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作物学报 ›› 2022, Vol. 48 ›› Issue (12): 2987-2993.doi: 10.3724/SP.J.1006.2022.13068

所属专题： 玉米：遗传育种·种质资源·分子遗传学

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

不依赖基因型的高效玉米遗传转化体系的建立

许洁婷1,2,3(), 刘相国4(), 金敏亮1,2, 潘弘2, 韩宝柱2, 李梦娇2, 岩说5, 胡国庆5, 严建兵1()   

  1. 1华中农业大学作物遗传改良国家重点实验室, 湖北武汉 430070
    2未米生物科技(江苏)有限公司, 江苏常州 213000
    3未米生物科技(海南)有限公司, 海南三亚 572000
    4吉林省农业科学院农业生物技术研究所, 吉林长春 130033
    5西双版纳傣族自治州农业科学研究所, 云南景洪 666100
  • 收稿日期:2021-11-26 接受日期:2022-03-25 出版日期:2022-12-12 网络出版日期:2022-04-20
  • 通讯作者: 严建兵
  • 作者简介:许洁婷, E-mail: xjt@wimibio.com;
    刘相国, E-mail: lxgyyj@cjaas.com第一联系人：

    **同等贡献

  • 基金资助:
    国家转基因生物新品种培育重大专项(2018ZX08010-04B);国家转基因生物新品种培育重大专项(2019ZX08010003-002-013);吉林省科技发展计划项目(20190201290JC)

Establishment of genotype-independent high-efficiency transformation system in maize

XU Jie-Ting1,2,3(), LIU Xiang-Guo4(), JIN Min-Liang1,2, PAN Hong2, HAN Bao-Zhu2, LI Meng-Jiao2, YAN Shuo5, HU Guo-Qing5, YAN Jian-Bing1()   

  1. 1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    2WIMI Biotechnology Co., Ltd., Changzhou 213000, Jiangsu, China
    3WIMI Biotechnology Co., Ltd., Sanya 572000, Hainan, China
    4Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130033, Jilin, China
    5Institute of Agricultural Sciences of Xishuangbanna Prefecture of Yunnan Province, Jinghong 666100, Yunnan, China
  • Received:2021-11-26 Accepted:2022-03-25 Published:2022-12-12 Published online:2022-04-20
  • Contact: YAN Jian-Bing
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    National Major Project for Developing New GM Crops(2018ZX08010-04B);National Major Project for Developing New GM Crops(2019ZX08010003-002-013);Science and Technology Innovation Program of Jilin Province(20190201290JC)

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摘要：

玉米遗传转化严重依赖于受体基因型, 目前绝大部分商业化玉米骨干自交系的遗传转化仍然十分困难。Baby boom (Bbm)和Wuschel2 (Wus2)是植物干细胞发育中的2个关键基因, 调节这2个基因的时空表达可以有效提高植物遗传转化效率。本研究以多个我国玉米骨干自交系为受体材料开展转化效率测试, 发现虽然Bbm和Wus2的超表达可以显著提高转化效率, 但对玉米的生长发育具有不利影响。本研究开发了一种新型辅助转化技术, 在辅助载体中加入致死基因表达元件, 并调节2个基因的时空表达。结果表明该辅助载体和目标载体混合转化时, 不但可以在T0代成功获得不含Bbm和Wus2辅助载体的正常生长发育的优质转化苗, 还显著提高转化效率(平均达19.5%)。本研究成功建立和完善了一套不依赖基因型的高效玉米遗传转化体系, 为提高生物育种效率和精准改良提供了有力支持。

关键词: 玉米, 遗传转化, Baby boom, Wuschel2, 辅助载体

Abstract:

The reliance on receptor genotype of genetic transformation made it difficult for the transformation of commercial maize lines. Expression regulation of two important genes in plant stem cell development, Baby boom (Bbm) and Wuschel2 (Wus2), was revealed to significantly improve transformation efficiency. Several Chinese core maize inbred lines were used as receptor materials to test the transformation efficiency. Although the overexpression of Bbm and Wus2 could significantly improve the transformation efficiency, it had a negative impact on the growth and development of T0 plants. Here, a new assisted transformation technology was developed, in which a lethal gene element was added to the assist vector and the spatial expression of the two genes were regulated. The results revealed that the hybrid transformation of the assist vector and the target vector could not only successfully obtain high-quality transformation seedlings without Bbm and Wus2 assist vector in T0 generation, but also significantly improve the transformation efficiency with an average 19.5%. The application of this improved genotype-independent genetic transformation system promises maize precise improvement with higher efficiency.

