牡丹授粉受精基因PoMYBs的VIGS体系的建立  

Establishing Virus Induced Gene Silencing(VIGS)System of Pollination and Fertilization Gene PoMYBs in Tree Peony

作　　者：王鹏摇 刘鹏 耿丹丹 王奎玲 郝青 WANG Pengyao;LIU Peng;GENG Dandan;WANG Kuiling;HAO Qing(College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao 266109, China)

机构地区：[1]青岛农业大学园林与林学院,山东青岛266109

出　　处：《青岛农业大学学报（自然科学版）》2021年第3期199-204,217,共7页Journal of Qingdao Agricultural University(Natural Science)

基　　金：国家自然科学基金项目(31601789);青岛农业大学高层次人才科研基金资助项目(6631114306);青岛农业大学研究生创新计划项目(QYC201923)。

摘　　要：授粉受精成败是影响牡丹结实和远缘杂交的关键生殖过程,但缺乏稳定的遗传转化体系严重影响了其基因功能验证。针对牡丹授粉受精基因功能鉴定,采用注射法分析了不同注射部位(雌蕊和花柄)和菌液浓度(1.0、1.5和2.0)对目的基因沉默效率的影响。结果表明,注射雌蕊、花柄PoMYB2基因沉默效率分别为62.5%、66.7%,注射法尤其是注射花柄的方法适用于牡丹植株病毒诱导的基因沉默(Virus induced gene silencing,VIGS)体系的构建;当菌液浓度OD600分别为1.0、1.5、2.0时,PoMYB2基因的沉默效率分别为100%、77.8%和0%,菌液OD600为1.0时沉默效率最高;注射花柄上部,菌液OD600为1.0时,PoMYB1和PoMYB2基因的表达量显著降低。由此获得了沉默PsMYBs的适宜体系:以PoMYBs基因的非保守区域为靶片段,以带花柄的紧实花蕾为材料,注射花柄上部,菌液OD600为1.0,置于去离子水中,暗培养1 d,转移至正常光照培养环境后可进行检测。本研究建立的VIGS沉默体系可有效沉默雌蕊中的内源基因,为进行牡丹雌雄蕊发育、授粉受精和结实方面的基因功能验证奠定了基础。Fertilization success is a bottleneck for tree peony seed setting and distant hybridization.However,the verification of gene function for tree peony is limited due to lack of the genetic transformation system.In this study,different injection sites(pistil and flower stalk)and bacterial concentrations(1.0,1.5 and 2.0)were conducted to test the VIGS effect of target genes by injection.Our results showed that the gene silencing efficiency of PoMYB2 injected pistils and flower stalks were 62.5%and 66.7%,respectively.The injection method especially injecting flower stalk was suitable for the construction of the Virus induced gene silencing(VIGS)system for tree peony.The silencing efficiency of PoMYB2 was 100%,77.8%and 0%,respectively,when the ODwas 1.0,1.5 and 2.0.The silencing efficiency was the highest when ODwas 1.0.The expression of PoMYB1 and PoMYB2 was significantly down-regulated when the upper part of the stalk was injected and ODwas 1.0.Therefore,an optimal VIGS system has been established in tree peony,namely,the nonconservative segment of PoMYBs gene was used as the target segment,and the flowers at bud stage were used as the material,the upper part of flower stalk were injected and OD was 1.0,then they were kept in dark for one day,afterwards,they were transferred to the culture with normal light,then,treated samples could be used for future analysis.The VIGS silencing system in this study can effectively silence the genes in the pistil,and will lay the foundation for the analysis of gene function related to pistil and stamen development,pollination and fertilization,and seed setting of tree peony.

关 键 词：牡丹 受精 VIGS体系 PoMYB 

分 类 号：S682.29[农业科学—观赏园艺]