农杆菌介导大麦无筛选标记转基因植株的获得

删除或更新信息，请邮件至freekaoyan#163.com(#换成@)

本站小编 Free考研考试/2021-12-26

龚强^,¹^,², 王轲^,², 叶兴国², 杜丽璞², 徐延浩^,¹

¹长江大学农学院,湖北荆州 434025

²中国农业科学院作物科学研究所,北京 100081

Generation of Marker-Free Transgenic Barley Plants by Agrobacterium-Mediated Transformation

GONG Qiang^,¹^,², WANG Ke^,², YE XingGuo², DU LiPu², XU YanHao^,¹

¹College of Agriculture, Yangtze University, Jingzhou 434025, Hubei

²Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081

通讯作者: 王轲,Tel：18500353117;E-mail： wangke03@caas.cn。徐延浩,Tel：18986663866;E-mail： xyh09@yangtzeu.edu.cn

责任编辑: 李莉
收稿日期:2019-11-15接受日期:2020-03-9网络出版日期:2020-09-16

基金资助:

国家自然科学基金.31201212

Received:2019-11-15Accepted:2020-03-9Online:2020-09-16
作者简介 About authors
龚强,Tel：13397221716;E-mail： gongqiang0206@163.com。

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.

Keywords：barley;transformation;marker-free transgenic;agrobacterium-mediated

PDF (3716KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex 收藏本文
本文引用格式
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 doi:10.3864/j.issn.0578-1752.2020.18.002
GONG Qiang, WANG Ke, YE XingGuo, DU LiPu, XU YanHao. Generation of Marker-Free Transgenic Barley Plants by Agrobacterium-Mediated Transformation[J]. Scientia Acricultura Sinica, 2020, 53(18): 3638-3649 doi:10.3864/j.issn.0578-1752.2020.18.002

0 引言

【研究意义】大麦（Hordeum vulgare L.）是世界第四大禾谷类作物,约占禾谷类作物总产量的8%^[1]。大麦营养成分丰富,富含蛋白质、抗性淀粉、纤维素和维生素等,而且大麦抗旱、耐贫瘠、耐盐碱性能较强,适应性广,在应对恶劣环境化和土壤盐渍化等逆境胁迫方面有着重要意义。目前,国内外大麦转基因研究大多以转化效率较高的品种Golden Promise为受体,利用其他商业化大麦进行转化难以获得转基因植株,或者转化效率很低,限制了大麦基因工程育种的步伐。近几年,大麦高质量参考基因组序列和部分基因功能注释不断完善^[2],需要对大麦进行功能基因组研究。另外,国内还未发现有关无筛选标记转基因大麦研究的报道。因此,提高大麦的遗传转化效率,拓宽大麦遗传转化的基因型,建立大麦无筛选标记遗传转化体系,对于大麦基因工程改良和产业化种植具有重要意义。【前人研究进展】长期以来,有关大麦遗传转化的研究较少,转化效率较低。1994年WAN等^[3]利用基因枪分别转化大麦Golden Promise幼胚、愈伤组织和小孢子,首次获得可育转基因株系。TINGAY等^[4]利用农杆菌介导法建立了大麦Golden Promise幼胚的转化体系,转化效率4.2%。TRIFONOVA等^[5]通过调整生长调节剂的种类、预培养时间和微损伤等方法,获得大麦转基因植株,转化效率1.7%—6.3%。FANG等^[6]将绿色荧光蛋白作为可视标记,以hpt作为筛选基因,利用农杆菌介导转化大麦Golden Promise幼胚,转化效率3.4%。TRAVELLA等^[7]依据荧光原位杂交技术（fluorescence in situ hybridization,FISH）检测结果,比较了农杆菌介导和基因枪介导对大麦转化效率和转基因拷贝数等的影响,发现农杆菌介导的转化效率是基因枪的2倍,且在利用农杆菌介导获得的转基因植株中目标基因表达稳定、沉默情况较少。BARTLETT等^[8]对农杆菌转化大麦后的再生过程进行了进一步优化,将hpt标记基因和luc荧光素报告基因转化大麦Golden Promise幼胚,转化效率提高到25%。HENSEL等^[9]进一步研究了外植体不同处理方法和共培养条件等因素对大麦转化效率的影响,进一步提高了大麦Golden Promise幼胚的转化效率（高达86.0%）,商业化春性大麦品种Optic、Helium和冬性大麦品种Igri、Tafeno等的转化效率也达到0.2%—7.8%。目前,国内有关大麦遗传转化的研究非常少,还处在建立高频再生体系和提高遗传转化效率的研究阶段^{[10,11,12,13,14,15,16,17,18,19,20]}。国内最早获得大麦转基因植株的研究报道出现于2004年,任江萍等^[21]以啤酒大麦品种晋引6号幼胚为材料,用基因枪法对1 200个幼胚进行了轰击,获得了7株阳性植株,转化效率为0.58%,在过表达TrxS的转基因植株中,α-淀粉酶和β-淀粉酶的活性有显著性提高^[22]。吕维涛等^[23]通过农杆菌介导法将反义磷脂酶Dγ转入大麦,获得了可耐0.7%NaCl的植株。李静雯等^[24]利用RNAi抑制B-hordein的合成,降低了大麦品种Golden Promise籽粒蛋白质的含量。随着转基因技术的发展, 转基因植物的安全性已经引起了公众的广泛关注,其中筛选标记基因的存在是转基因植物安全性评价的主要隐患。人们先后提出了几种获得无筛选标记转基因植物的技术,包括共转化法、定位重组体系、多元自动转化载体系统、转座子再定位系统和同源重组体系等,其中共转化法应用最为普遍^[25,26]。特别是农杆菌介导的双T-DNA载体共转化法,不但可以避免将细菌筛选标记转入植物,而且可以从转基因植物中排除nptII、Bar、hpt等筛选标记,在获得安全型转基因植物方面具有独特优势^[26]。农杆菌介导的共转化系统已经在很多植物如烟草^[27]、大豆^[28]、玉米^[29]、高粱^[30]、水稻^[31]和小麦^[26]中成功获得无筛选标记的转基因植株。【本研究切入点】虽然大麦Golden Promise的遗传转化效率较高,但是其他大麦品种转化效率仍然很低,需要拓展大麦基因型范围和提高转化效率。转基因大麦安全问题无疑会受到关注,在国内建立大麦无筛选标记转基因系统迫在眉睫。【拟解决的关键问题】本研究拟以优良大麦品种Vlamingh为受体,通过对培养基成分及培养步骤进行优化,建立其高效遗传转化体系。进一步利用农杆菌介导的共转化法将双T-DNA表达载体导入大麦,并通过后代自然分离获得无筛选标记转基因大麦植株。本研究将拓宽大麦转基因的受体基因型范围,为大麦基因功能解析和转基因育种提供高效的转化体系,为安全型转基因大麦材料创制提供技术保障。

1 材料与方法

1.1 植物材料和表达载体

供试材料为澳大利亚主栽大麦品种Vlamingh,播种于中国农业科学院作物科学研究所人工气候室,生长期间的温度为25℃,光周期为16 h光照、8 h黑暗。取授粉后12—14 d 的幼胚用于遗传转化。T₀代转基因大麦植株及时移栽,放置于人工气候室中生长和检测。T₁代转基因大麦植株2018年9月种植于中国农业科学院试作物科学研究所温室,用于筛选无筛选标记转基因植株。