Key words: maize, genetic transformation, Baby boom, Wuschel2, assist vector

图1

辅助载体和基因敲除载体示意图"

图2

郑58的愈伤诱导(A~C)、愈伤分化(D)和IL3 T0植株异常表型(E) 红色箭头所示为异常的雄穗。"

表1

玉米骨干自交系和常规受体材料中pWMDR001和p193412载体的转化效率"

试验编号
Test ID		 玉米自交系
Maize inbred line		 载体
Vector name		 起始胚数量
No. of embryos		 转化阳性苗数量
No. of positive transgenic seedlings		 转化效率
Transformation frequency (%)
1 郑58 Zheng 58 pWMDR001 100 24 24.0
2 X923-1 pWMDR001 50 3 6.0
3 IL3 pWMDR001 50 5 10.0
4 IL4 pWMDR001 50 9 18.0
5 京724 Jing 724 pWMDR001 50 6 12.0
6 KN5585 pWMDR001 50 9 18.0
7 B104 pWMDR001 100 18 18.0
8 郑58 Zheng 58 p193412 200 0 0
9 X923-1 p193412 100 0 0
10 IL3 p193412 100 0 0
11 IL4 p193412 100 0 0
12 京724 Jing 724 p193412 100 0 0
13 KN5585 p193412 200 21 10.5
14 B104 p193412 100 5 5.0

图3

混合转化后的基因型分离检测(A, B)和Waxy基因编辑情况(C) M: DL2000 marker; P: 阳性对照; N: 阴性对照。"

表2

3种玉米材料中pWMDR002和基因编辑载体混转的转化效率"

试验编号
Test ID		 玉米自交系
Maize
inbred line		 起始胚数量
No. of
embryos		 转化阳性苗数量
No. of positive
transgenic seedlings		 优质转化苗数量
No. of high-quality transformed
seedlings		 优质转化苗分离率
Separation rate of high-quality transformed seedling (%)		 优质转化效率
High-quality
transformation
frequency (%)
1 郑58 Zheng 58 313 24 8 33.3 2.6
2 郑58 Zheng 58 535 107 2 1.9 0.4
3 郑58 Zheng 58 828 129 56 43.4 6.8
4 郑58 Zheng 58 446 103 14 13.6 3.1
5 KN5585 210 40 5 12.5 2.4
6 KN5585 256 65 18 27.7 7.0
7 KN5585 260 51 16 31.4 6.2
8 B104 189 59 13 22.0 6.9
9 B104 242 70 11 15.7 4.5
10 B104 150 48 10 20.8 6.7

表3

4个玉米自交系中pWMDR003和基因编辑载体混合转化的转化效率"

试验编号
Test ID		 玉米自交系
Maize inbred
line		 起始胚数量
No. of
embryos		 转化阳性苗数量
No. of positive transgenic
seedlings		 优质转化苗数量
No. of high-quality transformed seedlings		 优质转化苗分离率
Separation rate of high-quality
transformed
seedling (%)		 优质转化效率
High-quality transformation frequency (%)		 平均优质转化效率
Average high-quality
transformation frequency (%)
1 B104 430 133 123 92.5 28.6 31.0
2 B104 100 39 39 100.0 39.0
3 B104 150 45 38 84.4 25.3
4 郑58
Zheng 58		 160 18 14 77.8 8.8 16.4
5 郑58
Zheng 58		 200 56 48 85.7 24.0
6 京724
Jing 724		 50 6 5 83.3 10.0 11.5
7 京724
Jing 724		 100 15 13 86.7 13.0
8 糯IN12
Nuo IN12		 50 7 7 100.0 14.0 13.3
9 糯IN12
Nuo IN12		 200 27 25 92.6 12.5
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