百合斑驳病毒在卷丹顶端分生组织中的分布

doi:10.13560/j.cnki.biotech.bull.1985.2024-0229

生物技术通报 ›› 2024, Vol. 40 ›› Issue (8): 212-220.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0229

百合斑驳病毒在卷丹顶端分生组织中的分布

许雪飞¹^,²(), 杨盼盼², 张文亮³, 边光亚³, 徐雷锋², 刘会超¹(), 明军²()

1.河南科技学院园艺园林学院，新乡 453003
2.中国农业科学院蔬菜花卉研究所蔬菜生物育种全国重点实验室，北京 100081
3.石家庄市农林科学研究院，石家庄 050021

收稿日期:2024-03-08 出版日期:2024-08-26 发布日期:2024-06-27
通讯作者: 明军，男，博士，研究员，研究方向：观赏植物资源与育种；E-mail: mingjun@caas.cn；
刘会超，男，博士，教授，研究方向：观赏植物资源与创新利用；E-mail: huichaoliu2012@163.com
作者简介:许雪飞，女，硕士研究生，研究方向：园林植物与应用；E-mail: 2378472302@qq.com
第一联系人：杨盼盼为本文共同第一作者
基金资助:
国家重点研发计划项目(2022YFD1602206);国家重点研发计划项目(2019YFD1001002);湘西州技术攻关“揭榜挂帅”项目(2022JBGS0008);石家庄市农业科技项目(23005)

Distribution of Lily Mottle Virus in the Shoot Tips and Root Tips of Lilium lancifolium

XU Xue-fei¹^,²(), YANG Pan-pan², ZHANG Wen-liang³, BIAN Guang-ya³, XU Lei-feng², LIU Hui-chao¹(), MING Jun²()

1. School of Horticulture and Landscape Architecture, Henan Institute of Science and Technology, Xinxiang 453003
2. State Key Laboratory of Vegetable Biobreeding，Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
3. Shijiazhuang Academy of Agriculture and Forestry Sciences，Shijiazhuang 050021

Received:2024-03-08 Published:2024-08-26 Online:2024-06-27

摘要/Abstract

摘要：

【目的】百合斑驳病毒（lily mottle virus, LMoV）是危害重要食药用百合卷丹的主要病毒之一，每年给百合种植业造成数十亿的损失。通过原位杂交技术明确LMoV在卷丹顶端分生组织中的分布，为百合脱毒培养提供支撑。【方法】采用种植于贵州省清镇市的栽培卷丹作为试验材料，利用RT-PCR并于NCBI网站下载不同地方LMoV分离物构建进化树鉴定卷丹所携带的病毒种类，据检测结果选取LMoV为对象，基于LMoV基因组序列设计并制备RNA荧光探针，开展卷丹茎尖和根尖石蜡切片组织的RNA原位杂交，观察卷丹茎尖和根尖中LMoV的分布。【结果】RT-PCR检测表明卷丹材料侵染了LMoV，构建进化树发现本实验分离物与吉林和辽宁的LMoV分离物关系最近，原位杂交进而表明卷丹茎尖中LMoV杂交信号主要分布在顶端分生组织下方0.15-0.2 mm的组织中，靠近紧挨分生组织叶原基的初生叶基部有较弱的病毒杂交信号，靠近初生叶的成熟叶顶端中病毒荧光信号强烈，感染LMoV的卷丹分生组织中心纵切面无毒区大小为（0.4-0.6）mm ×（0.15-0.2）mm；在根尖0.25 mm × 0.2 mm分生组织中无杂交信号分布，在皮层细胞中信号最强烈，在根冠和根伸长区信号较弱。【结论】卷丹顶端分生组织及最接近顶端的一个叶原基[大小约（0.4-0.6）mm×0.2 mm]未发现病毒信号，可作为茎尖脱毒的起始材料。根顶端分生组织外侧根冠有较强病毒信号，不适合作为卷丹脱毒培养的外植体。

关键词: 卷丹, 百合斑驳病毒, 病毒分布, 原位杂交, 茎尖, 根尖, RT-PCR

Abstract:

【Objective】Lily mottle virus（LMoV）is one of the major viruses that jeopardize the important food and medicinal lily Lilium lancifolium Thunb, causing billions RMB yuan losses to the lily cultivation industry every year. We clarified the distribution of LMoV in the apical meristematic tissues of L. lancifolium by in situ hybridization to provide support for the cultivation of lily detoxification.【Method】Cultivated L. lancifolium planted in Qingzhen city, Guizhou province, was used as the test material. We identified the virus species carried by the test material by RT-PCR and LMoV isolates from different places were downloaded from the NCBI website to construct an evolutionary tree using the material of L. lancifolium. RNA flourescent probes were designed and prepared based on the LMoV genome sequence, and in situ hybridization was carried out in the stem tip and root tip regions to observe the distribution of LMoV.【Result】RT-PCR showed that the L. lancifolium material was infested with LMoV, and the construction of evolutionary tree revealed that the isolates in this experiment were most closely related to the LMoV isolates from Jilin and Liaolin, and in situ hybridization showed that the LMoV hybridization signals were mainly distributed in the tissues 0.15-0.2 mm below the shoot apical meristem, and there were weak viral hybridization signals in the base of the primary leaf adjacent to the first leaf primordium. The fluorescence signals of the virus were strong in the apices of mature leaves close to the primary leaves, and the size of the avirulent zone in the center of the LMoV-infected L. lancifolium meristem was (0.4-0.6) mm×(0.15-0.2) mm in longitudinal section. There was no distribution of hybridization signals in the 0.25 mm×0.2 mm meristem of the root tip. The signals were the strongest in the cortex and weaker in the root crown and root elongation zone.【Conclusion】No viral signals were found in the apical meristem and the closest apical leaf primordium[（0.4-0.6）mm×0.2 mm in size]. These regions can be used as a starting material for stem tip detoxification. The outer root crown of the apical meristem had virus signals and was not suitable for use as an explant in the culture of L. lancifolium detoxification.