植物表达载体pWMB123由叶兴国实验室构建^[26],包含2段独立的T-DNA区域,一段T-DNA区域含有Bar,另一段T-DNA区域含有GUS（图1）。将表达载体pWMB123转入农杆菌菌株C58C1后用于大麦转化。

图1

新窗口打开|下载原图ZIP|生成PPT
图1双T-DNA表达载体pWMB123结构

Fig. 1The structure of pWMB123 vector with double T-DNA regions

1.2 培养基

大麦遗传转化所用的基本培养基为MS,在MS培养基中添加不同有机物质（表1）,所有培养基的pH调整为5.8,121℃灭菌15 min。

Table 1
表1
表1农杆菌介导转化大麦幼胚所用培养基及其组成
Table 1Composition of the media used for Agrobacterium-mediated barley immature embryo transformation (g·L^-1)

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

^a需过滤除菌,培养基灭菌完成后再添加。IM：侵染液;CM：共培养培养基;SM1：第一次筛选培养基;SM2：第二次筛选培养基;TM：过渡培养基;DM：分化培养基
^a Need to filter sterilization, add the medium after sterilization. IM: Infection medium; CM: Co-culture medium; SM1: First selection medium; SM2: Second selection medium; TM: Transition medium; DM: Differentiation medium

新窗口打开|下载CSV

将大麦幼胚诱导的愈伤组织分为3份,1份转移到过渡培养基（transition medium,TM）,另外2份分别转移到分化培养基（differentiation medium,DM）和改良的分化培养基（modified differentiation medium,DMM：DM培养基添加kinetin（KT）1 mg·L^-1、6- benzylaminopurine（6-BA）0.5 mg·L^-1和1-naphthaleneacetic acid（NAA）0.05 mg·L^-1）,光照培养2周,诱导绿芽分化。将完整的大麦幼胚接种到1/2MS培养基上,进行成苗培养,利用该方法探索吲哚乙酸（indole-3-acetic acid,IAA）、吲哚丁酸（indole butyric acid,IBA）和萘乙酸（NAA）不同配比对大麦植株生根的影响。共利用15种不同生长素配比的培养基对大麦植株的生根效果进行比较（表2）。此外,根据已发表的文献^[9,32],探索添加IBA,且没有其他生长素的第一次筛选培养基（first selection medium,SM1）、DM和大麦再生培养基（barley regeneration medium,BRM,NH₄NO₃ 0.32 g·L^-1、KNO₃ 3.64 g·L^-1、KH₂PO₄ 0.325 g·L^-1、CaCl₂·2H₂O 0.441 g·L^-1、MgSO₄·7 H₂O 0.246 g·L^-1、NaFeEDTA 27.53 mg·L^-1、MnSO₄·H₂O 84 μg·L^-1、H₃BO₃ 31 μg·L^-1、ZnSO₄·7 H₂O 72 μg·L^-1、Na₂ MoO₄·2H₂O 1.2 μg·L^-1、CuSO₄·5 H₂O 0.25 μg·L^-1、CoCl₂·6 H₂O 0.24 μg·L^-1、KI 1.7 μg·L^-1、Vitamin B5 112 mg·L^-1、6-BAP 0.225 mg·L^-1、L-glutamine 146.4 mg·L^-1、36 g·L^-1 maltose stock, and 196 μL·L^-1和CuSO₄·5 H₂O 0.245 mg·L^-1）的生根效果。

Table 2
表2
表2不同激素配比的15种1/2MS生根培养基
Table 2Composition of 15 rooting media with different hormone ratios on the basis of 1/2 MS medium (mg·L^-1)

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

新窗口打开|下载CSV

1.3 农杆菌介导转化大麦幼胚

参考BARTLETT等^[8]方法进行农杆菌介导的大麦转化,并进行较大改动,具体步骤如下：

1.3.1 取样和灭菌分批次种植大麦受体材料,取开花授粉后14 d左右的未成熟大麦籽粒,用70%乙醇表面灭菌1 min,然后用15%次氯酸钠震荡灭菌15 min,最后用无菌水洗4—5次。在显微镜下用解剖刀掀开大麦种皮,用尖镊子小心剥取大麦完整胚放置于没有乙酰丁香酮的侵染液（infection medium,IM）培养基中。

1.3.2 农杆菌侵染和共培养将携带表达载体pWMB123的C58C1农杆菌28℃过夜培养,取2 mL菌液5 000 r/min离心,然后加入2 mL IM培养基,侵染大麦幼胚约10 min,然后将胚放入共培养培养基（co-culture medium,CM）,23℃黑暗共培养。

1.3.3 愈伤组织的诱导共培养2 d后,切除胚轴,盾片面向上转移到SM1,进行愈伤的诱导和第一次筛选培养,25℃黑暗培养14 d左右,然后将愈伤组织转移到SM2进行第二次筛选培养,25℃黑暗培养21 d左右。

1.3.4 愈伤组织的分化,转基因苗的生根及移栽将愈伤组织转移到DM,25℃光照条件下培养2—3周,诱导绿芽分化。然后及时将绿芽转到1/2MS生根培养基（rooting medium,RT）继续25℃光照培养,根系健壮的再生小植株直接移栽到花盆中。

1.4 T₀代转基因植株的鉴定

从移栽成活的抗性再生植株上取少量叶片,利用Bar蛋白抗体试纸条法（取1 cm长的叶片,液氮速冻破碎;加入0.4 mL Extractin buffer,将试纸条标有指示箭头的一端浸入buffer内,静止1 min,观察Bar蛋白抗体表达结果）检测Bar,利用组织化学染色法结合PCR扩增检测GUS。

1.5 T₁代中无筛选标记转基因植株筛选

种植T₀代Bar和GUS均为阳性的转基因植株,利用新型植物基因组提取试剂盒CW0531（康为世纪生物科技有限公司）从叶片中提取基因组DNA,然后分别用特异引物Bar-F：5′-ACCATCGTCAACCACTACATCG-3′,Bar-R：5′-GCTGCCAGAAACCCACGTCATG-3′和GUS-F：5′-CAAGGAAATCCGCAACCATATC-3′,GUS-R：5′-TCAAACGTCCGAATCTTCTCCC-3′对基因组中的Bar和GUS进行PCR检测。PCR扩增体系为2×Taq Master Mix（诺唯赞生物科技有限公司,南京）7.5 μL、10 pmol·L^-1引物各0.5 μL和DNA模板50—200 ng,补充ddH₂O至15 μL。反应程序为95℃ 5 min;95℃ 30 s,60℃ 30 s,72℃ 1 min,35个循环;72℃ 5 min。取5 μL PCR产物用1%琼脂糖凝胶电泳分析。统计T₁代转基因植株中Bar和GUS的分离情况,筛选只含有GUS不含有Bar的无筛选标记转基因植株。

