS,2个T-DNA的共转化效率为71.43%。选取4个同时含有Bar和GUS的转基因植株,对其自交后代进行检测,在BL8株系中筛选到2株只含GUS而不含Bar的转基因植株,无筛选标记效率为6.9%。在T₁代转基因植株中对Bar和GUS进行了Southern blot鉴定,发现在多数转基因植株中Bar和GUS均为多拷贝整合,进一步证实BL8-15和BL8-19为无筛选标记的转基因植株。【结论】利用大麦品种Vlamingh为转化材料可以较高效率获得转基因植株,提高愈伤组织分化效率和转基因植株生根效率,降低转基因植株白化现象。利用农杆菌介导双T-DNA表达载体转化大麦,成功获得了无筛选标记转基因植株。

关键词: 大麦, 遗传转化, 无筛选标记, 农杆菌介导法

Abstract:

【Objective】Genotype dependence is a very serious problem in barley genetic transformation in which Golden Promise has been mainly used. In addition, the transformation efficiency of barley is still very low in China. Moreover, the generation of marker-free transgenic barley plants is very important to the future commercialization of genetically modified barley due to the considerably increased public concerns. It is necessary to establish an efficient genetic transformation system for generating marker-free transgenic barley plants, expand available barley genotypes for genetic transformation, and provide technical support for functional genomics study and breeding and potential commercialization of genetically modified varieties of barley. 【Method】The composition and auxin adding ratio in the medium used for barley transformation as well as culture regime was optimized to establish an efficient Agrobacterium-mediated genetic transformation system for barley using the immature embryos approximately 14 days post anthesis of a good agronomic trait variety Vlamingh. A double T-DNA vector pWMB123 containing the Bar gene and the GUS gene cassette was introduced into barley by Agrobacterium-mediated transformation to obtain marker-free transgenic barley plants in T₁ generation. 【Result】 It is indicated that the shoot induction medium supplemented with 1.0 mg·L^-1 KT, 0.5 mg·L^-1 6-BA and 0.05 mg·L^-1 NAA significantly promoted the differentiation ability of barley transformed calli. The adding of 2.5 mg·L^-1 copper sulfate in shoot induction medium lightened the albinism of transgenic barley plantlets. Through the exploration of different hormone ratios and medium components, it was found that the SM1 supplemented with 1.0 mg·L^-1 IBA without other auxin had the best rooting effect. A total of 138 immature embryos were transformed with Agrobacterium containing pWMB123 vector, and fourteen transgenic barley plants were obtained with a transformation efficiency of 10.14%. Detection by PCR, Quick Stix strips and histochemical staining assays confirmed that all T₀ plants contained Bar gene, among which four plants did not contain GUS gene. The co-transformation efficiency of the two T-DNAs is 71.43%. In the T₁ populations derived from the four T₀ transgenic lines containing Bar and GUS genes, two transgenic plants with GUS gene and without Bar gene were screened out in line BL8, and the efficiency for marker-free transgenic plants was 6.9%. The two foreign genes of Bar and GUS were identified to be integrated in most T₁transgenic barley plants be multiple copies using Southern blot. It was further confirmed that BL8-15 and BL8-19 were marker-free transgenic plants. 【Conclusion】An efficient genetic transformation system mediated by Agrobacterium-mediated for generating marker-free transgenic plants with healthy roots and less albinism were successfully established in barley.

Key words: barley, transformation, marker-free transgenic, agrobacterium-mediated

龚强,王轲,叶兴国,杜丽璞,徐延浩. 农杆菌介导大麦无筛选标记转基因植株的获得[J]. 中国农业科学, 2020, 53(18): 3638-3649.

GONG Qiang,WANG Ke,YE XingGuo,DU LiPu,XU YanHao. Generation of Marker-Free Transgenic Barley Plants by Agrobacterium-Mediated Transformation[J]. Scientia Agricultura Sinica, 2020, 53(18): 3638-3649.

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组分 Composition	侵染液 IM	共培养培养基 CM	第一次筛选培养基 SM1	第二次筛选培养基 SM2	过渡培养基 TM	分化培养基 DM
MS	0.434	0.434	4.340	4.340	2.700	2.170
麦芽糖 Maltose			30	30	20	20
葡萄糖 Glucose	10	10
乙酰丁香酮Acetosyringone	0.02745	0.03924
谷氨酰胺 Glutamine					0.75
酪蛋白水解物Casein hydrolysate			1	1
肌醇 Myoinositol			0.35	0.35
脯氨酸 Proline			0.69	0.69
盐酸硫胺Thiamine HCl			0.001	0.001	0.004
麦草畏Dicamba			0.0025	0.0025
二氯苯氧乙酸2, 4-D					0.0025
硫酸铜CuSO₄			0.00125	0.00125	0.00125	0.00125
6-苄氨基嘌呤6-BA					0.0001
琼脂糖Agarose		8
植物凝胶Phytagel			3.5	3.5	3.5	3.0
草铵膦Phosphinothricin^a			0.005	0.010	0.010	0.005
羧苄青霉素Carbenicillin^a			0.25	0.25	0.25	0.25
头孢噻肟Cefotaxime^a			0.25

培养基编号 Medium ID	IAA	IBA	NAA
SR1	1	1	1
SR2	1	1	2
SR3	1	2	1
SR4	2	1	1
SR5	1	1	0
SR6	1	0	1
SR7	0	1	1
SR8	1	0	0
SR9	2	0	0
SR10	0	0	1
SR11	0	0	1.5
SR12	0	0	2
SR13	0	1	0
SR14	0	1.5	0
SR15	0	2	0

株系 Lines	Bar⁺GUS⁺	Bar⁺GUS^—	Bar^—GUS⁺
BL1	19	1	0
BL6	23	0	0
BL-7	24	2	0
BL-8	27	0	2