Expression of hairpin RNA (hpRNA) targeting the three CTV-silencing suppressor genes confers sweet orange with stem-pitting CTV tolerance
Expression of hairpin RNA (hpRNA) targeting the three CTV-silencing suppressor genes confers sweet orange with stem-pitting CTV tolerance
复制标题
针对三个 CTV 沉默抑制基因的发夹 RNA (hpRNA) 的表达赋予甜橙茎点蚀 CTV 耐受性
DOI：
10.1080/14620316.2017.1304166
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发表时间：
2017-01-01
影响因子：
1.9
通讯作者：
Zhong, Yun
中科院分区：
文献类型：
--
作者：
Cheng, Chunzhen;Zhang, Yongyan;Zhong, Yun
摘要利用RNA干扰（RNAi）技术沉默病毒的沉默抑制基因已被证明是一种有效的防治植物病毒病的方法。柑橘衰退病毒（Citrus tristeza virus，CTV）具有三个沉默抑制基因p20、p23和p25。本研究采用重叠延伸PCR（SOE-PCR）技术，构建了两个靶向p25、p20和p23的RNAi载体pCAMBIA 2301 G-hp 252023和pCAMBIA 2301 G-hp 252020。通过农杆菌介导法转化甜橙子，获得了7个表达hp 2520（p25和p20的发夹RNA）和hp 252023（p25、p20和p23的发夹RNA）的转基因植株。用CTV严重茎点病株系CT 14 A对转基因植株进行嫁接接种，结果表明，没有转基因植株对CTV严重茎点病株系CT 14 A产生免疫。然而，在hp 2520的两个株系和hp 252023的唯一株系中检测到较低至低得多的CTV滴度。hp 252023表达系的无性后代在接种CT 14 A后2年没有表现出茎点蚀症状，尽管在它们的叶片中仍然检测到低水平的CT 14 A。我们的研究结果表明，表达CTV沉默抑制基因的hpRNA赋予甜橙子对茎痘CTV的高耐受性。
ABSTRACT Silencing the silencing suppressor genes of viruses by RNA interference (RNAi) has been demonstrated to be an efficient way to combat viral diseases in plants. Citrus tristeza virus (CTV) has three silencing suppressor genes, p20, p23, and p25. To silence them, the splicing by overlap extension by PCR (SOE-PCR) was applied in this study for construction of two RNAi vectors, pCAMBIA2301G-hp2520 (to target p25 and p20) and pCAMBIA2301G-hp252023 (to target p25, p20 and p23). Following Agrobacterium-mediated transformation, seven and one independent sweet orange transgenic lines were obtained for expressing the fusion transgenes, hp2520 (hairpin RNA of p25 and p20) and hp252023 (hairpin RNA of p25, p20 and p23), respectively. Challenging the transgenic plants by graft-inoculation with severe stem-pitting CTV strain CT14A showed that no transgenic line was immune to the strain. However, lower to much lower CTV titer was detected in two lines of hp2520 and the only line of hp252023. Vegetative progenies of the hp252023-expressing line showed no stem-pitting symptoms 2 years post CT14A inoculation, although a low level of CT14A was still detectable in their leaves. Our results show that expressing the hpRNAs of CTV silencing suppressor genes conferred sweet orange with high tolerance to stem-pitting CTV.