1.6 Southern blot杂交

利用CTAB法从转基因大麦叶片中提取高质量基因组DNA,对30 μg基因组DNA用限制性内切酶EcoRⅠ进行过夜酶切,然后利用1%的琼脂糖凝胶在30 V电压下电泳16 h。将凝胶置于20%盐酸溶液中变性处理10 min,用ddH₂O清洗2—3次。通过真空泵转膜仪将酶切后的DNA片段转移至带正电的尼龙膜上。以pWMB123质粒DNA为模板,分别用Bar和GUS特异引物（Bar-F、Bar-R、GUS-F和GUS-R）扩增的片段作为探针,探针标记和杂交过程参考DIG High Prime DNA labelling and Detection Starter Kit II（Roche,美国）说明书进行。利用Tanon 5200化学发光成像系统显影。

2 结果

2.1 不同培养基对大麦愈伤组织分化植株和生根的影响

将SM2培养基上的大麦幼胚诱导的愈伤组织分为3份,1份按照BARTLETT等^[8]方法转移到TM上,在弱光照条件下（光照条件下用纸遮盖培养皿）培养2周;另外2份分别转移到DM和DMM上。大麦幼胚愈伤组织经在DM和DMM上正常光照培养2周后,发现DMM上大多数愈伤组织的能产生绿芽点或绿苗（图2-A）,而DM上仅有少数愈伤组织产生绿芽点（图2-B）,表明DMM比DM更适合大麦愈伤组织分化。将在TM弱光条件下继代培养的愈伤组织转移到DMM培养2周后发现,虽然有绿芽的产生（图2-C）,但分化率明显不及直接分化的愈伤组织。

图2

新窗口打开|下载原图ZIP|生成PPT
图2大麦愈伤组织在不同培养基上的分化情况

A：愈伤组织不经过TM培养直接在DMM分化情况;B：愈伤组织不经过TM培养直接在DM分化情况;C：愈伤组织经过TM培养后在DMM分化情况
Fig. 2Differentiation performance of barley callus on different shoot induction medium

A: Differentiation on DMM without the culture step on TM; B: Differentiation on DM without the culture step on TM; C: Differentiation on DMM after the culture step on TM

将分化培养基上产生大麦再生小植株直接转移到生根培养基1/2MS上,发现再生植株产生白化现象（图3-A）。然后在培养基中添加2.5 mg·L^-1的CuSO₄,发现可以显著降低白化现象（图3-B）。因此,后续所有使用的生根培养基都添加了2.5 mg·L^-1的CuSO₄,但是转基因苗在1/2MS培养基上仍然不生根。

图3

新窗口打开|下载原图ZIP|生成PPT
图3CuSO₄对转基因大麦小植株白化现象的影响

A：不添加CuSO₄;B：添加CuSO₄
Fig. 3The effect of copper sulfate on the albinism of transgenic barley plants

A: 1/2MS medium without copper sulfate; B: 1/2MS medium with 2.5 mg·L^-1 copper sulfate

为了解决大麦转基因植株生根困难的问题,首先利用一步成苗培养方法激素不同配比对大麦植株生根的影响。共利用15种不同生长素配比的培养基对大麦植株的生根效果进行了比较（表2）,培养10 d后发现单加IBA的生根效果最好（图4）。不同浓度的IBA都可以显著促进大麦的生根,其中1 mg·L^-1的生根效果最佳,因此,生根培养基中选择添加到1 mg·L^-1的IBA。

图4

新窗口打开|下载原图ZIP|生成PPT
图4大麦再生植株在不同激素配比培养基上的生根情况

Fig. 4Rooting status of transgenic barley plantlets on 15 media with different hormone ratios

图5

新窗口打开|下载原图ZIP|生成PPT
图5不同培养基成分对大麦再生植株生根的影响

A：添加IBA的DM;B：添加IBA的BRM;C：只添加IBA（没有添加其他生长素）的SM1
Fig. 5Effect of different media containing various auxin ratios on the rooting of transgenic barley plantlets

A: DM with IBA; B: BRM with IBA; C: SM1 with IBA and without other auxins

虽然一步成苗的大麦植株在添加IBA的1/2MS培养基上生根效果很好,但转基因大麦植株在该培养基上生根效果并不理想。因此,又探索了添加IBA的没有其他生长素的SM1、DM和BRM的生根效果。结果表明,大麦转基因植株在仅添加IBA无其他生长

2.2 大麦转基因T₀代阳性植株的获得和检测

利用携带pWMB123表达载体的C58C1农杆菌共转化了大麦品种Vlamingh的138个幼胚,经过愈伤组织诱导和2次筛选培养,获得了125个抗性愈伤组织,愈伤组织诱导率为91%。抗性愈伤组织经过在优化过的培养基上分化和生根,共获得14棵候选转基因植株（BL1—BL14）,转化效率为10.14%。将幼苗及时移栽至花盆,在温室中生长。然后对T₀代转基因植株进行试纸条检测,并提取基因组DNA进行目标基因的PCR检测。Bar蛋白抗体试纸条检测结果显示全部转基因植株中均含有Bar（图6-A）;组织化学染色和PCR扩增结果显示,其中4株转基因株（BL2—BL5）不含有GUS（图6-B和图6-C）,说明携带GUS和Bar的2个T-DNA的共转化效率为71.43%。

图6

新窗口打开|下载原图ZIP|生成PPT
图6T₀代转基因大麦植株鉴定

A：Bar蛋白抗体试纸条检测;B：GUS组织化学染色;C：GUS的PCR检测。1—14是大麦T₀代转基因植株;WT：Vlamingh;P：pWMB123质粒;M：DL5000 marker
Fig. 6Identification of T₀ transgenic barley plants

A: Detection of Bar protein by Quick Stix strips; B: Detection of GUS gene by histochemical staining; C: Detection of GUS gene by PCR. 1-14: T₀transgenic barley plants; WT: Vlamingh; P: pWMB123 plasmid; M: DL5000 marker

2.3 T₁代中无筛选标记转基因大麦植株的获得

为了获得无筛选标记大麦转基因植株,选取4个Bar和GUS均为阳性的T₀代大麦转基因植株BL1、BL6、BL7和BL8,形成T₁代株系,研究T₁代中外源转入基因的分离情况。理论上,T₁代应该分离出4种类型：Bar⁺GUS^—、Bar^—GUS⁺、Bar^—GUS^—和Bar⁺GUS⁺。利用PCR方法共检测了98株T₁代转基因植株（图7）,在BL8株系获得了2株无筛选标记（Bar^—GUS⁺）植株（表3）,共检测到3株Bar⁺GUS^—类型植株,没有检测到Bar^—GUS^—类型植株,其他植株全部为Bar⁺GUS⁺类型（表3）。根据PCR检测结果,选取部分T₁代转基因植株,分别利用Bar和GUS作探针进行Southern blot鉴定（图8）,进一步证实BL8-15和BL8-19为无筛选标记的转基因植株。在BL-8株系中无筛选标记植株的获得效率为6.9%,在全部T₁代植株中无筛选标记植株的获得效率仅有2.04%。以上结果也证实,Bar和GUS可以在大麦转基因后代中稳定遗传和表达。

