Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (6): 1839-1849.DOI: 10.1016/j.jia.2023.06.021

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利用TaWOX5基因建立小麦近缘物种高效遗传转化体系的研究

  

  • 收稿日期:2023-03-07 接受日期:2023-05-18 出版日期:2024-06-20 发布日期:2024-05-29

Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Yanan Chang1, 2*, Junxian Liu2, 3*, Chang Liu4*, Huiyun Liu1, Huali Tang1, Yuliang Qiu1, Zhishan Lin1, Ke Wang1#, Yueming Yan2#, Xingguo Ye1# #br#   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

    2 Beijing Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environment Improvement/College of Life Science, Capital Normal University, Beijing 100048, China

    3 College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China 4State Key Laboratory of Plant Cell and Chromosome Engineering/Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

  • Received:2023-03-07 Accepted:2023-05-18 Online:2024-06-20 Published:2024-05-29
  • About author:#Correspondence Xingguo Ye, E-mail: yexingguo@caas.cn; Yueming Yan, E-mail: yanym@cnu.edu.cn; Ke Wang, E-mail: wangke03@caas.cn * These authors contributed equally to this study.
  • Supported by:

    This research was supported by grants from the National Natural Science Foundation of China (31971946) and the Technology Innovation Program of the Chinese Academy of Agricultural Sciences, China (2060302-2-23 and ASTIP-2060302-2-19).

摘要:

小麦近缘物种作为珍贵的遗传资源,对于农业生产、小麦功能基因组研究和品质改良具有重要的作用。在本研究中,我们利用来自小麦的再生相关基因TaWOX5,通过农杆菌转化一粒小麦,六倍体小黑麦和黑麦幼胚,以较高效率获得了转基因植株;进一步对转基因植株中的GUS基因和bar基因进行了PCR检测,对GUS蛋白和bar蛋白分别进行了组织化学染色以及bar试纸条检测,计算了转化效率。结果表明,一粒小麦基因型PI428182的转化效率为94.4%4六倍体小黑麦基因型Lin456、ZS3297、ZS1257和ZS3224的转化效率分别为52.1%、41.2%、19.4%和16.0%,黑麦基因型兰州黑麦的转化效率为7.8%。对转基因植株进行的荧光原位杂交(FISH)检测和基因组原位杂交(GISH)检测结果证实,在一粒小麦和六倍体小黑麦转基因植株中GUS基因倾向于整合到染色体末端,也有整合到近染色体着丝粒区域的情况;在六倍体小黑麦转基因植株中,外源DNA片段随机整合到了AABB基因组和RR基因组;转入的外源基因在转基因植株T1代中基本符合孟德尔遗传规律。本研究结果将为利用基因工程对一粒小麦、六倍体小黑麦和黑麦的遗传改良奠定基础,促进粮食和饲料生产,同时加快了包括小麦在内的麦类植物的功能基因组学研究。

Abstract:

Species closely related to wheat are important genetic resources for agricultural production, functional genomics studies and wheat improvement.  In this study, a wheat gene related to regeneration, TaWOX5, was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos.  Transgenic plants were efficiently generated.  During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS).  Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for the presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and the QuickStix strip assay for bar protein.  The transformation efficiency of Tmonococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%.  Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analyses indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) regions of the chromosomes in transgenic Tmonococcum and hexaploid triticale plants.  In the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB and RR genomes.  Furthermore, the transgene was almost stably inherited in the next generation by Mendel’s law.  The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

Key words: Triticum monococcum  , hexaploid triticale ,  rye ,  TaWOX5 ,  Agrobacterium , transformation efficiency