Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMM...Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.展开更多
本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度...本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度,以测试基因沉默的有效性。结果表明,真空渗透和种子吸胀的接种方式能够在瓜类作物中产生最高的基因沉默率(FGS)。在22℃和25℃的培养环境下,黄瓜、甜瓜和西瓜PDS沉默有效性(EGSL)较高。黄瓜、甜瓜和西瓜生长1个月后,EGSL达到100%,显著高于30℃的培养环境下EGSL。生长3个月后,甜瓜和西瓜在22℃培养环境下EGSL分别为89.7%和95%;在25℃培养环境下EGSL分别为85.6%和86.1%,均显著性高于30℃下EGSL。在相对湿度为30%和50%环境下,黄瓜的EGSL分别为70%和72%,甜瓜的EGSL分别为76%和73%,显著高于相对湿度为80%的培养环境下EGSL。西瓜在相对湿度为30%的培养环境下的EGSL为69%,显著高于相对湿度为50%时的EGSL(38%)和相对湿度在80%时的EGSL(33%)。综上所述,通过优化了瓜类作物的CGMMV-VIGS的技术体系中接种方式和培养环境参数,提高了基因沉默率和有效性,并且能够快速获得整个植株基因沉默的种质资源,为作物优质和抗逆基因功能的研究提供有效途径。展开更多
The coat protein gene and its 3′ noncoding region of a watermelon isolate of Cucumber green mottle mosaic virus (CGMMV-LN) in Liaoning, China was determined and compared with other isolates. The gene encoding the coa...The coat protein gene and its 3′ noncoding region of a watermelon isolate of Cucumber green mottle mosaic virus (CGMMV-LN) in Liaoning, China was determined and compared with other isolates. The gene encoding the coat protein of CGMMV-LN comprises of 486 nucleotides and encodes a putative protein of 161 amino acids. Sequence comparisons showed that the coat protein of this isolate was identical to that of seven CGMMV isolates infecting cucurbits in Europe and Asia. The 3′ noncoding region of CGMMV-LN consists of 176 nucleotides, which is same as that of CGMMV-KOM, CGMMV-KW and CGMMV–SH.展开更多
为了揭示黄瓜绿斑驳病毒（Cucumber green mottle mosaic virus,CGMMV）河北分离物分子变异及系统进化情况。本研究自河北省5个代表性西瓜产区采集病样,分离纯化获得5个CGMMV河北分离物,经RTPCR扩增、基因克隆获各分离物的外壳蛋白（Coa...为了揭示黄瓜绿斑驳病毒（Cucumber green mottle mosaic virus,CGMMV）河北分离物分子变异及系统进化情况。本研究自河北省5个代表性西瓜产区采集病样,分离纯化获得5个CGMMV河北分离物,经RTPCR扩增、基因克隆获各分离物的外壳蛋白（Coat protein,CP）基因序列。使用Vector NTI 10.0软件（Informax,Frederick,MD,USA）和MEGA5.0软件（Koichiro,Tokyo,Japan）对河北省5个分离物及GenBank已登录其它分离物CP进行序列分析,构建系统发育树。系统进化关系分析表明：所有CGMMV分离物可划分为5个组,CGMMV河北分离物与中、日、韩三国分离物亲缘关系较近,位于同一组别。与希腊2个分离物（CGMMV-GR5、GR3）亲缘关系最远。CGMMV CP序列相似性分析结果表明：河北省5个分离物CP核苷酸、氨基酸序列相似性在99%~100%之间,与其它17个分离物序列相似性在92%~100%之间。与中国LN及Liaoning两个分离物及韩国Watermelon分离物相似性最高为100%,与希腊2个分离物（GR5、GR3）序列相似性最低为92%~99%。揭示了CGMMV河北分离物的分类地位及其与不同来源分离物之间的遗传进化关系,为进一步研究CGMMV株系划分、基因遗传变异提供了理论基础。展开更多
基金supported by the National Natural Science Foundation of China (31571247)the grants from the earmarked fund for the China Agriculture Research System (CARS-26-13)the Agricultural Science and Technology Innovation Program (ASTIP), Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2018-ZFRI-08)
文摘Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.
文摘本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度,以测试基因沉默的有效性。结果表明,真空渗透和种子吸胀的接种方式能够在瓜类作物中产生最高的基因沉默率(FGS)。在22℃和25℃的培养环境下,黄瓜、甜瓜和西瓜PDS沉默有效性(EGSL)较高。黄瓜、甜瓜和西瓜生长1个月后,EGSL达到100%,显著高于30℃的培养环境下EGSL。生长3个月后,甜瓜和西瓜在22℃培养环境下EGSL分别为89.7%和95%;在25℃培养环境下EGSL分别为85.6%和86.1%,均显著性高于30℃下EGSL。在相对湿度为30%和50%环境下,黄瓜的EGSL分别为70%和72%,甜瓜的EGSL分别为76%和73%,显著高于相对湿度为80%的培养环境下EGSL。西瓜在相对湿度为30%的培养环境下的EGSL为69%,显著高于相对湿度为50%时的EGSL(38%)和相对湿度在80%时的EGSL(33%)。综上所述,通过优化了瓜类作物的CGMMV-VIGS的技术体系中接种方式和培养环境参数,提高了基因沉默率和有效性,并且能够快速获得整个植株基因沉默的种质资源,为作物优质和抗逆基因功能的研究提供有效途径。
文摘The coat protein gene and its 3′ noncoding region of a watermelon isolate of Cucumber green mottle mosaic virus (CGMMV-LN) in Liaoning, China was determined and compared with other isolates. The gene encoding the coat protein of CGMMV-LN comprises of 486 nucleotides and encodes a putative protein of 161 amino acids. Sequence comparisons showed that the coat protein of this isolate was identical to that of seven CGMMV isolates infecting cucurbits in Europe and Asia. The 3′ noncoding region of CGMMV-LN consists of 176 nucleotides, which is same as that of CGMMV-KOM, CGMMV-KW and CGMMV–SH.
文摘为了揭示黄瓜绿斑驳病毒（Cucumber green mottle mosaic virus,CGMMV）河北分离物分子变异及系统进化情况。本研究自河北省5个代表性西瓜产区采集病样,分离纯化获得5个CGMMV河北分离物,经RTPCR扩增、基因克隆获各分离物的外壳蛋白（Coat protein,CP）基因序列。使用Vector NTI 10.0软件（Informax,Frederick,MD,USA）和MEGA5.0软件（Koichiro,Tokyo,Japan）对河北省5个分离物及GenBank已登录其它分离物CP进行序列分析,构建系统发育树。系统进化关系分析表明：所有CGMMV分离物可划分为5个组,CGMMV河北分离物与中、日、韩三国分离物亲缘关系较近,位于同一组别。与希腊2个分离物（CGMMV-GR5、GR3）亲缘关系最远。CGMMV CP序列相似性分析结果表明：河北省5个分离物CP核苷酸、氨基酸序列相似性在99%~100%之间,与其它17个分离物序列相似性在92%~100%之间。与中国LN及Liaoning两个分离物及韩国Watermelon分离物相似性最高为100%,与希腊2个分离物（GR5、GR3）序列相似性最低为92%~99%。揭示了CGMMV河北分离物的分类地位及其与不同来源分离物之间的遗传进化关系,为进一步研究CGMMV株系划分、基因遗传变异提供了理论基础。
