发根农杆菌介导文冠果（Xanthoceras sorbifolium）毛状根遗传转化体系的建立

doi:10.13560/j.cnki.biotech.bull.1985.2025-1162

摘要/Abstract

摘要：

目的建立高效的文冠果毛状根遗传转化体系，为文冠果基因功能验证和通过分子育种技术定向培育稳产高产优良品种提供技术支持与研究基础。方法以4种优异文冠果种质萌发幼苗的下胚轴和5种发根农杆菌为材料，采用切-浸-芽法诱导转基因毛状根至2 cm以上，通过表型观察、PCR及测序技术来评估本技术体系基因的表达和编辑效率。结果以RUBY基因作为报告基因，筛选出最适发根农杆菌菌株为K599，其转基因毛状根的植株诱导率达81.67%，阳性率达51.64%以上，并通过PCR扩增和RT-qPCR分析，检测RUBY基因成功整合到文冠果毛状根基因组中。以GUS-GFP基因作为报告基因，转基因毛状根经GUS染色变蓝并在显微镜下观测到明显的绿色荧光，进一步证实本技术体系可用于基因的表达和功能定位研究。此外，构建XsPDS3基因的CRISPR/Cas9基因编辑载体，经测序检测其靶点编辑效率为20%，证明了该系统在基因编辑方面的潜力。结论建立了简单高效的文冠果毛状根遗传转化体系，为文冠果的基因功能验证提供了关键技术平台，并有望通过根再生建立文冠果遗传转化体系用于遗传改良。

关键词: 文冠果, 毛状根, 转化体系, 基因功能鉴定

Abstract:

Objective Xanthoceras sorbifolium is a woody oil crop with both ecological restoration and economic value, and characteristic of Northwest China. Establishing an efficient hairy root genetic transformation system for X. sorbifolium may provide technical support and research foundation for gene function validation and the targeted breeding of stable, high-yielding superior varieties through molecular breeding techniques. Method Using the hypocotyls of four superior X. sorbifolium seedling genotypes and five Agrobacterium rhizogenes strains as experimental materials, the cut-soak-bud method was employed to induce transgenic hairy roots to > 2 cm in length. Phenotypic observation, PCR and sequencing techniques were utilized to evaluate the expression and editing efficiency of the gene in this technical system. Result Using the RUBY gene as the reporter gene, the selected most suitable strain of A. rhizogenes was K599, with the plant induction rate of 81.67% and the positive rate of over 51.64% for the transgenic hairy roots. The successful integration of the RUBY gene into the genome of X. sorbifolium hairy roots were detected by PCR amplification and RT-qPCR quantitative methods. Using the GUS-GFP gene as the reporter gene, the transgenic hairy root was stained to be blue with GUS staining solution and observed with obvious green fluorescence under the microscope, further confirming that this technical system can be used for gene expression and functional localization research. In addition, the CRISPR/Cas9 gene editing vector for the XsPDS3 gene was constructed, and its target editing efficiency was detected to be 20% through sequencing, demonstrating the potential of this system in gene editing. Conclusion This study established a simple and efficient genetic transformation system for the hairy roots of Wenguan fruit, providing a key technical platform for gene function verification of Wenguan fruit. It is expected to establish a genetic transformation system for Wenguan fruit through root regeneration for genetic improvement.

Key words: Xanthoceras sorbifolium, hairy roots, transformation system, gene function identification

周韬, 李亚强, 何慧娟, 虎海防, 陈同森, 潘越, 王天斌. 发根农杆菌介导文冠果（Xanthoceras sorbifolium）毛状根遗传转化体系的建立[J]. 生物技术通报, 2026, 42(7): 97-104.

ZHOU Tao, LI Ya-qiang, HE Hui-juan, HU Hai-fang, CHEN Tong-seng, PAN Yue, WANG Tian-bin. Establishment of Hairy Root Genetic Transformation System in Xanthoceras sorbifolium Mediated by Agrobacterium rhizogenes[J]. Biotechnology Bulletin, 2026, 42(7): 97-104.

图/表 7

表1 引物序列

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Usage
RUBY-PF RUBY-PR	AGACACTCCGCCTCCATCC	RUBY基因片段扩增
RUBY-PF RUBY-PR	TTCTACATCACCTGCCTGCTT	RUBY基因片段扩增
RUBY-RT-PF RUBY-RT-PR	ACGCATGAAGGTGAAGAAACA	RUBY基因表达检测
RUBY-RT-PF RUBY-RT-PR	CCATCCGCAGTGGGTGAG	RUBY基因表达检测
XsPDS3-T1	TTTCGAGGGAGTGAATCCATGGG	靶点序列
F2-tgRNA1-T1 R2-tgRNA1-T1	tgcagTTTCGAGGGAGTGAATCCAT	基因编辑载体构建
F2-tgRNA1-T1 R2-tgRNA1-T1	aaacATGGATTCACTCCCTCGAAAC	基因编辑载体构建
T1-PF T1-PR	GTCGGAGATGATGGTGATGAAT	靶点片段扩增
T1-PF T1-PR	AGTCCAGGCAAACAACCTGATA	靶点片段扩增

图1 农杆菌菌株对文冠果毛状根转化效率的影响不同字母表示差异显著（P<0.05），下同

Fig. 1 Influence of Agrobacterium strain types on the transformation efficiency of hairy roots in X. sorbifoliumDifferent letters indicate significant difference (P<0.05). The same below

图2 文冠果毛状根转化技术流程

Fig. 2 Process of hairy root transformation technology for X. sorbifolium

图3 文冠果红色转基因根表型和PCR检测A：转RUBY基因产生红色转基因毛状根表型；B：RUBY基因的PCR扩增电泳鉴定

Fig. 3 Phenotype and PCR detection of red transgenic roots in X. sorbifoliumA: Transforming the RUBY gene to produce a red transgenic hairy root phenotype. B: PCR amplification and electrophoresis identification of RUBY gene

图4 文冠果红色转基因根的表达检测A：RT-qPCR表达水平检测；B：GUS染色检测；C：GFP荧光信号检测

Fig. 4 Transgenic expression detection of red transgenic roots of X. sorbifoliumA: Detection of RT-qPCR expression level. B: Detection of GUS staining. C: Detection of GFP fluorescence signal

图5 转基因毛状根诱导效率的优化A：不同幼苗萌发时间的转基因毛状根阳性率；B：不同幼苗萌发时间的转基因毛状根植株诱导效率；C：不同品种的转基因毛状根阳性率；D：不同品种的转基因毛状根植株诱导效率

Fig. 5 Optimization of induction efficiency of transgenic hairy rootsA: Positive rate of transgenic hairy roots at different seedling germination times. B: Induction efficiency of transgenic hairy root at different seedling germination times. C: Positive rate of transgenic hairy roots across different varieties. D: Induction efficiency of transgenic hairy root across different varieties

图6 文冠果毛状根的CRISPR-Cas9基因编辑A：XsPDS3的基因结构和选定的靶点，蓝色字符表示sgRNA，红色字符表示PAM序列；B：WT为野生型测序结果；C：M1为靶点基因突变类型1的测序结果；D：M2为靶点序列突变类型2的测序结果

Fig. 6 CRISPR-Cas9 gene editing of X. sorbifolium hairy rootsA: The gene structure and selected targets of XsPDS3, with blue characters for sgRNA and red characters for PAM sequences. B: WT is wild-type sequencing result. C: Sequencing results of M1 as the target gene mutation type 1. D: M2 is the sequencing result of target sequence mutation type 2

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