发根农杆菌介导文冠果毛状根遗传转化体系的构建

doi:10.11707/j.1001-7488.LYKX20250763

摘要/Abstract

摘要：

目的: 探究发根农杆菌介导的毛状根遗传转化体系在文冠果中的应用潜力，为文冠果遗传改良和基因功能验证提供参考。方法: 以文冠果高产抗逆品种‘中石4号’幼苗为材料，分别利用携带35S::Ruby和35S::eYFP质粒的发根农杆菌介导植株转化，构建文冠果毛状根转化体系。转化促根系发育基因XsXTH32，验证该体系在文冠果功能基因验证中的有效性。结果: 发根农杆菌成功诱导文冠果幼苗产生毛状根。在侵染2周后，伤口形成明显愈伤组织，约第4周时发育成毛状根。不同类型发根农杆菌毛状根诱导效率高达68.67%~78.67%。K599菌株的毛状根诱导率为78.67%，阳性率达到50.39%。常规PCR和RT-qPCR检测确定外源基因Ruby整合至文冠果毛状根基因组中，并呈现肉眼可见的红色表型；携带eYFP荧光蛋白标记的转基因毛状根呈现清晰黄色荧光表型。对转化35S::XsXTH32-eYFP的阳性毛状根进行RT-qPCR分析，其表达水平较野生型毛状根显著提高，达9.04倍。结论: 建立了发根农杆菌介导的文冠果毛状根遗传转化体系，K599菌株具有最高的阳性根诱导率和毛状根诱导率，并实现外源基因的稳定整合与高水平表达，在短周期内完成基因功能验证。

关键词: 文冠果, 发根农杆菌, 毛状根, 遗传转化体系

Abstract:

Objective: Xanthoceras sorbifolium is a woody oilseed species endemic to China. Investigating the potential applications of Agrobacterium rhizogenes-mediated hairy root genetic transformation system in X. sorbifolium fills a current gap in its genetic transformation research and provides a foundation for genetic improvement and gene function validation in X. sorbifolium. Method: The high-yield and stress-tolerant cultivar 'zhongshi 4' seedlings of X. sorbifolium were used as materials. Transformation was mediated by A. rhizogenes strains carrying the 35S::Ruby and 35S::eYFP reporter plasmids, respectively, to establish a hairy root transformation system for X. sorbifolium. The root development-promoting gene XsXTH32 was transformed to validate the effectiveness of this system for functional gene verification in X. sorbifolium. Result: A. rhizogenes successfully induced positive hairy roots in X. sorbifolium seedlings. Results showed that distinct callus was formed at the root base of seedlings in two weeks post-infection. The callus was further developed into hairy roots between the third and fourth weeks. The hairy root induction efficiency by different strains reached 68.67% to 78.67%. Specifically, the hairy root induction rate of strain K599 was 78.67%, with a positive rate of 50.39%. Conventional PCR and RT-qPCR analyses confirmed the integration of the foreign gene Ruby into the hairy root genome of X. sorbifolium, which exhibited a visually red phenotype. Transformation with A. rhizogenes carrying the eYFP fluorescent marker gene produced transgenic hairy roots exhibiting distinct yellow fluorescence. RT-qPCR analysis was performed on positive hairy roots transformed with 35S::XsXTH32-eYFP. The expression level was significantly increased compared with that in wild-type hairy roots, reaching 9.04-fold. Conclusion: An A.rhizogenes-mediated hairy root transformation system was established for X.sorbifolium. Among the strains tested, K599 exhibited the highest hairy root and transgene-positive root induction rates. The system enabled the stable integration and high-level expression of exogenous genes, thereby facilitating rapid gene function validation.

Key words: Xanthoceras sorbifolium, Agrobacterium rhizogenes, hairy roots, genetic transformation system

中图分类号:

S727.32

张薇,张鑫,毕泉鑫,刘肖娟,姜国维,宁楚龙,赵海艳,王利兵. 发根农杆菌介导文冠果毛状根遗传转化体系的构建[J]. 林业科学, 2026, 62(8): 120-128.

Wei Zhang,Xin Zhang,Quanxin Bi,Xiaojuan Liu,Guowei Jiang,Chulong Ning,Haiyan Zhao,Libing Wang. Construction of Agrobacterium rhizogenes-mediated Genetic Transformation System for Xanthoceras sorbifolium Hairy Roots[J]. Scientia Silvae Sinicae, 2026, 62(8): 120-128.

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引物名称 Primer name	引物序列 Primer sequences（5′→3′）	引物用途 Primer purpose
eYFP-F/Ruby–F	GGAAGTTCATTTCATTTGGAGAGA	基因克隆与验证 Gene cloning and verification
eYFP–R	TGTGGCCGTTTACGTCGCC
Ruby–R	GTGAATCTTGGCGAGGTTGGC
XsXTH32–F	ATGGCTTCCATTATTGTGTA
XsXTH32–R	TTAACATTCTGGTATCTGGGT
35S::XsXTH32–F	ccatggtagatctgactagtATGGCTTCCATTATTGTGTA
35S::XsXTH32–R	ggcctcctaggtacacgcgtACATTCTGGTATCTGGGTAT
qRT-Ruby–F	AACAGCATCCTTGAGTCTCTTCG	荧光定量 Fluorescence quantitative
qRT-Ruby–R	TTCTCTTTGGAGATCTCGCCTTC
qRT-eYFP–F	CCACATGAAGCAGCACGACT
qRT-eYFP–R	GTTGTACTCCAGCTTGTGCC
qRT-XsXTH32–F	ACCTTCCCCAGGCTACAACC
qRT-XsXTH32–R	ATTGCAACTTAATGGCCGCA
qRT-XsTip41–F	AAGATGAATTGGCTGATAACGGA
qRT-XsTip41–R	AGCTCTCACGAAGAATGACTGGG

菌株类型 Strain type	侵染外植体数 Number of the explant inoculated	产生阳性毛状根平均外植体数 Number of average positive rooting explants	阳性根诱导率 Positive root induction rate （%）	毛状根转化率 Hairy root transformation efficiency （%）
MSU440	50	34.33±1.53b	27.81±6.42b	68.67±2.49b
ArA4	50	37.67±2.08ab	37.91±6.53b	75.33±3.40ab
C58C1	50	35.33±2.52ab	35.64±5.77b	70.67±4.11ab
Ar1193	50	36.67±1.53ab	36.07±5.89b	73.33±2.49ab
K599	50	39.33±3.06a	51.39±14.25a	78.67±4.99a

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