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。
【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.
作者
许雪飞
杨盼盼
张文亮
边光亚
徐雷锋
刘会超
明军
XU Xue-fei;YANG Pan-pan;ZHANG Wen-liang;BIAN Guang-ya;XU Lei-feng;LIU Hui-chao;MING Jun(School of Horticulture and Landscape Architecture,Henan Institute of Science and Technology,Xinxiang 453003;State Key Laboratory of Vegetable Biobreeding,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081;Shijiazhuang Academy of Agriculture and Forestry Sciences,Shijiazhuang 050021)
出处
《生物技术通报》
CAS
CSCD
北大核心
2024年第8期212-220,共9页
Biotechnology Bulletin
基金
国家重点研发计划项目(2022YFD1602206,2019YFD1001002)
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)。
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
Lilium lancifolium
lily mottle virus
virus distribution
in situ hybridization
shoot tip
root tip
RT-PCR
