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农业生物技术学报  2020, Vol. 28 Issue (6): 974-983    DOI: 10.3969/j.issn.1674-7968.2020.06.003
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
基于VIGS基因沉默体系的换锦花LsMYBs基因功能研究
周洋丽, 侯朔, 郑正权, 高燕会*, 童再康
浙江农林大学 亚热带森林培育国家重点实验室,临安 311300
Study on LsMYBs Gene Function in Lycoris sprengeri Based on VIGS Gene Silencing System
ZHOU Yang-Li, HOU Shuo, ZHENG Zheng-Quan, GAO Yan-Hui*, TONG Zai-Kang
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an 311300, China
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摘要 换锦花(Lycoris sprengeri)是石蒜属(Lycoris)多年生球根类非模式植物,花色多为红蓝复色花,观赏价值极高,利用基因工程技术探讨换锦花花色形成机制对换锦花新品种选育具有重要的指导意义。本研究采用无缝克隆技术构建换锦花LsMYBs病毒介导的基因沉默(virus-induced gene silencing, VIGS)载体,利用真空渗透法侵染换锦花花苞,并对侵染后换锦花花瓣花色苷含量和花色形成相关基因的表达进行检测,建立换锦花VIGS技术体系并初步分析LsMYBs的基因功能。研究结果表明LsMYBs沉默后换锦花花瓣LsMYBs基因的表达明显下降,而花瓣尖端的蓝色加深,花色苷含量明显增加;VIGS载体的插入片段为400 bp时诱导基因沉默后LsMYBs的表达量相对稳定,是未沉默的LsMYBs基因表达量的1/2,而插入cDNA全长片段基因沉默后LsMYBs表达不稳定。LsMYB4基因沉默后花色苷形成过程中结构基因(早期形成基因(early biosynthesis gene, EBG)和晚期形成基因(late biosynthesis gene, LBG))查尔酮合成酶基因(chalcone synthase, LsCHS)、查尔酮异构酶基因(chalcone isomerase, LsCHI)、黄烷酮3-羟化酶基因(flavanone 3-hydroxylase, LsF3H)、类黄酮-3'-羟化酶基因(flavonoid 3'-hydroxylase, LsF3'H)、二氢黄酮醇-4-还原酶基因(dihydroflavonol-4-reductase, LsDFR)、花青素合酶基因(anthocyanidin synthaes, LsANS)、类黄酮3-O-葡萄糖基转移酶基因(flavonoid 3-o-glycosyltransferase, LsUFGT)的表达量明显上升,但变化程度不同;而LsMYB5沉默后仅LBGs基因(LsANS, LsDFR3LsUFGT)表达量明显上升,其他基因表达变化不明显,说明LsMYB4LsMYB5在换锦花花色形成的过程中可能起着负调控的作用,但调控的靶基因对象不完全相同。本研究结果对进一步解析LsMYBs转录因子对换锦花花色苷形成的调控机制提供了重要的信息。
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周洋丽
侯朔
郑正权
高燕会
童再康
关键词 换锦花病毒介导的基因沉默(VIGS)LsMYBs花色苷转录抑制    
AbstractLycoris sprengeri is a kind of non-model perennial bulbous plant of Lycoris, whose flower color is bicolor with red and blue, and which is great ornamental value. It is of great significance to explore the mechanism of flower color formation by using genetic engineering technology for the breeding of new varieties of L. sprengeri. Virus-induced gene silencing (VIGS) is a new reverse genetics technology, which could be used to study the gene function of non-model plants. In this study, LsMYBs VIGS vectors were constructed by seamless cloning technology , the buds of L. sprengeri were infected using vacuum permeation method, and the expression of LsMYBs and anthocyanin contents of the petals in L. sprengeri after infection were detected, which would establish the VIGS technology system in L. sprengeri and analyzed the gene function of LsMYBs. The results showed that the expression of LsMYBs genes decreased significantly and the total contents of anthocyanin increased significantly after LsMYBs genes silencing. The relative expression of LsMYBs gene was relatively stable by 400 bp insertion fragment in VIGS vector, while the relative expression of LsMYBs was unstable after inserting a full length fragment of cDNA. The relative expression of structural genes ((chalcone synthase (LsCHS), chalcone isomerase (LsCHI), flavanone 3-hydroxylase (LsF3H), flavonoid 3'-hydroxylase (LsF3'H), dihydroflavonol-4-reductase (LsDFR), anthocyanidin synthaes (LsANS), flavonoid 3-o-glycosyltransferase (LsUFGT)) related to anthocyanin formation was affected after LsMYB4 gene silencing, the relative expression of all of the structural genes increased significantly during the whole anthocyanin biosynthesis process (early biosynthesis genes (EBGs) and late biosynthesis genes (LBGs)) and the degree of change was varying; while the expression of LBGs genes (LsANS, LsDFR3 and LsUFGT) increased significantly when LsMYB5 silencing, however, the expression of other genes did not change significantly. It could be inferred that LsMYB4 and LsMYB5 might play a negative regulatory role in the regulation of anthocyanin formation, while the target genes may be different. The results in this study provide a technical and theoretical basis for further analysis of the regulatory mechanism of LsMYBs transcription factors on the biosynthesis of anthocyanin in L. sprengeri.
Key wordsLycoris sprengeri    Virus-induced gene silencing    LsMYBs    Anthocyanin    Transcriptional inhibition
收稿日期: 2019-12-21     
ZTFLH:  S682.2  
基金资助:国家自然科学基金(31670696); 浙江省十三五育种专项(2016C02056-13)
通讯作者: *,gaoyanhui408@126.com   
引用本文:   
周洋丽, 侯朔, 郑正权, 高燕会, 童再康. 基于VIGS基因沉默体系的换锦花LsMYBs基因功能研究[J]. 农业生物技术学报, 2020, 28(6): 974-983.
ZHOU Yang-Li, HOU Shuo, ZHENG Zheng-Quan, GAO Yan-Hui, TONG Zai-Kang. Study on LsMYBs Gene Function in Lycoris sprengeri Based on VIGS Gene Silencing System. 农业生物技术学报, 2020, 28(6): 974-983.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.06.003     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I6/974
 
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