图7

新窗口打开|下载原图ZIP|生成PPT
图7T₁代转基因大麦株系BL8中Bar（A）和GUS（B）的PCR检测

1—22：T₁转基因株系BL8;23：Vlamingh;P：pWMB123质粒;M：DL2000 marker;15和19为筛选到的无筛选标记转基因植株
Fig. 7PCR detection of Bar (A) and GUS (B) genes in T₁ transgenic barley plants

1-22: BL-8 of T₁transgenic barley plants; 23: Vlamingh; P: pWMB123 plasmid; M: DL2000 marker; 15 and 19 were marker-free transgenic plants

Table 3
表3
表3T₁代株系中Bar和GUS的分离情况
Table 3Genotyping detection for the Bar and GUS genes in the selected T₁ lines by PCR

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

新窗口打开|下载CSV

图8

新窗口打开|下载原图ZIP|生成PPT
图8T₁代转基因大麦植株的Southern blot鉴定

A：Bar的Southern blot鉴定;B：GUS的Southern blot鉴定。WT：受体对照;M：DL15000 marker;BL8-15和BL8-19为筛选到的无筛选标记转基因植株
Fig. 8Southern blot detection of T₁ transgenic barley plants

A: Southern blot of Bar; B: Southern blot of GUS. WT: Wild type; M: Marker; BL8-15 and BL8-19were marker-free transgenic plants

3 讨论

3.1 大麦遗传转化体系优化

目前,国内外大麦遗传转化的受体品种主要为Golden Promise,基因型范围非常窄。本研究利用了再生能力比较强的大麦品种Vlamingh,拓展了大麦遗传转化的范围。尤其重要的是,该大麦品种为春性,在温室条件下其生长周期比Golden Promise短一个月以上, Vlamingh作为受体材料不但可以缩短转化周期,还可以减少在温室培养材料的水电等消耗。

本研究参考BARTLETT等^[8]描述的大麦遗传转化体系,并根据之前的文献报道和叶兴国实验室在小麦遗传转化方面积累的经验,对大麦遗传转化体系进行了优化。首先,发现同样的大麦愈伤组织经过在TM上培养后分化能力下降（图2）,因此认为可以省略TM这一步骤,这样不但缩短了大麦的遗传转化过程,也节省了时间和成本。另外,还对分化培养基进行了优化发现DMM分化效果最好（图2）。通过调节生根培养基中不同激素比例（图4）和培养基关键成分（图5）,筛选到了生根效果较好的SM1。利用优化后的转化体系植株再生率很高,但是由于改良分化培养基和生根培养基用了一些愈伤组织和再生植株,减少了器官齐全转基因植株的数量,最终的转化效率只有10.14%,该体系实际转化效率应该更高。

3.2 无筛选标记转基因大麦植株获得效率

WANG等^[26]对农杆菌转化双T-DNA载体的12个小麦T₁株系进行了无筛选标记转基因植株筛选,在3个株系中获得了无筛选标记转基因植株,无筛选标记转基因植株的获得效率分别为40.0%、14.3%和7.5%;而在全部T₁代群体（共12个株系）中无筛选标记转基因植株的获得效率仅为4.3%。MATTHEWS等^[33]利用农杆菌转化双T-DNA载体,也成功获得了3个只含目标基因的无筛选标记转基因大麦植株,无筛选标记转基因植株获得效率0.83%—18.55%。根据孟德尔遗传定律,如果筛选标记基因和目标基因在T₀代转基因植株中都以单拷贝形式存在,T₁代中无筛选标记植株的获得效率应为18.75%,而本研究中BL8株系T₁代中无筛选标记转基因植株的频率仅为6.9%。本研究T₁代大麦转基因植株的Southern blot鉴定结果显示（图8）,Bar和GUS在转基因植株中整合的拷贝数较高,尤其Bar的拷贝数在多数转基因植株中高达10个以上,显著降低了无筛选标记转基因植株的频率。因此,为了提高无筛选标记转基因植株获得效率,需要降低转基因植株中Bar整合的拷贝数。

MCCORMAC等^[27]发现双T-DNA载体中2个T-DNA区的大小影响外源转入基因整合的拷贝数。WANG等^[26]研究结果也表明,携带Bar的T-DNA区段较小导致其在转基因植株中整合的拷贝数较多。在本研究中,含Bar的T-DNA区段长度仅为1.7 kb左右,而含GUS的T-DNA区段长度为4.4 kb,Southern结果也显示GUS基因在转基因植株中的拷贝数确实低于Bar。因此,增加含Bar的T-DNA区段长度可以降低Bar在T₀代转基因植株的拷贝数,从而达到增加T₁代无筛选标记转基因植株的获得效率。

3.3 获得无筛选标记转基因植物的意义

目前,转基因植物的生物安全性越来越引起了公众关注,成为了转基因植物商业化种植的主要限制。实际上选择标记的存在是转基因作物商业化的主要障碍之一。早期消除选择标记和不必要的载体骨架将有助于克服生物安全性限制,并有助于环境释放转基因大麦。因此,开发无标记的转基因大麦对于大麦转基因育种和大麦转基因商业化有重要意义。

在植物遗传转化研究中使用的标记基因主要有2种类型：第一种是细菌选择性标记,如bla、kan和aadA等;第二种是植物选择标记,如nptII、Bar和hpt。尽管标记基因应用于遗传转化过程显著提高了植物的转化效率,但是由于大多数选择标记编码抗生素或除草剂的基因,获得转基因植株后这些基因在植物基因组中的存在和表达就变得多余。基因枪介导的共转化可以去除植物选择标记,但是由于细菌选择标记与载体中的目标基因紧密连接,因此不能去除细菌选择标记。基因枪转化线性植物表达盒可以成功去除转基因植物中的所有标记基因^[34,35]。因此,无筛选标记转基因植株通常利用基因枪介导或农杆菌介导的共转化方法获得。但是,无筛选标记转基因植株的获得效率与筛选标记基因整合的拷贝数密切相关,而一般情况下基因枪转化产生的拷贝数显著高于农杆菌转化。所以,农杆菌介导的共转化法是获得无筛选标记转基因植株的最优方法。

4 结论

本研究发现改良的DMM能显著提高大麦愈伤组织的分化能力;添加1.0 mg·L^-1的IBA的SM1（无其他生长素）的生根效果最佳;利用大麦品种Vlamingh建立了高效遗传转化体系且转化效率可达10.14%,进一步利用双T-DNA的表达载体在T₁代中通过自然分离成功获得了无筛选标记大麦转基因植株。

参考文献原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1]

国家大麦青稞产业技术体系. 中国现代农业产业可持续发展战略研究·大麦青稞分册, 北京: 中国农业出版社, 2018.

