发根农杆菌介导的柑橘黄龙病抗性快速评价体系优化及应用

doi:10.3864/j.issn.0578-1752.2024.16.007

摘要/Abstract

摘要：

【目的】黄龙病（Huanglongbing，HLB）是一种毁灭性柑橘病害，主要由韧皮部杆菌属亚洲种（Candidatus Liberibacter asiaticus，CLas）侵染引起。本研究旨在探究侵染初期CLas在柑橘毛状根中的增长规律，优化发根农杆菌（Agrobacterium rhizogenes）介导的黄龙病抗性快速评价体系并应用于抗菌肽的抗性评价。【方法】基于植物表达载体pGNGM1300，通过发根农杆菌K599介导的毛状根转化体系转化不同柑橘品种，筛选毛状根诱导快、转化率高的品种；接种CLas后，利用实时荧光定量PCR（real-time quantitative PCR，qPCR）对毛状根中的CLas进行定期检测，揭示其增长规律；在优化黄龙病抗性评价体系的基础上，通过CLas和相关基因的（RT-）qPCR、胼胝质含量检测、显微结构观察和症状观察等开展抗菌肽的黄龙病抗性评价。【结果】供试的11个柑橘品种中，香橼（Citrus medica）的毛状根诱导最快（15 d）、诱导率最高（73.75%），并且毛状根中转基因阳性率高（53.54%）。qPCR定期监测结果显示，在接种后第20—30天（days post inoculation，dpi），CLas开始在根中定殖；在30—50 dpi，CLas含量呈显著上升的趋势；CLas含量在60—120 dpi时略有波动，但与50 dpi时差异不显著。抗菌肽MaSAMP（stable antimicrobial peptide）、HBD-4（homo sapiens defensin beta 4）和CB（cecropin B）的抗性评价结果分析显示，在50—120 dpi，MaSAMP和CB表达植株中CLas含量均低于对照，且差异显著，除90 dpi外HBD-4表达植株中CLas含量也显著低于对照。3种抗菌肽表达植株中的胼胝质含量均显著低于对照（60 dpi），韧皮部细胞壁增厚不明显，未出现明显的淀粉粒和胼胝体沉积，并且均未发现根部死亡等现象（90 dpi）。【结论】揭示了侵染早期CLas在柑橘毛状根中的增长规律；优化了发根农杆菌介导的黄龙病抗性快速评价体系；明确转基因过表达抗菌肽MaSAMP、HBD-4和CB可显著抑制CLas的增殖，减少胼胝质沉积，减轻黄龙病症状，具有用于黄龙病防控实践的潜力。

关键词: 柑橘黄龙病, 抗菌肽, 发根农杆菌, 毛状根转化, 抗性评价

Abstract:

【Objective】Citrus Huanglongbing (HLB) is a destructive citrus disease primarily caused by Candidatus Liberibacter asiaticus (CLas). The objective of this research is to investigate the growth pattern of CLas in citrus hairy roots at the initial stage of infection, optimize the rapid evaluation system of Agrobacterium rhizogenes-mediated HLB resistance and apply it to the resistance evaluation of antimicrobial peptides.【Method】A plant expression vector, pGNGM1300, was employed to transform different citrus varieties via the hairy root transformation system, which was mediated by A. rhizogenes K599. Subsequently, varieties with rapid induction and high transformation rate were then selected. Following inoculation with CLas, the presence of CLas in hairy roots was consistently demonstrated through the use of real-time quantitative PCR (qPCR), which allowed for the monitoring of its growth pattern. Further, the optimization of the HLB resistance evaluation system permitted the HLB resistance evaluation of antimicrobial peptides to be conducted via (RT-) qPCR analysis of CLas and related genes, quantification of callose deposition, microstructure observation, and symptom assessment.【Result】Among the 11 tested citrus varieties, citron (Citrus medica) exhibited the fastest induction of hairy roots (15 d), accompanied by the highest induction rate (73.75%) and a substantial transgene positivity rate (53.54%) in hairy roots. The results of regular qPCR monitoring indicated that CLas began to colonize roots on 20-30 days post-inoculation (dpi). At 30-50 dpi, there was a notable increase in the CLas content. The content of the CLas exhibited a slight fluctuation at a range of 60 to 120 dpi, though the discrepancy with 50 dpi was not statistically significant. The results of the antimicrobial peptide resistance evaluation, conducted on MaSAMP (stable antimicrobial peptide), HBD-4 (homo sapiens defensin beta 4), and CB (cecropin B), revealed that at 50-120 dpi, the content of CLas in both MaSAMP- and CB-expressing plants was significantly lower than that of the control. However, except that at 90 dpi, the CLas content was also significantly lower in HBD-4-expressing plants than the control. The callose content in all three plants expressing antimicrobial peptides was significantly lower than that of the control (60 dpi). There was no visible cell wall thickening in the phloem, and no significant deposition of starch grains or callus was observed. Furthermore, none of the plants exhibited signs of root death at 90 dpi.【Conclusion】This study elucidates the growth dynamics of CLas in citrus hairy roots during the early stages of infection and optimizes a rapid scoring system for A. rhizogenes-mediated HLB resistance. In addition, the results show that overexpression of MaSAMP, HBD-4 and CB effectively suppresses CLas proliferation, reduces callose deposition and alleviates HLB symptoms. These findings have the potential to be used in the prevention and control of HLB.

