摘要
以经过改造的烟草脆裂病毒(Tobacco rattle virus,TRV)载体PYL156为工具,采用叶片注射法侵染茄子(Solanum melongena L.)叶片,通过表型比较、TRV病毒分子检测和荧光定量PCR技术分析,探究环境温度、植株大小、目的基因插入片段大小对茄子TRV-VIGS沉默体系的影响。结果表明,昼夜温度为(25±3)℃和(20±2)℃时,接种茄子幼苗子叶的植株沉默效果明显,侵染后植株叶片中目的基因表达量与阴性对照相比明显降低;早春日光温室和秋季日光温室条件下,侵染植株表型性状和基因表达量差异不显著。沉默片段大小为200 bp左右时目的基因的沉默效果最好;侵染茄子幼苗子叶期植株出现明显病毒斑,而接种6周龄真叶则无明显表型差异。用茄子生长素诱导基因(SmIAA19)为靶基因对其进行验证,结果表明显著抑制了SmIAA19的表达,其在叶片中的表达量和生长素含量均显著降低,表明Sm IAA19是1个与生长素代谢途径相关的基因。
Virus-induced gene silencing（VIGS）system is a very useful tool for evaluating gene function and molecular mechanism. Many factors,such as vector,viral proliferation and systemic movement throughout the plant,affect the efficiency of VIGS. In order to improve the VIGS silence efficiency in eggplant（Solanum melongena L.）,three parameters（environment temperature,seedling age and gene fragment size）were optimized. The results showed that the silencing efficiency was higher in temperature controlled greenhouse[（25 ± 3）℃,daytime and（20 ± 2）℃,night]than in room temperature greenhouse. After inoculation of the cotyledons of eggplant,the plants had obvious silence symptom,but there was no obvious silence effect when the six-week-old true leaves were inoculated. The silence frequencies of the gene fragments 597 bp,442 bp and 205 bp were 56.67%,62.5% and 68.75% respectively,indicating that about 200 bp might be the most suitable fragment size for VIGS. Then,the function of Sm IAA19 from eggplant IAA family genes was evaluated using this VIGS system. The Sm IAA19 silence plants presented obvious phenotypic symptoms after inoculation for 4 weeks. The transcript levels of Sm IAA19 and auxin content in the Sm IAA19 silenced plants were significantly reduced compared to the control. It showed that Sm IAA19 was an auxin metabolic related gene in eggplant,and TRV-VIGS system was a reliable method for the function identification of eggplant gene.
作者
齐东霞
张映
赵祯
张伟伟
陈钰辉
连勇
田时炳
刘富中
QI Dongxia;ZHANG Ying;ZHAO Zhen;ZHANG Weiwei;CHEN Yuhui;LIAN Yong;TIAN Shibing;LIU Fuzhong(Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China;The Institute of Vegetables and Flowers, Chongqing Academy of Agricultural Sciences, Chongqing 400055, China)
出处
《园艺学报》
CAS
CSCD
北大核心
2018年第4期691-701,共11页
Acta Horticulturae Sinica
基金
国家自然科学基金项目(31572122)
中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS)
农业部中央级公益性科研院所基本科研业务费专项(1610102016025)
农业部园艺作物生物学与种质创制重点实验室项目
重庆市院士引导专项(cstc2017jcyj-yszx X0005)
