中國農業科學 ?? 2020, Vol. 53 ?? Issue (5): 955-964.doi: 10.3864/j.issn.0578-1752.2020.05.008

? 植物保護 ? 上一篇    下一篇

侵染廣東連州葫蘆的黃瓜綠斑駁花葉病毒的分子特征 及致病性分析

李正剛,農媛,湯亞飛,佘小漫,于琳,藍國兵,鄧銘光,何自福()   

  1. 廣東省農業科學院植物保護研究所/廣東省植物保護新技術重點實驗室,廣州510640
  • 收稿日期:2019-09-04 接受日期:2019-11-19 出版日期:2020-03-01 發布日期:2020-03-14
  • 通訊作者: 何自福 E-mail:[email protected]
  • 作者簡介:李正剛,E-mail:[email protected]
  • 基金資助:
    國家重點研發計劃(2018YFD0201209);廣東省現代農業產業共性關鍵技術研發創新團隊建設項目(2019KJ134);廣東省重點領域研發計劃(2018B020202006);廣東省重點領域研發計劃(2018B020202007)

Molecular Characteristic and Pathogenicity Analyses of Cucumber green mottle mosaic virus (CGMMV) Infecting Bottle Gourd in Lianzhou, Guangdong

LI ZhengGang,NONG Yuan,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,DENG MingGuang,HE ZiFu()   

  1. Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640
  • Received:2019-09-04 Accepted:2019-11-19 Online:2020-03-01 Published:2020-03-14
  • Contact: ZiFu HE E-mail:[email protected]

摘要:

【目的】黃瓜綠斑駁花葉病毒(Cucumber green mottle mosaic virus,CGMMV)是侵染瓜類作物的主要病毒之一,對瓜類產業造成巨大的危害。本研究旨在探明侵染廣東省連州市葫蘆的CGMMV分離物(CGMMV-GDLZ)分子特征及其在系統進化中的地位,并測定其對黃瓜、葫蘆和西瓜的致病性,為CGMMV的防控提供理論依據。【方法】從廣東省連州市葫蘆種植基地采集2個疑似CGMMV侵染的病樣以及1個無癥狀樣品,提取總RNA,根據CGMMV參考序列(GenBank登錄號:KX883801)設計引物進行RT-PCR檢測,引物序列為F:CCACGAGTTGTTTCCTAATGCTG/R:TTTGCTAGGCGTGATCGGATTGT,退火溫度53℃,擴增長度890 bp。將CGMMV全長序列分為前后兩段,前半段1—3 511 nt,擴增引物序列為F:AAGTTCATTTCATTTGGAGAGGGTTTTAATTTTTATAA TTAAACAAA/R:AGTTCTGCATTAATTGCTATTTGGTAGGCACAGTGGTAG;后半段3 301—6 423 nt,擴增引物序列為F:GTGCGTGCTACCCCGACTCCAATAGGTTTGATTGCCCGTG/R:GGTGGAGATGCCATGCCGACCCTGGGCCCCTACCCGGGGAAAGG。將前后兩段PCR產物通過同源重組的方法克隆到pCB301雙元載體上,測序得到CGMMV-GDLZ分離物全長序列。利用CGMMV-GDLZ分離物全長序列在NCBI中進行Blast分析,然后通過MEGA7軟件對CGMMV-GDLZ以及其他已經報道的CGMMV分離物進行系統進化樹分析。將構建好的pCB301-CGMMV侵染性克隆注射接種本生煙驗證其侵染性,然后再注射接種黃瓜、葫蘆和西瓜的子葉,測定CGMMV-GDLZ分離物的致病性。【結果】RT-PCR結果證實,廣東省連州市葫蘆病樣感染了CGMMV。CGMMV-GDLZ分離物全長序列為6 423 nt,編碼4個蛋白,分別為129K復制酶(61—3 495 nt)、186K復制相關蛋白(61—5 007 nt)、運動蛋白MP(4 994—5 788 nt)和外殼蛋白CP(5 763—6 248 nt)。CGMMV-GDLZ核苷酸序列與CGMMV-eWT分離物(GenBank登錄號:KY753928)同源性最高,為99.97%。系統進化樹分析結果顯示,CGMMV-GDLZ分離物與日本、韓國等東亞CGMMV分離物同屬Group 1,在遺傳距離上與山東、浙江和河南的CGMMV分離物最接近。pCB301-CGMMV侵染性克隆可以系統侵染本生煙,造成本生煙上部葉片出現皺縮、斑駁、凸起等癥狀,RT-PCR和Western blot進一步確認了侵染性克隆的侵染性。注射接種CGMMV-GDLZ分離物后15 dpi,葫蘆和西瓜即可產生斑駁、花葉、突起、生長遲緩等癥狀,24 dpi時癥狀更明顯。而15 dpi時,CGMMV-GDLZ分離物在黃瓜上的癥狀不明顯,與未接種對照植株幾乎沒有區別;將植株從控溫接種室移入網室中,30 dpi時,黃瓜植株上部葉片開始出現斑駁和花葉,40 dpi時,癥狀已經非常明顯。RT-PCR和Western blot檢測進一步確認了上述結果。【結論】侵染廣東省連州市葫蘆的CGMMV-GDLZ分離物與山東、浙江和河南的CGMMV分離物很可能具有相同的傳染源;CGMMV-GDLZ分離物可以侵染本生煙、黃瓜、葫蘆和西瓜等作物,但對這些作物的致病性存在差異。