[本文引用: 1]

China Agriculture Research System Of Barley. China's Modern Agricultural Industry Sustainable Development Strategy Research · Barley Volume. Beijing: China Agriculture Press, 2018: 2-8. (in Chinese)

[本文引用: 1]

[2]

MASCHER

, GUNDLACH

, HIMMELBACH

, BEIER

, TWARDZIOK

S O

, WICKER

, RADCHUK

, DOCKTER

, HEDLEY

P E

, RUSSELL

, BAYER

, RAMSAY

, LIU

, HABERER

, ZHANG

X Q

, ZHANG

, BARRERO

R A

, LI

, TAUDIEN

, GROTH

, FELDER

, HASTIE

, SIMKOVA

, STANKOVA

, VRANA

, CHAN

, MUNOZ-AMATRIAIN

, OUNIT

, WANAMAKER

, BOLSER

, COLMSEE

, SCHMUTZER

, ALIYEVA-SCHNORR

, GRASSO

, TANSKANEN

, CHAILYAN

, SAMPATH

, HEAVENS

, CLISSOLD

, CAO

, CHAPMAN

, DAI

, HAN

, LI

, LIN

, MCCOOKE

J K

, TAN

, WANG

, YIN

, ZHOU

, POLAND

J A

, BELLGARD

M I

, BORISJUK

, HOUBEN

, DOLEZEL

, AYLING

, LONARDI

, KERSEY

, LANGRIDGE

, MUEHLBAUER

G J

, CLARK

M D

, CACCAMO

, SCHULMAN

A H

, MAYER

KF X

, PLATZER

, CLOSE

T J

, SCHOLZ

, HANSSON

, ZHANG

, BRAUMANN

, SPANNAGL

, LI

, WAUGH

, STEIN

. A chromosome conformation capture ordered sequence of the barley genome
Nature, 2017,544(7651):427-433.

DOI:10.1038/nature22043 URLPMID:28447635 [本文引用: 1]

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

[3]

WAN

, LEMAUX

P G

. Generation of large numbers of independently transformed fertile barley plants
Plant Physiology, 1994,104(1):37-48.

DOI:10.1104/pp.104.1.37 URLPMID:12232059 [本文引用: 1]

A rapid, efficient, and reproducible system to generate large numbers of independently transformed, self-fertile, transgenic barley (Hordeum vulgare L.) plants is described. Immature zygotic embryos, young callus, and microspore-derived embryos were bombarded with a plasmid containing bar and uidA either alone or in combination with another plasmid containing a barley yellow dwarf virus coat protein (BYDVcp) gene. A total of 91 independent bialaphos-resistant callus lines expressed functional phosphinothricin acetyltransferase, the product of bar. Integration of bar was confirmed by DNA hybridization in the 67 lines analyzed. Co-transformation frequencies of 84 and 85% were determined for the two linked genes (bar and uidA) and for two unlinked genes (bar and the BYDVcp gene), respectively. More than 500 green, fertile, transgenic plants were regenerated from 36 transformed callus lines on bialaphos-containing medium; albino plants only were regenerated from 41 lines. T0 plants in 25 lines (three plants per line) were analyzed by DNA hybridization, and all contained bar. Most contained the same integration patterns for the introduced genes (bar, uidA, and the BYDVcp gene) as their parental callus lines. Transmission of the genes to T1 progeny was confirmed in the five families analyzed by DNA hybridization. A germination test of immature T1 embryos on bialaphos-containing medium was useful for selecting individuals that were actively expressing bar, although this was not a good indicator of the presence or absence of bar. Expression of bar in some progeny plants was indicated by resistance to the herbicide Basta. The T1 plants were in soil approximately 7 months after bombardment of the immature embryo.

[4]

TINGAY

, MCELROY

, KALLA

, FIEG

, WANG

, THORNTON

, BRETTELL

. Agrobacterium tumefaciens-mediated barley transformation
The Plant Journal, 1997,11(6):1369-1376.

DOI:10.1046/j.1365-313X.1997.11061369.x URL [本文引用: 1]

[5]

TRIFONOVA

, MADSEN

, OLESEN

. Agrobacterium -mediated transgene delivery and integration into barley under a range of in vitro culture conditions
Plant Science, 2001,161(5):871-880.

DOI:10.1016/S0168-9452(01)00479-4 URL [本文引用: 1]

[6]

FANG

Y D

, AKULA

, ALTPETER

. Agrobacterium -mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA: barley genomic DNA junctions
Journal of Plant Physiology, 2002,159(10):1131-1138.

DOI:10.1078/0176-1617-00707 URL [本文引用: 1]

[7]

TRAVELLA

, ROSS

S M

, HARDEN

, EVERETT

, SNAPE

J W

, HARWOOD

W A

. A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques
Plant Cell Reports, 2005,23(12):780-789.

DOI:10.1007/s00299-004-0892-x URL [本文引用: 1]

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

[8]

BARTLETT

J G

, ALVES

S C

, SMEDLEY

, SNAPE

J W

, HARWOOD

W A

. High-throughput Agrobacterium -mediated barley transformation
Plant Methods, 2008,4(1):22.

DOI:10.1186/1746-4811-4-22 URL [本文引用: 4]

[9]

HENSEL

, VALKOV

, MIDDLEFELL-WILLIAMS

, KUMLEHN

. Efficient generation of transgenic barley: The way forward to modulate plant-microbe interactions
Journal of Plant Physiology, 2008,165(1):71-82.

DOI:10.1016/j.jplph.2007.06.015 URLPMID:17905476 [本文引用: 2]

Stable genetic transformation represents the gold standard approach to the detailed elucidation of plant gene functions. This is particularly relevant in barley, an important experimental model widely employed in applied molecular, genetic and cell biological research, and biotechnology. Presented are details of the establishment of a protocol for Agrobacterium-mediated gene transfer to immature embryos, which enables the highly efficient generation of transgenic barley. Advancements were achieved through comparative experiments on the influence of various explant treatments and co-cultivation conditions. The analysis of representative numbers of transgenic lines revealed that the obtained T-DNA copy numbers are typically low, the generative transmission of the recombinant DNA is in accordance with the Mendelian rules and the vast majority of the primary transgenics produce progeny that expresses the respective transgene product. Moreover, the newly established protocol turned out to be useful to transform not only the highly amenable cultivar (cv.) 'Golden Promise' but also other spring and winter barley genotypes, albeit with substantially lower efficiency. As a major result of this study, a very useful tool is now available for future functional gene analyses as well as genetic engineering approaches. With the aim to modify the expression of barley genes putatively involved in plant-fungus interactions, numerous transgenic plants have been generated using diverse expression cassettes. These plants represent an example of how transformation technology may contribute to further our understanding of important biological processes.

[10]

马春业. 农杆菌介导miR396基因对大麦愈伤组织的遗传转化
[D]. 武汉: 华中农业大学, 2016.

[本文引用: 1]

C Y

. Genetic transformation of miR396 gene into barley callus by Agrobacterium tumefaciens infection
[D]. Wuhan: Huazhong Agricultural University, 2016. (in Chinese)

[本文引用: 1]

[11]

马玲珑, 任盼荣, 汪军成, 孟亚雄, 马小乐, 李葆春, 王化俊. 啤酒大麦品种甘啤4号成熟胚再生体系的建立
分子植物育种, 2015,13(3):663-669.

[本文引用: 1]

L L

, REN

P R

, WANG

J C

, MENG

Y X

, MA

X L

, LI

B C

, WANG

H J

. Establishing regeneration system from mature embryos of beer barley variety Ganpi 4
Molecular Plant Breeding, 2015, 2015,13(3):663-669. (in Chinese)

[本文引用: 1]

[12]

余桂红, 张旭, 孙晓波, 马鸿翔. 大麦苏啤4号幼胚愈伤组织的诱导及植株的高频再生
江苏农业学报, 2013,29(5):953-956.

