灰比诺葡萄病毒内蒙古分离物全基因组分析  

Complete genome sequence analysis of grapevine pinot gris virus isolates from Inner Mongolia

作　　者：郭孟泽 徐磊[1] 闫雨婷 孙平平 张磊[1] 李正男 GUO Mengze;XU Lei;YAN Yuting;SUN Pingping;ZHANG Lei;LI Zhengnan(College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Hohhot 010018,Inner Mongolia,China)

机构地区：[1]内蒙古农业大学园艺与植物保护学院,呼和浩特010018

出　　处：《果树学报》2024年第12期2425-2435,共11页Journal of Fruit Science

基　　金：内蒙古自治区科技计划项目(2022YFDZ0029);内蒙古自治区自然科学基金项目(2022QN03018);内蒙古自治区高等学校“青年科技英才支持项目”(NJYT23079)。

摘　　要：【目的】获取灰比诺葡萄病毒(grapevine pinot gris virus,GPGV)内蒙古分离物全基因组序列,并对该病毒群体进行序列一致性、系统发育、基因重组以及群体遗传多样性等分析。【方法】以GPGV阳性样品为试验材料,通过RT-PCR技术和cDNA末端快速扩增技术(rapid amplification of cDNAends,RACE)克隆GPGV内蒙古分离物的全基因组序列,并通过分子生物学分析软件对其进行基因组序列分析。【结果】成果克隆了2条GPGV内蒙古分离物(20IM-Vi-Vi1和20IM-ViVi2)的全基因组序列,序列全长均为7250 nt,且均编码3个ORFs;序列一致性分析结果显示,20IM-Vi-Vi1与20IM-ViVi2基因组序列核苷酸一致率为96.4%,与其他分离物的全基因组序列一致率分别为79.7%~96.8%、79.5%~97.7%;系统进化分析表明,GPGV所有全基因组分离物可划为4个分支,其中本研究中所获得的2个分离物20IM-ViVi1与20IM-ViVi2均聚集在第Ⅰ分支,并均与中国夏黑分离物SRR2845691-GPGV亲缘关系最近;遗传多样性分析结果表明,GPGV具有较高的遗传多样性,其中GPGV亚洲分离物遗传多样性最高。【结论】首次获得GPGV内蒙古分离物的全基因组序列,并阐述了2个GPGV内蒙古分离物与已知病毒之间的进化关系,可为中国GPGV株系划分、遗传进化研究奠定理论基础。【Objective】The objective of this study was to acquire the complete genomic sequence of grapevine pinot gris virus(GPGV)isolates from Inner Mongolia and to perform a comprehensive analy-sis of the GPGV population,encompassing sequence identity,phylogenetic relationships,gene recombi-nation,sequence similarity and genetic diversity.【Methods】Grape leaf samples that had previously test-ed positive for GPGV served as the experimental materials for total RNA extraction.A total of 100 mg of GPGV-infected grape samples were processed in accordance with the Spectrum™Plant Total RNA Kit instructions.The quality and concentration of the extracted RNA were evaluated via 1%agarose gel electrophoresis and microspectrophotometry,respectively,and the RNA was preserved at-80℃for fu-ture use.Vector NTI software was utilized to align all the full-length genomic sequences of GPGV re-ported in the NCBI GenBank database.Three primer pairs(GPGV-1F/GPGV-1R,GPGV-2F/GPGV-2R and GPGV-3F/GPGV-3R)were designed within the conserved regions to amplify the complete genom-ic sequence of GPGV,ensuring that overlapping fragments between adjacent amplification products ex-ceeded 200 bp.Subsequently,primers(GPGV3 and GPGV1)were designed for amplifying the termi-nal sequences of GPGV.Total RNA was employed as a template to synthesize cDNA using the Super-Script™ⅢReverse Transcriptase Kit under the conditions of 50℃for 1 hour,followed by 70℃for 15 minutes.The cDNA template was then used to amplify the nucleotide sequences of GPGV with Q5 High-Fidelity 2×Master Mix,employing thermal cycling parameters of denaturation at 98℃for 30 sec-onds,annealing at 56℃for 30 seconds,and extension at 72℃for 2 minutes over 35 cycles.The SMARTer®RACE 5'/3'Kit was used to amplify the 5'and 3'terminal sequences of GPGV.PCR prod-ucts were identified through 1%agarose gel electrophoresis,and the target fragments were purified us-ing a gel DNA purification kit.The amplified and RACE-obtained GPGV genomic sequences were as-sembled using Vector NTI

关 键 词：灰比诺葡萄病毒 系统进化分析 序列一致性 遗传多样性 

分 类 号：S663.1[农业科学—果树学] S436.631[农业科学—园艺学]