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作物学报 ›› 2018, Vol. 44 ›› Issue (10): 1423-1432.doi: 10.3724/SP.J.1006.2018.01423

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

以抗除草剂Bar基因稳定转化谷子技术研究

陈倩楠1,2,王轲1,汤沙1,杜丽璞1,智慧1,贾冠清1,赵宝华2,叶兴国1,刁现民1,*()   

  1. 1中国农业科学院作物科学研究所, 北京 100081
    2河北师范大学生命科学学院, 河北石家庄 050024
  • 收稿日期:2017-11-23 接受日期:2018-07-20 出版日期:2018-10-10 网络出版日期:2018-08-01
  • 通讯作者: 刁现民
  • 基金资助:
    本研究由国家自然科学基金项目(31501324);本研究由国家自然科学基金项目(31522040);中国农业科学院基本科研业务费(Y2017JC15);中国农业科学院基本科研业务费(CAAS-XTCX2016001-5);中国农业科学院基本科研业务费(CAAS-XTCX2016002);国家现代农业产业技术体系建设专项(CARS-06-13.5-A04);中国农业科学院创新工程杂粮团队项目资助

Use of Bar Gene for the Stable Transformation of Herbicide-resistant Foxtail Millet Plants

Qian-Nan CHEN1,2,Ke WANG1,Sha TANG1,Li-Pu DU1,Hui ZHI1,Guan-Qing JIA1,Bao-Hua ZHAO2,Xing-Guo YE1,Xian-Min DIAO1,*()   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2 College of Life Science, Hebei Normal University, Shijiazhuang 050024, Hebei, China
  • Received:2017-11-23 Accepted:2018-07-20 Published:2018-10-10 Published online:2018-08-01
  • Contact: Xian-Min DIAO
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31501324);This study was supported by the National Natural Science Foundation of China(31522040);the Fundamental Research Funds of CAAS(Y2017JC15);the Fundamental Research Funds of CAAS(CAAS-XTCX2016001-5);the Fundamental Research Funds of CAAS(CAAS-XTCX2016002);the China Agriculture Research System(CARS-06-13.5-A04);the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences

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摘要：

农杆菌介导的谷子遗传转化效率一直是制约谷子功能基因研究及转基因育种效率的主要因素。本研究以冀谷11谷子幼穗为外植体, 选取0.5~1.0 cm的幼穗, 切成0.5 cm左右的小段, 置改良后的MS培养基上诱导15~20 d形成胚性愈伤组织3120块。愈伤组织侵染转化前用侵染液悬浮浸泡, 并经45°C热激预处理3 min, 可以有效提高26.1%的瞬时遗传转化效率。将转化后的愈伤组织经草丁膦(PPT)筛选后获得513块抗性愈伤组织, 抗性愈伤组织获得率为16.4%。抗性愈伤组织经分化、壮苗培养后获得7株抗性植株, 经PCR和Southern杂交检测得到6株T0代转基因阳性株, 对T3代转化植株叶片进行PPT抗性分析, 并结合Bar蛋白抗体试纸条鉴定, 表明Bar基因已稳定整合到谷子基因组中。本研究建立了农杆菌介导转化谷子优良品种的遗传转化体系, 对提高谷子转基因育种效率和谷子模式研究系统的建立有重要意义。

关键词: 谷子, 根癌农杆菌, 遗传转化, 热激, 抗除草剂

Abstract:

Efficiency of Agrobacterium-mediated genetic transformation has been the main factor that restricts the study of functional genes and transgenic breeding in foxtail millet. In this study, foxtail millet variety Jigu 11 panicle primordia were used as explants. Young panicles of 0.5-1.0 cm in length were picked and cut into small pieces. The young embryos were cultured in modified MS medium for inducing embryogenic calli, totally forming 3120 embryogenic calli in 15-20 days. Soaking the calli in the infection suspension prior to transformation, and the heat treatment at 45°C for 3 min could effectively improve the transient genetic transformation efficiency by 26.1%. The transformed calli were screened with phosphinothricin (PPT), in which 513 were resistant calli, with the resistant calli rate of 16.4%. Seven herbicide-resistant plants were obtained after resistant calli differentiation and seedling culture. Six T0 transgenic positive plants were identified by PCR and Southern blot. PPT resistance analysis was carried out on leaves in T3 generation of transformed plants, and combined with Bar protein antibody test strip identification, the results confirmed that the Bar gene stably incorporated into the genome of foxtail millet seedlings. This study established a stable genetic transformation system in foxtail millet, which is of great significance in improving the efficiency of molecular breeding, and prompting foxtail millet as a new model plants.