[本文引用: 1]

G H

, ZHANG

X B

, MA

H X

. Calli induction from immature embryos and plantlet high frequency regeneration of Hordeum vulgare L. cv.Supi 4
Jiangsu Journal of Agricultural Science, 2013,29(5):953-956. (in Chinese)

[本文引用: 1]

[13]

王兴珍, 林国梁, 赖勇, 杨轲, 孟亚雄, 李葆春, 马小乐, 尚勋武, 王化俊. 西北地区特色大麦品种成熟胚离体培养的初步研究
麦类作物学报, 2013,33(2):286-289.

DOI:10.7606/j.issn.1009-1041.2013.02.013 URL [本文引用: 1]

为筛选组织培养特性优良的大麦品种并建立高效的再生体系，选用西北地区主栽品种甘啤3号、甘啤4号、甘啤6号及德国引进品种玛俐、博乐共5个大麦品种，分别研究了基因型、培养基成分、种子处理方式等因素对大麦成熟胚组织培养的影响。结果表明，不同大麦品种的出愈率、绿点率和绿苗率均存在极显著差异，其中博乐的综合效应最好。脱分化培养基A3、分化培养基C2是适合进行再生体系构建的培养基。胚刮碎与纵切处理的愈伤组织诱导率显著高于横切。甘啤3号、甘啤4号、博乐的愈伤组织诱导频率和分化绿苗率均较高，是适合进行遗传转化的受体材料。

WANG

X Z

, LIN

G L

, LAI

, YANG

, MENG

Y X

, LI

B C

, MA

X L

, SHANG

X W

, WANG

H J

. Preliminary study of tissue culture for mature embryo from local feature barley
Journal of Triticeae Crops, 2013,33(2):286-289. (in Chinese)

DOI:10.7606/j.issn.1009-1041.2013.02.013 URL [本文引用: 1]

黎冬华, 廖玉才, 李和平. 根癌农杆菌介导的大麦茎尖转化研究
麦类作物学报, 2012,32(1):44-47.

DOI:10.7606/j.issn.1009-1041.2012.01.008 URL [本文引用: 1]

为了建立农杆菌介导的大麦茎尖遗传转化技术，以我国大麦主推品种鄂大麦9号、鄂大麦32122为材料，以农杆菌介导法转化大麦茎尖，经PPT筛选获得了转抗除草剂基因的大麦株系。PCR鉴定表明，以鄂大麦9号茎尖为受体，筛选获得4个转基因植株，阳性率为0.59%；以鄂大麦32122茎尖为受体，筛选获得10个阳性植株，阳性率为1.96%。转基因T₁代Southern杂交分析证实，外源基因确已整合到大麦基因组中，已鉴定的转基因株系均含单个转基因拷贝，不同转基因株系整合的位点不同。因此大麦茎尖可作为农杆菌转化的受体。本研究结果为拓宽大麦遗传转化受体提供了技术和方法。

D H

, LIAO

Y C

, LI

H P

. Transformation of shoot apical meristems of elit barley cultivars via agribacterium-mediated transformation
Journal of Triticeae Crops, 2012,32(1):44-47. (in Chinese)

DOI:10.7606/j.issn.1009-1041.2012.01.008 URL [本文引用: 1]

高润红, 杜志钊, 郭桂梅, 邹磊, 何婷, 陈志伟, 李梁, 陆瑞菊, 黄剑华. 优良大麦品种花30幼胚遗传转化体系的优化
植物遗传资源学报, 2012,13(5):901-906.

[本文引用: 1]

GAO

R H

, DU

Z Z

, GUO

G M

, ZOU

, HE

, CHEN

Z W

, LI

, LU

R J

, HUANG

J H

. Optimization of genetic transformation system for immature embryos of improved barley( Hordeum vulgare L.) variety Hua 30
Journal of Plant Genetic Resources, 2012,13(5):901-906. (in Chinese)

[本文引用: 1]

[16]

闫永荣, 张正英, 李静雯, 李淑洁. 甘啤4号大麦幼胚愈伤组织诱导及植株再生的研究
分子植物育种, 2010,8(1):89-93.

[本文引用: 1]

YAN

Y R

, ZHANG

Z Y

, LI

J W

, LI

S J

. Studies on callus induction and plant regeneration from immature embryos of Ganpi4 hao
Molecular Plant Breeding, 2010,8(1):89-93. (in Chinese)

[本文引用: 1]

[17]

李静雯, 张正英. 根癌农杆菌介导的大麦幼胚遗传转化影响因素研究
大麦与谷类科学, 2010(2):1-6.

URL [本文引用: 1]

为建立和优化根癌农杆菌介导的大麦遗传转化体系,以大麦幼胚为转化受体,研究了根癌农杆菌介导法转化大麦的主要影响因素.结果表明,以gus基因瞬时表达率、抗性愈伤组织诱导率为转化指标,筛选出了对大麦幼胚感染力比较强的农杆菌菌株EHA105,以及高敏感受体基因型甘啤4号和秀81-47等.实验结果还表明,预培养1 d的大麦幼胚具有较高的瞬时表达率(91%),是较好的转化受体.菌液侵染浓度OD600=0.5,侵染时间为30 min时转化效率最高.在此基础上,侵染后幼胚与农杆菌采用液体共培养方式可明显提高转化效率,gus基因瞬时表达率最高可达90%.在优化条件下,转化约30 d后抗选择剂潮霉素(Hyg)的愈伤组织占受体组织总数的34.2%.

J W

, ZHANG

Z Y

. Study on factors influencing the genetic transformation of immature barley embryos mediated byAgrobacterium tumefaciens
Barley and Cereal Science, 2010(2):1-6. (in Chinese)

URL [本文引用: 1]

郭晓琳, 张红伟, 刘欣洁, 刘亚娟, 张锋, 孙东发, 谭振波. 大麦成熟胚愈伤组织的诱导和植株再生的研究
植物遗传资源学报, 2005,6(4):418-422.

[本文引用: 1]

GUO

X L

, ZHANG

H W

, LIU

X J

, LIU

Y J

, ZHANG

, SUN

D F

, TAN

Z B

. Study on callus induction and plant regeneration from mature embryos of barley elite cultivars
Journal of Plant Genetic Resources, 2005,6(4):418-422. (in Chinese)

[本文引用: 1]

[19]

李会勇, 尹钧, 刘雷, 任江萍, 郭向云, 李志远. Cu²⁺浓度对啤酒大麦幼胚组织培养与植株再生的影响
麦类作物学报, 2003,23(2):27-29.

