芦笋病毒诱导基因沉默体系的初步构建  

Preliminary Construction of a Virus-induced Gene Silencing System in Asparagus officinalis

作　　者：吴贺 李云宾 董陈文华 林春[1,2] 刘正杰[1,2] 毛自朝[1,2] WU He;LI Yunbin;DONG Chenwenhua;LIN Chun;LIU Zhengjie;MAO Zichao(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;Institute of Improvement and Utilization of Characteristic Resource Plants,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学农学与生物技术学院,云南昆明650201 [2]云南农业大学特色小宗作物研究中心,云南昆明650201

出　　处：《云南农业大学学报(自然科学版)》2025年第1期31-41,共11页Journal of Yunnan Agricultural University:Natural Science

基　　金：云南省农业联合专项重点项目(202101BD070001-027);云南省外专引智项目(202305AO350034);云南省基础研究计划面上项目(202201AT070254)。

摘　　要：【目的】在多年生雌雄异株植物芦笋(Asparagus officinalis L.)中建立病毒诱导基因沉默(virus-induced gene silencing,VIGS)体系,以快速探究芦笋中关键基因的功能。【方法】以绿芦笋品种‘Guelph Millennium’为试验材料,选用八氢番茄红素脱氢酶编码基因(AoPDS)和镁螯合酶Ⅰ亚基编码基因(AoCH42)为报告基因,基于苹果潜伏球形病毒(apple latent spherical virus,ALSV)沉默载体,采用真空渗透法通过农杆菌对芦笋露白种子进行侵染,经表型和qRT-PCR检测,构建芦笋的VIGS体系,并进一步验证编码芦笋NADH-脱氢酶铁硫蛋白亚基S8基因(AoNDUFS8)的功能。【结果】侵染21 d后,AoPDS和AoCH42沉默植株分别出现白化和黄化表型;AoNDUFS8沉默植株长势瘦弱,发育迟缓。qRT-PCR结果显示:与对照组相比,植株侵染后,目的基因表达均下降,预示成功构建芦笋VIGS体系。【结论】基于ALSV的VIGS体系能有效沉默芦笋中的目的基因,为后续芦笋功能基因组分析提供了实践基础。[Purpose]To establish a virus-induced gene silencing(VIGS)system in the perennial dioecious plant of Asparagus officinalis L.,exploring the function of key genes.[Methods]The green A.officinalis variety‘Guelph Millennium’was used as material,the phytoene dehydrogenase(AoPDS)and the magnesium chelatase subunitⅠ(AoCH42)were selected as report genes,and the apple latent spherical virus(ALSV)was used as silencing vector,the geminating A.officinalis seeds were infected by the resulted Agrobacterium tumefaciens with vacuum infiltration method.The VIGS system of A.officinalis was constructed by phenotype and qRT-PCR,and the function of AoNDUFS8 gene encoding A.officinalis NADH-dehydrogenase ferrithione subunit was further verified.[Results]After 21 days of infection,both the AoPDS and AoCH42 silenced plants had an albino and etiolation phenotypes,while the AoNDUFS8 silenced plants grew thin and stunted.qRT-PCR results showed that:compared with the control group,the expression of the target gene decreased after VIGS,indicating that the silencing system was successfully established.[Conclusion]The VIGS system can effectively silence the target gene(s)in A.officinalis,providing a practical basis for subsequent functional genomic analyses of A.officinalis.

关 键 词：芦笋 病毒诱导基因沉默(VIGS)体系 PDS CH42 基因功能 

分 类 号：S435.672[农业科学—农业昆虫与害虫防治]