根癌农杆菌介导的荔枝体细胞胚遗传转化体系的建立和LcMYB1抑制荔枝体细胞胚发生的转录组研究

doi:10.1016/j.jia.2024.03.007

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (2): 594-609.DOI: 10.1016/j.jia.2024.03.007

根癌农杆菌介导的荔枝体细胞胚遗传转化体系的建立和LcMYB1抑制荔枝体细胞胚发生的转录组研究

收稿日期:2023-09-24 接受日期:2024-01-25 出版日期:2025-02-20 发布日期:2025-01-22

Development of an Agrobacterium tumefaciens-mediated transformation system for somatic embryos and transcriptome analysis of LcMYB1’s inhibitory effect on somatic embryogenesis in Litchi chinensis

Yaqi Qin^*, Bo Zhang^*, Xueliang Luo, Shiqian Wang, Jiaxin Fu, Zhike Zhang, Yonghua Qin, Jietang Zhao^#, Guibing Hu^#

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs/Guangdong Litchi Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou 510642, China

Received:2023-09-24 Accepted:2024-01-25 Online:2025-02-20 Published:2025-01-22
About author:Yaqi Qin, E-mail: qyq19930102@126.com; Bo Zhang, E-mail: bozhang@scau.edu.cn; #Correspondence Jietang Zhao, E-mail: jtzhao@scau.edu.cn; Guibing Hu, E-mail: guibing@scau.edu.cn *These authors contributed equally to this study.
Supported by:
This study was supported by the National Natural Science Foundation of China (31872066 and 32272663), the Science and Technology Planning Project of Guangzhou, China (2023B01J2002), the Key Research and Development Program of Hainan, China (ZDYF2023XDNY052), the Seed Industry Engineering Project of Department of Agriculture and Rural Affairs of Guangdong, China (2022-NPY-00-004 and 2022-NBH-00-001), and the Litchi Industry Science and Technology Special Mission of Yunnan, China (202204BI090021).

摘要/Abstract

摘要： 荔枝作为一种全球水果作物，具有极其重要的经济意义。然而，由于荔枝难以实现稳定的遗传转化，其功能基因组学的研究遇到了实质性的阻碍。本研究中，我们建立了一个有效的根癌农杆菌介导‘黑叶’荔枝体细胞胚的遗传转化系统。通过外植体类型、菌株种类、菌液浓度、侵染时间和侵染方式的选择对该遗传转化体系进行了优化。随后通过LcMYB1基因的异位表达验证了遗传转化体系在荔枝中的可行性，并得到了转基因愈伤组织。但是，在转基因愈伤组织向体细胞胚分化的过程中遇到了巨大挑战。为了深入探究LcMYB1在体细胞胚诱导过程中抑制作用的分子基础，对胚型愈伤组织（EC）、球形胚（G）和转基因愈伤组织（TC）进行了全面的转录组分析。在C-vs-G和C-vs-TC中共鉴定得到1166个共同差异表达基因，GO富集和KEGG通路分析表明这些共同差异表达基因主要与植物激素信号转导通路相关。此外，RT-qPCR验证了转基因愈伤组织中许多与体细胞胚诱导相关的基因表达显著下调。综上所述，本遗传转化体系为荔枝的功能基因组学研究提供了有力的支持。

Abstract:

Litchi has great economic significance as a global fruit crop. However, the advancement of litchi functional genomics has encountered substantial obstacles due to its recalcitrance to stable transformation. Here, we present an efficacious Agrobacterium tumefaciens-mediated transformation system in somatic embryos of ‘Heiye’ litchi. This system was developed through the optimization of key variables encompassing explant selection, A. tumefaciens strain delineation, bacterium concentration, infection duration, and infection methodology. The subsequent validation of the transformation technique in litchi was realized through the ectopic expression of LcMYB1, resulting in the generation of transgenic calli. However, the differentiation of transgenic calli into somatic embryos encountered substantial challenges. To delineate the intricate molecular underpinnings of LcMYB1’s inhibitory role in somatic embryo induction, a comprehensive transcriptome analysis was conducted that encompassed embryogenic calli (C), globular embryos (G), and transgenic calli (TC). A total of 1,166 common differentially expressed genes (DEGs) were identified between C-vs.-G and C-vs.-TC. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these common DEGs were mostly related to plant hormone signal transduction pathways. Furthermore, RT-qPCR corroborated the pronounced down-regulation of numerous genes that are associated with somatic embryo induction within the transgenic calli. The development of this transformation system provides valuable support for functional genomics research in litchi.

Key words: Litchi chinensis , A. tumefaciens , transformation , LcMYB1 , somatic embryo

Yaqi Qin, Bo Zhang, Xueliang Luo, Shiqian Wang, Jiaxin Fu, Zhike Zhang, Yonghua Qin, Jietang Zhao, Guibing Hu.

根癌农杆菌介导的荔枝体细胞胚遗传转化体系的建立和LcMYB1抑制荔枝体细胞胚发生的转录组研究 [J]. Journal of Integrative Agriculture, 2025, 24(2): 594-609.

Yaqi Qin, Bo Zhang, Xueliang Luo, Shiqian Wang, Jiaxin Fu, Zhike Zhang, Yonghua Qin, Jietang Zhao, Guibing Hu. Development of an Agrobacterium tumefaciens-mediated transformation system for somatic embryos and transcriptome analysis of LcMYB1’s inhibitory effect on somatic embryogenesis in Litchi chinensis[J]. Journal of Integrative Agriculture, 2025, 24(2): 594-609.

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根癌农杆菌介导的荔枝体细胞胚遗传转化体系的建立和LcMYB1抑制荔枝体细胞胚发生的转录组研究

Development of an Agrobacterium tumefaciens-mediated transformation system for somatic embryos and transcriptome analysis of LcMYB1’s inhibitory effect on somatic embryogenesis in Litchi chinensis

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