DOI:10.7606/j.issn.1009-1041.2003.02.047 URL [本文引用: 1]

为了提高大麦幼胚胚性愈伤组织和植株再生频率,本试验以引进的4个啤酒大麦品种为材料,在诱导、分化与生根培养基上分别进行了不同浓度Cu2+处理.结果表明,MS诱导培养基中增加Cu2+浓度对啤酒大麦愈伤组织的诱导无明显影响,但对胚性愈伤组织的形成有明显的促进作用;在分化与生根培养基中不同浓度Cu2+处理的各品种再生率也存在显著差异,都表现为在5.0～10.0μmol@L-1的浓度下明显提高了出愈率和再生率,而且每个胚性愈伤组织的平均再生率也有显著提高.通过切片技术证明Cu2+浓度主要影响胚性愈伤组织的发生.供试品种在出愈率和再生率上也存在一定的差异,同时Cu2+浓度和基因型之间表现出正互作效应.

H Y

, YIN

, LIU

, REN

J P

, GUO

X Y

, LI

Z Y

. Effect of Cu²⁺concentration on tissue culture and plant regeneration of beer barley
Journal of Triticeae Crops, 2003,23(2):27-29. (in Chinese)

DOI:10.7606/j.issn.1009-1041.2003.02.047 URL [本文引用: 1]

严华军, 王君晖. 大麦成熟胚胚性愈伤组织的高频诱导和植株再生
作物学报, 1996,22(1):58-65.

URL [本文引用: 1]

YAN

H J

, WANG

J H

. Efficeient induction of embryogenic callus and regeneration from barley mature embryos
Acta Agronomoca Sinica, 1996,22(1):58-65. (in Chinese)

URL [本文引用: 1]

[21]

REN

, LIU

, YIN

, WANG

, LI

. Optimization of transgenic system of barley and transformation of TrxS gene by means of particle bombardment
Acta Botanica Boreali-Occidentalia Sinica, 2004,24(9):1662-1668.

[本文引用: 1]

[22]

孔维威. TrxS的表达及其对啤酒大麦发芽种子水解酶和贮藏物质降解的影响
[D]. 郑州: 河南农业大学, 2006.

[本文引用: 1]

KONG

W W

. The expression and the effects of TrxS gene on hydrolytic enzyme and degration of storage substances during malting barley seed germination
[D]. Zhengzhou: Henan Agricultural University, 2006. (in Chinese)

[本文引用: 1]

[23]

吕维涛, 刘芳, 崔德才, 赵檀方. 农杆菌介导法获得转反义磷脂酶Dγ基因大麦
生物技术, 2005,15(5):11-13.

[本文引用: 1]

Lü

W T

, LIU

, CUI

D C

, LIU

T F

. Production of transgenic barley with Anti -PLDγ gene via Agrobacterium tumefaciens
Biotechnology, 2005,15(5):11-13. (in Chinese)

[本文引用: 1]

[24]

李静雯, 张正英, 令利军, 李淑洁. 利用RNAi抑制B-hordein合成降低大麦籽粒蛋白质含量
中国农业科学, 2014,47(19):3746-3756.

DOI:10.3864/j.issn.0578-1752.2014.19.003 URL [本文引用: 1]

【目的】通过RNAi策略抑制大麦籽粒B-hordein的表达，获得高氮肥水平下籽粒蛋白质含量降低的转基因大麦新种质，探索大麦品质改良新途径。【方法】采用同源PCR技术克隆大麦B-hordein核心保守序列，通过Gateway技术构建大麦B-hordein的RNAi植物表达载体pBract207-zz-gp4，利用农杆菌介导法转化大麦Golden Promise幼胚，获得转基因植株。通过PCR检测、Southern杂交验证RNAi构件在转基因大麦及其后代基因组的整合情况。采用半定量RT-PCR分析转基因大麦花后不同发育时期B-hordein转录表达情况。基于近红外谷物分析仪测定蛋白质含量，并进行醇溶蛋白SDS-PAGE检测，以筛选蛋白质含量降低的转基因大麦株系。开展氮肥运筹相关试验，检验RNAi效率及高氮肥水平下转基因大麦蛋白质含量变化情况。【结果】获得大麦B-hordein片段2条（Gp4和Gp5），测序结果表明该片段大小为349 bp，聚类分析表明该序列跟已知大麦醇溶蛋白基因同源性最高达92%，与该基因已登录的mRNA序列同源性高于98%，确认所得片段为大麦B醇溶蛋白基因片段。利用Gateway技术将Gp4片段正反向插入载体pBract207，反向重复序列用i18 和 iv2 intron连接，构建了Ubi启动子驱动的大麦B-hordein RNAi植物表达载体 pBract207-zz-gp4。通过农杆菌介导转化大麦Golden Promise幼胚，结合目标基因及筛选标记基因的PCR验证，获得含有RNAi构件及筛选标记基因的转基因大麦株系11个。Southern杂交检测表明RNAi构件已经稳定整合到T₂/T₃转基因大麦基因组。随机选取6个T₃转基因大麦株系，半定量RT-PCR结果表明花后不同发育时期转基因大麦籽粒B-hordein表达水平明显低于非转基因对照，20 d时转基因株系表达量不仅相比同期对照降低28.19%—55.19%，而且在各时期中也最低。利用随机选取的T₁和T₃籽粒进行蛋白质含量测定表明，8个转基因大麦株系的蛋白质含量相比非转基因对照显著降低，SDS-PAGE电泳结果显示与非转基因相比，转基因各株系B-醇溶蛋白比率有不同程度降低，其中3个株系B-醇溶蛋白比率降低明显，平均减幅为49.42%。采用蛋白质含量降低的转基因大麦株系RNAi-20开展氮肥运筹试验，结果表明，高氮肥水平HN₂及HN₃处理，该株系蛋白质含量显著低于非转基因对照；施以等量高氮肥时，伴随氮肥后移（HN₂到HN₃），该株系总蛋白含量相对非转基因对照逐渐降低。SDS-PAGE电泳结果显示B醇溶蛋白含量降低，电泳带型发生变化。【结论】RNAi技术能抑制籽粒B-hordein表达，降低B-醇溶蛋白含量，高氮肥水平下能显著降低大麦蛋白质含量。

J W

, ZHANG

Z Y

, LING

L J

, LI

S J

. Creating low grain protein content barley by suppressing b-hordein synthesis through RNA interference
Scientia Agricultura Sinica, 2014,47(19):3746-3756. (in Chinese)

DOI:10.3864/j.issn.0578-1752.2014.19.003 URL [本文引用: 1]

叶兴国, 陈明, 杜丽璞, 徐惠君. 小麦转基因方法及其评述
遗传, 2011,33(5):422-430.

[本文引用: 1]

X G

, CHEN

, DU

L P

, XU

H J

. Description and evaluation of transformation approaches used in wheat
Hereditas, 2011,33(5):422-430. (in Chinese)

[本文引用: 1]

[26]

WANG

, LIU

, DU

, YE

X G

. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co- transformation strategy in commercial Chinese wheat varieties
Plant Biotechnology Journal, 2016,15(5):614-623.

DOI:10.1111/pbi.12660 URLPMID:27862820 [本文引用: 6]

Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker-free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium-mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker-free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T-DNA regions. The average co-integration frequency of the gus and the bar genes located on the two independent T-DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker-free plants was related to the number of bar gene copies integrated in the genome. Marker-free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.

[27]

MCCORMAC

A C

, FOWLER

M R

, CHEN

D F

, ELLIOTT

M C

. Efficient Co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation
Transgenic Research, 2001,10(2):143-155.