關鍵詞: 葫蘆, 黃瓜綠斑駁花葉病毒, 分子特征, 侵染性克隆, 致病性

Abstract:

【Objective】Cucumber green mottle mosaic virus (CGMMV) is one of the main viruses that infect melon crops, which causes great harm to the melon industry. The objective of this study is to identify the molecular characteristic and phylogenetic status of CGMMV-GDLZ isolate, analyze the pathogenicity of CGMMV-GDLZ isolate on cucumber, bottle gourd, and watermelon, and to provide a theoretical basis for the prevention and control of CGMMV. 【Method】Two symptomatic samples and one asymptomatic sample were collected from a farm located in Lianzhou City of Guangdong Province and were subjected to total RNA extraction. RT-PCR detection was performed using primer pair F: CCACGAGTTGTTTCCTAATGCTG/R: TTTGCTAGGCGTGATCGGATTGT, which was designed according to CGMMV reference genome (GenBank accession number KX883801). The annealing temperature is 53℃, and the product is 890 bp in length. To construct pCB301-CGMMV infectious cDNA clones, two primer pairs were designed to amplify the first half (1-3 511 nt) and the second half (3 301-6 423 nt) of the nucleotide sequence of CGMMV-GDLZ isolate. The primer pair used to amplify the first half is F: AAGTTCATTTCATTTGGA GAGGGTTTTAATTTTTATAATTAAACAAA/R: AGTTCTGCATTAATTGCTATTTGGTAGGCACAGTGGTAG, and the primer pair used to amplify the second half is F: GTGCGTGCTACCCCGACTCCAATAGGTTTGATTGCCCGTG/R: GGTGGAGATGC CATGCCGACCCTGGGCCCCTACCCGGGGAAAGG. Then the acquired products were cloned into pCB301 by homologous recombination, followed by Sanger DNA sequencing. Blast analysis was performed in NCBI using the full-length sequence of CGMMV-GDLZ isolate, then the phylogenetic tree was constructed by MEGA7 software using the sequences of CGMMV-GDLZ and other reported CGMMV isolates. Agrobacterium containing pCB301-CGMMV was infiltrated into Nicotiana benthamiana leaves to verify the infectivity of CGMMV-GDLZ isolate. Subsequently, pCB301-CGMMV was agroinfiltrated into the cotyledon of cucumber, bottle gourd, and watermelon to analyze the pathogenicity of CGMMV. 【Result】RT-PCR detection verified that the two symptomatic samples were infected by CGMMV. Sanger sequencing reveals that CGMMV-GDLZ isolate contains 6 423 nt and encodes four proteins, 129K replicase (61-3 495 nt), 186K replication-associated protein (61-5 007 nt), movement protein (4 994-5 788 nt), and coat protein (5 763-6 248 nt). CGMMV-GDLZ isolate has the highest nucleotide similarity (99.97%) with CGMMV-eWT isolate (GenBank accession number KY753928). Phylogenetic analysis showed that CGMMV-GDLZ and other CGMMV isolates from Japan and Korea were clustered into Group 1, and CGMMV-GDLZ was closest to isolates from Shandong, Zhejiang, and Henan. In addition, the pCB301-CGMMV infectious cDNA clones could successfully infect N. benthamiana, causing crinkle, mottle, and mosaic symptoms in the upper leaves. RT-PCR and Western blot detection further verified the infection of the infectious cDNA clones. Moreover, CGMMV-GDLZ isolate could also infect bottle gourd and watermelon, causing mottle, mosaic, and growth retardation at 15 days post-infiltration (dpi), and showed more severe symptoms at 24 dpi. However, the upper leaves of cucumber agro-infiltrated with pCB301-CGMMV did not show obvious symptoms compared with control plants. Further studies found the agro-infiltrated plants began to appear mottle and mosaic symptoms at 30 dpi after transferred from the greenhouse to artificial net-room without temperature control. The symptoms were more obvious at 40 dpi. RT-PCR and Western blot detection further verified the above results. 【Conclusion】CGMMV-GDLZ isolate from Lianzhou City of Guangdong Province may have the same infection source with CGMMV isolates from Shandong, Zhejiang, and Henan provinces. CGMMV-GDLZ isolate can infect N. benthamiana, cucumber, bottle gourd, and watermelon, but the pathogenicity varies in different crops.

Key words: bottle gourd, Cucumber green mottle mosaic virus (CGMMV), molecular characteristic, infectious cDNA clones, pathogenicity

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