Key words: foxtail millet, Agrobacterium, genetic transformation, heat shock, herbicide-resistant

表1

农杆菌介导谷子愈伤组织转化培养基配方"

组分
Composition		 诱导培养基
IM		 侵染液
IL		 共培养基
CM		 恢复培养基RM 筛选培养基
SM		 分化培养基DM 壮苗培养基
SSM
MS 4.43 0.443 0.443 4.43 4.43 4.43 2.215
L-Glu 0.5 — — 0.5 0.5 — —
CEH 0.8 0.8 0.8 0.8 0.8 — —
MES 1.95 0.5 0.5 1.95 1.95 — —
inositol 0.1 — — 0.1 0.1 0.1 —
VCa — — — — — 0.01 0.01
Glucose — 36 10 — — — —
Sucrose — 68.5 — — — 20 30
Maltose 40 — — 40 40 — —
CuSO4a — 0.0006 — — — — —
AgNO3a 0.005 — 0.005 0.005 0.005 — —
2,4-Da 0.002 — 0.002 0.002 0.002 — —
KTa 0.0005 — 0.005 0.0005 0.0005 0.002 —
NAAa — — — — — — 0.0005
ASa — — 0.1962, L-1 — — — —
Cefa — — — 0.1 — — —
Carba — — — 0.25 0.25 — —
Tima — — — — — 0.15 0.15
PPTa — — — — 0.005/0.01 — 0.001
Agar 5 — 8 5 5 — —
Phytagel — — — — — 2.5 2

表2

遗传转化热激处理方式"

热激温度
Heat temperature (°C)		 热激时间
Heat time (min)		 冰浴时间
Ice bath time (min)
25(CK) — —
37 3 1
45 3 1
47 3 1

图1

幼穗大小及愈伤组织状态 A: 诱导7 d后愈伤组织; B: 诱导20 d后愈伤组织; C: 诱导40 d后愈伤组织; D: 不同长度幼穗。"

表3

幼穗诱导培养15 d后愈伤组织诱导效率的比较"

幼穗大小
Panicle size (cm)		 愈伤组织数量
Calli number		 幼穗数量
Panicle number		 愈伤组织诱导率
Calli induction rate (%)a
≤ 0.5 23 30 74.35±2.35
18 25
20 27
0.5-1.0 181 198 92.74±1.13
190 204
207 221
1.1-1.5 104 137 75.14±4.11
97 123
77 109
1.6-2.0 43 96 39.16±4.98
39 103
31 89
≥ 2.0 17 75 21.54±1.85
13 68
21 92

图2

愈伤组织诱导培养时间对转化效率的影响 A: 诱导7 d后愈伤组织; B: 诱导15 d后愈伤组织; C: 诱导30 d后愈伤组织; D: GUS瞬时转化效率; E: 诱导15 d愈伤组织GUS染色; F: 诱导30 d愈伤组织GUS染色。*, **分别表示在P ≤ 0.05和P ≤ 0.01水平上的显著差异。"

图3

谷子愈伤组织热激处理GUS瞬时转化 A: 不同温度下热激3 min的GUS瞬时表达率; B: 25°C (CK) GUS染色; C: 45°C热激下GUS染色; D: 45°C热激温度下不同热激时间的GUS瞬时表达率; E: 45°C热激1.5 min GUS染色; F: 45°C热激3 min GUS染色。*和**分别表示在P ≤ 0.05和P ≤ 0.01水平上显著差异。"

图4

T0代转基因植株鉴定 A: 愈伤组织分化; B: 再生植株; C: 壮苗; D: 再生植株移栽; E: Bar基因PCR检测, 1~7为T0代转化单株; F: T0代转化株Southern blot, 1~5: 经Sac I酶切, 7~11: 经Hind III酶切, 6: 野生型。M: marker DL2000; H: 水对照; WT: 野生型; P: 质粒。"

图5

T3代转基因株bar基因抗性试验 A: 2.5 g L-1 PPT溶液涂抹叶片; B: 2.5 g L-1 PPT 溶液喷洒叶片, 左侧转化株, 右侧未转化株; C: Bar蛋白抗体试纸条鉴定结果。CK: 未转化阴性对照; Vegative: 转基因阴性株; Positive: 转基因阳性株。"

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