DOI:10.1023/A:1008909203852 URL [本文引用: 2]

The co-transformation of a single plant genome with two independent T-DNA regions provides opportunities for genetic separation in subsequent generations. In an effective strategy, co-delivery events must form a high proportion of the total transformed population. In this study, using the model plant species tobacco (Nicotiana tabacum), it was shown that the frequency of co-transformation within a given T₀ population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fold that of the designated T-DNA region for co-transfer. Overall, 40–50% of T₀ lines demonstrated the capacity for segregational separation of co-transformed T-DNA regions. Hence, the estimate of the required number of total transformants for such an independent strategy may seem to be as little as 2-fold that for a conventional, single T-DNA strategy, but we strongly temper such estimates with indications that high co-transformation frequencies may be associated with a higher incidence of linkage. In this co-transformation study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a selectable marker. This arrangement could reveal that lsquo

read-through rsquo

events within the Agrobacterium cells, resulting in the co-transfer of adjacent T-DNA regions as a single linked unit, accounted for up to 20% of co-transformed plant lines. Such read-through co-delivery appeared to be more frequent from the lsquo

supervirulent rsquo

EHA101 A. tumefaciens strain, compared to the lsquo

ordinary

LBA4404 strain. By using the binary plasmid with three selectable T-DNA regions, we have been able to consider the frequency of co-integration of a third independent T-DNA within a T₀ subpopulation of co-transformants. This was found to be higher than expected. These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed that screening against such sequences may add a significant factor in achieving the desired, final genotype.

[28]

XING

, ZHANG

, SATO

, STASWICK

, CLEMENTE

. The use of the two T-DNA binary system to derive marker-free transgenic soybeans
Vitro Cellular & Developmental Biology Plant, 2000,36(6):456-463.

[本文引用: 1]

[29]

MILLER

, TAGLIANI

, WANG

, BERKA

, BIDNEY

, ZHAO

Z Y

. High efficiency transgene segregation in Co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system
Transgenic Research, 2002,11(4):381-396.

DOI:10.1023/A:1016390621482 URL [本文引用: 1]

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the beta

-glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R₀ plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R₀ events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R₁ generation. Of these R₀ events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R₁ generation. A total of 64.4% of the R₀ 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R₁ progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R₀ plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R₁ phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.

[30]

, WU

, YIN

, MORRAND

, CHEN

, FOLK

W R

, ZHANG

Z J

. evelopment of marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] using standard binary vectors with bar as a selectable marker
Plant Cell Tissue & Organ Culture, 2009,99(1):97-108.

[本文引用: 1]

[31]

MANGU

V R R

, CHIDAMBARAM

, RAJASEKARAN

, KARUPPANNAN

. Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene
Plant Cell Reports, 2011,30(7):1241-1252.

DOI:10.1007/s00299-011-1033-y URL [本文引用: 1]

Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260a center dot pSSJ1 and a multi-copy binary vector pBin19a dagger nptII-ap24 in the same cell. pGV2260a center dot pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. pBin19a dagger nptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T(0) plants (67%). Segregation of hph from ap24 was accomplished in the T(1) generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 transgenes and the absence of the hph gene in the SMG-eliminated T(1) plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap24 gene. Homozygous, SMG-free T(2) plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani. Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacterium-mediated co-transformation with the same SMG.

[32]

ISHIDA

, TSUNASHIMA

, HIEI

, KOMARI

. Wheat (Triticum aestivum L.). Methods in Molecular Biology, Agrobacterium Protocols, 3rd edn
New York: Springer, 2014: 189-198.

[本文引用: 1]

[33]

MATTHEWS

P R

, WANG

M B

, WATERHOUSE

P M

, THORNTON

, FIEG

S J

, GUBLER

, JACOBSEN

J V

. Marker gene elimination from transgenic barley, using co-transformation with adjacent `twin T-DNAs' on a standard Agrobacterium transformation vector
Molecular Breeding, 2001,7(3):195-202.

DOI:10.1023/A:1011333321893 URL [本文引用: 1]

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

[34]

YAO

, CONG

, CHANG

J L

, LI

K X

, YANG

G X

, HE

G Y

. Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment
Journal of Experimental Botany, 2006,57(14):3737-3746.

DOI:10.1093/jxb/erl145 URLPMID:17032730 [本文引用: 1]

Two groups of linear gene constructs (gus and bar, and 1Ax1 and bar) lacking vector backbone sequences were independently transferred into the elite wheat (Triticum aestivum L.) variety EM12, and genetically stable transgenic plants with low copy number transgene integration were recovered. Co-transformation experiments were carried out in parallel using either circular whole plasmid(s) or linear gene cassettes which were purified from the same plasmid by restrictive digestion, each cassette consisting of a promoter, an open reading frame, and a terminator. Six transgenic wheat lines transformed with 1Ax1 plus bar gene cassettes, five lines with gus plus bar gene cassettes, three lines with p1Ax1 plus pAHC20, and two lines with pAHC25 were regenerated with transformation frequencies of 0.6, 0.5, 0.3, and 0.2%, respectively. Southern blotting analysis showed that there were 1-4 hybridizing bands in transgenic lines carrying gene cassettes, of which most lines displayed single-copy transgene insertion. Expression analyses showed that 50.5% of the T1 lines carrying gus plus bar gene cassettes have the expression signals of two genes. SDS-PAGE analysis of the T1 generation revealed that 71% of herbicide-resistant plants carrying 1Ax1 plus bar gene cassettes expressed the high molecular weight subunit 1Ax1 in the endosperm. Gene cassettes were transmitted and segregated in the subsequent generations, in simple Mendelian ratios. In addition, reverse transcription-polymerase chain reaction (RT-PCR) results confirmed that 1Ax1 gene cassettes were expressed specifically in the endosperm of the transgenic wheat plant. It is proposed that gene transfer using multiple gene cassettes offers an efficient and rapid method to obtain the single-copy transgenic wheat.

[35]

YAO

, CONG

, HE

, CHANG

, LI

, YANG

. Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency
Molecular Biology Reports, 2007,34(1):61-67.

DOI:10.1007/s11033-006-9016-8 URL [本文引用: 1]

Genetic manipulation using gene cassettes was applied to the elite wheat variety EM12 via particle bombardment, which allows an improvement in transformation frequency. We simultaneously transferred to wheat immature embryos with two non-linked genes, gus and bar, on either separate gene cassettes or one plasmid. The linear gene cassettes were excised and purified by restriction digestion of the plasmid, and consisted of promoters, open reading frames and terminators. No difference was observed in GUS transient expression of between gene cassettes and single whole plasmid. However, the stable transformation frequency was significantly increased to 1.1% using gene cassettes, compared with 0.4% when using single plasmid. Procedures of the efficient co-transformation with gene cassettes were developed. Factors influencing on the transformation frequency were also studied in order to optimize the procedure. These were acceleration pressure, target distance, gold particle size, the quantity ratio of gene cassettes and the age of target explants. Based on the transient and stable expression of the gus gene cassettes, optimization of transformation parameters improved the reproducibility of transformation in the elite wheat variety.