Key words: Lilium lancifolium, lily mottle virus, virus distribution, in situ hybridization, shoot tip, root tip, RT-PCR

许雪飞, 杨盼盼, 张文亮, 边光亚, 徐雷锋, 刘会超, 明军. 百合斑驳病毒在卷丹顶端分生组织中的分布[J]. 生物技术通报, 2024, 40(8): 212-220.

XU Xue-fei, YANG Pan-pan, ZHANG Wen-liang, BIAN Guang-ya, XU Lei-feng, LIU Hui-chao, MING Jun. Distribution of Lily Mottle Virus in the Shoot Tips and Root Tips of Lilium lancifolium[J]. Biotechnology Bulletin, 2024, 40(8): 212-220.

图/表 6

图1 卷丹原位杂交试验材料 A：花；B：采收后的鳞茎；C：包埋后抽茎的鳞茎。标尺=1 cm

Fig. 1 In situ hybridization material of L. lancifolium A: Flower; B: bulbs after harvesting; C: bulbs after embedding. Bar = 1 cm

表1 病毒检测引物序列

Table 1 Primer sequences for virus detection

引物名称Primer name	引物序列Primer sequence（5'-3'）	产物大小Product size/bp	参考文献Reference
CMV- Forward primer	ATGGACAAATCTGAATCAACCAG	657	[15]
CMV- Reverse primers	TCAGACTGGGAGCACTCCAG	657
LSV- Forward primer	GAAGAAGCACGCTGGACTG	171
LSV- Reverse primers	CGCCTGATGATCCCCTC	171
LMoV- Forward primer	GAAGAAGCACGCTGGACTG	428
LMoV - Reverse primers	CGCCTGATGATCCCCTC	428
Lily Actin- Forward primer	GCATCACACCTTCTACAACG	257	[31]
Lily Actin - Reverse primers	GAAGAGCATAACCCTCATAGA	257	[31]

图2 三种病毒RT-PCR检测电泳图 1-4分别为4株卷丹百合样品； P：阳性对照；N：阴性对照；A：actin（257 bp）；B：CMV（657 bp）；C：LSV（171 bp）；D：LMoV（428 bp）；M2：DNA marker

Fig. 2 Electrophoresis diagram of three viruses via RT-PCR 1-4 are 4 lily samples; P: positive control; N: negative control

图3 LMoV扩增片段与其他LMoV分离物的系统进化树

Fig. 3 Phylogenetic tree of LMoV-amplified fragments and other isolates

图4 LMoV在卷丹茎尖中的原位杂交结果 A：阴性对照（未感染LMoV 的卷丹根尖）；B：茎尖中LMoV信号；C：茎尖分生组织及其周围组织中病毒分布（为B中白色虚线框放大图）；D：分生组织及其最靠近顶端的一个叶原基无病毒信号（为C中白色虚线框放大图）；E：顶端分化新的叶原基图，原先第一片靠近顶端的叶原基发育来的幼叶基部开始感染（为B中蓝色虚线框放大图）；SAM：顶端分生组织；Le：叶片；Lp：叶原基；Bpl：初生叶基部；Bml：成熟叶基部；Tml：成熟叶顶端；Pi：髓细胞。B比例尺=500 μm；C和E比例尺=200 μm，A和D标尺=100 μm。蓝色为细胞核染色，红色为LMoV阳性信号

Fig. 4 In situ hybridization of LMoV in the shoot tips of L. lancifolium A: Negative control（LMoV-uninfected stem tips of L. lancifolium）; B: LMoV signal in the stem tip; C: virus distribution in the apical meristem and its surrounding tissues（enlarged white dashed box in B）; D: non-virus signal in the meristem and its closest apical leaf primordium（enlarged white dashed box in C）; E: image of the apical meristem dividing into new leaf primordia, where the base of the young leaves developed from the original first near-apical leaf primordium began to be infected（enlarged blue dashed box in B）; SAM: apical meristematic tissue; Le: leaf blade; Lp: leaf primordium; Bpl: the base of the primary leaf; Bml: the base of the mature leaf; Tml: the tip of a mature leaf; Pi: pith cell. B scale bar = 500 μm; C and E scale bar = 200 μm; A and D scale bar = 100 μm. Blue is nuclear staining and red is LMoV positive signal

图5 LMoV在卷丹根尖中的原位杂交结果 A：阴性对照（未感染LMoV的卷丹根尖）；B：根尖中心纵剖面中LMoV分布图；C：LMoV在根尖皮层细胞和维管束中分布；D：LMoV在根顶端组织中的分布；VB：维管束；Pi：髓细胞；Co：皮层细胞；Ez：伸长区。蓝色为细胞核染色，红色为LMoV阳性信号。A, B标尺=200 μm；C、D标尺=100 μm

Fig. 5 In situ hybridization of LMoV in the root tip of L. lancifolium A: Negative control（LMoV-uninfected stem tips of L. lancifolium）; B: LMoV distribution in the longitudinal profile of the apical center; C: distribution of LMoV in root tip cortical cells and vascular bundles; D: distribution of LMoV in root apical meristem（Am）and a small amount invades the root cap（RC）; VB: vascular bundles; Pi: pith cells; Co: cortex cells; Ez: elongation zone. The blue color is the nuclei staining, and the red color is the LMoV positive signal. A, B bars = 200 μm, C, D bars = 100 μm

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百合斑驳病毒在卷丹顶端分生组织中的分布

Distribution of Lily Mottle Virus in the Shoot Tips and Root Tips of Lilium lancifolium

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