基于牡丹类黄酮糖基转移酶基因建立VIGS技术体系

Establishing Virus Induced Gene Silencing(VIGS)System in Tree Peony Using PsUFGT Genes

牡丹(Paeonia suffruticosa Andrews)花色的化学基础研究较为深入,但因其无遗传转化体系,导致花色相关基因的功能验证只能利用其他植物进行旁证。利用保守区段长分别为413和418 bp的牡丹花瓣类黄酮糖基转移酶基因PsUF3GT和PsUF5GT,通过VIGS表达载体,采用二因素三水平的试验体系对目的基因进行沉默。结果表明,PsUF3GT和PsUF5GT的表达量在花斑中(盛开期花瓣,真空抽滤15 min)和非斑中(蕾期花瓣,真空抽滤10 min)分别比空载体处理的花斑中(盛开期花瓣,真空抽滤10 min)和非斑中(蕾期花瓣,真空抽滤15 min)降低了65.0%和85.0%;花斑中总花青苷含量分别降低了24.2%和28.2%,尤其是盛开期花瓣(真空抽滤15 min)处理后3G型糖苷降低了92.2%,蕾期花瓣(真空抽滤10 min)处理后3G5G型糖苷降低了54.9%。由此获得了沉默PsUF3GT和PsUF5GT的适宜体系:以盛开期或者花蕾期带花柄的花朵为材料,抽真空渗透10~15 min后,置于去离子水中暗培养1 d(8℃,湿度60%);之后转移至23~25℃、湿度60%的环境,光照培养3 d后可用于检测和分析。本研究中建立的VIGS体系可有效沉默花瓣中的内源基因,为牡丹基因功能验证及其花色形成的分子机制研究奠定了基础。

Considerable progress has been made on the phytochemical basis of flower color formation on tree peony（Paeonia suffruticosa Andrews）. However,due to lack of genetic transformation system,function of the genes related to flower color formation has to be characterized using other model plant systems. We utilized virus induced gene silencing（VIGS）for establishing the functional characterizing technique to study UDP-glucose：flavonoid glycosyltransferases（UFGTs）genes in tree peony. The fragments with 413 bp and 418 bp length with a putatively conserved domain of PsUF3GT and PsUF5GT were selected for constructing VIGS recombinant vectors,respectively. Nine treatments with two factors and three levels were conducted to test the gene silencing effects. The results showed that the expression level of PsUF3GT after infiltrating petals of blooming for 15 min,or the expression level of PsUF5GT after infiltrating petals of bud stage for 10 min,were reduced 65.0% and 85.0% as compared with that of control petals of blooming or bud stage infiltrated using empty vectors for 10 min or 15 min,respectively. Meanwhile,total anthocyanins content in petal blotch were also decreased 24.2% and 28.2% after treatment of flowers at blooming or bud stage by infiltration for 15 min or 10 min,respectively,in which,cyanidin 3-O-glucoside reduced 92.2% in petals at blooming stage infiltrated for 15 min and cyanidin 3,5-O-diglucoside reduced 54.9 % in petals at bud staged infiltrated for 10 min. Therefore,an optimal VIGS system has been established in tree peony,namely,flowers at blooming or bud stage were subjected to vacuum infiltration for 10–15 min,then kept in dark for 1 d at 8 ℃ with relative 60% humidity,afterwards,kept at 23–25 ℃ with normal light and relative 60% humidity for 3 d,then,treated samples could be used for further functional characterization of the silenced genes. Our VIGS method would be beneficial to functional characterization of the genes related to flower color formation,and study the molecular mechanism of flower color variation.