Key words: citrus Huanglongbing, antimicrobial peptide, Agrobacterium rhizogenes, hairy root transformation, resistance evaluation

曹鹏, 周金环, 王新亮, 李楚欣, 李佳欣, 姜培, 刘金香, 宋震. 发根农杆菌介导的柑橘黄龙病抗性快速评价体系优化及应用[J]. 中国农业科学, 2024, 57(16): 3182-3191.

CAO Peng, ZHOU JinHuan, WANG XinLiang, LI ChuXin, LI JiaXIN, JIANG Pei, LIU JinXiang, SONG Zhen. Optimization and Application of Rapid Evaluation System for Citrus Huanglongbing Resistance Mediated by Agrobacterium rhizogenes[J]. Scientia Agricultura Sinica, 2024, 57(16): 3182-3191.

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引物名称Primer name	引物序列Primer sequence (5′-3′)	用途Usage
CLas16S-F	TGAGTGCTAGCTGTTGGGTG	CLas16S基因的qPCR分析 qPCR analysis for CLas16S gene
CLas16S-R	CTGCGCGTTGCATCGAATTA	CLas16S基因的qPCR分析 qPCR analysis for CLas16S gene
Cs18S-F	AATTGTTGGTCTTCAACGAGGAA	柑橘内参基因 Citrus reference gene
Cs18S-R	AAAGGGCAGGGACGTAGTCAA	柑橘内参基因 Citrus reference gene
CsCalS7-qF	GACGCCTAACCGAGTACCTGC	CsCalS7的qPCR分析 qPCR analysis for CsCalS7
CsCalS7-qR	GTGCAGCTGGTGATCCATCA	CsCalS7的qPCR分析 qPCR analysis for CsCalS7
CsPP2B15-qF	GCTCTGAGATCTAGGGTTTCCG	CsPP2B15的qPCR分析^[24] qPCR analysis for CsPP2B15
CsPP2B15-qR	CCCCTTTCAGATGCTGACCC	CsPP2B15的qPCR分析^[24] qPCR analysis for CsPP2B15
2OG oxygenase-qF	CGAAAGCACATGATGGAAAG	2OG oxygenase的qPCR分析^[24] qPCR analysis for 2OG oxygenase
2OG oxygenase-qR	GCCACGAGTGAAGAAATGGT

品种 Variety	枝条总数 Total number of branches	生根枝数 Number of branches with hairy roots	生根时间 Hairy root induction time (d)	生根总数 Total number of hairy roots	阳性根数 Total number of positive hairy roots	生根率 Hairy root induction rate (%)	阳性根率 Hairy root positive rate (%)
锦橙C. sinensis	60	13	30	58	2	21.67	3.44
尤力克柠檬C. limon	262	179	20	722	313	68.32	43.35
香橼C. medica	160	118	15	480	257	73.75	53.54
枳壳P. trifoliata	40	6	60	19	9	15.00	47.36
卡里佐枳橙 C. sinensis×P. trifoliata	40	14	30	53	8	35.00	15.09
粗柠檬C. limonia	40	29	20	116	46	72.50	39.65
墨西哥莱檬C. aurantiifolia	40	25	20	267	76	62.50	28.46
摩洛哥酸橙C. aurantium	40	20	25	88	48	50.00	54.54
甜橙C. sinensis	40	6	60	27	14	15.00	51.85
沙田柚* C. grandis	345	5	180	11	4	1.45	36.37
纽荷尔脐橙* C. sinensis	385	24	120	40	15	6.23	37.50
锦橙* C. sinensis	50	10	60	13	10	20.00	76.92

处理 Treatment	植株编号 Plant number	20 dpi		30 dpi		40 dpi
处理 Treatment	植株编号 Plant number	CLas	Ct18S	CLas	Ct18S	CLas	Ct18S
pGNGM1300	cp2333-3	37.87	18.43	35.43	19.63	28.01	17.08
	cp2333-5	38.87	17.15	36.61	19.06	31.72	18.87
	cp2333-9	35.51	19.48	33.79	18.04	29.4	16.31
	cp2333-12	36.96	18.77	31.68	17.64	29.41	17.62
	cp2333-16	37.49	17.72	35.84	18.67	28.53	18.79
	cp2333-17	37.71	18.57	33.31	17.86	27.21	18.94
CLas阳性对照 Positive control	+CK	22.89	16.11	22.56	15.63	21.81	15.05
CLas阴性对照 Negative control	-CK	—	17.61	—	17.47	—	18.06