State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling, Shaanxi 712100, China.
BMC Plant Biol. 2012 Aug 10;12:141. doi: 10.1186/1471-2229-12-141.
The Barley stripe mosaic virus (BSMV)-based vector has been developed and used for gene silencing in barley and wheat seedlings to assess gene functions in pathogen- or insect-resistance, but conditions for gene silencing in spikes and grains have not been evaluated. In this study, we explored the feasibility of using BSMV for gene silencing in wheat spikes or grains.
Apparent photobleaching on the spikes infected with BSMV:PDS at heading stage was observed after 13 days post inoculation (dpi), and persisted until 30 dpi, while the spikes inoculated with BSMV:00 remained green during the same period. Grains of BSMV:PDS infected spikes also exhibited photobleaching. Molecular analysis indicated that photobleached spikes or grains resulted from the reduction of endogenous PDS transcript abundances, suggesting that BSMV:PDS was able to induce PDS silencing in wheat spikes and grains. Inoculation onto wheat spikes from heading to flowering stage was optimal for efficient silencing of PDS in wheat spikes. Furthermore, we used the BSMV-based system to reduce the transcript level of 1Bx14, a gene encoding for High-molecular-weight glutenin subunit 1Bx14 (HMW-GS 1Bx14), by 97 % in the grains of the BSMV:1Bx14 infected spikes at 15 dpi, compared with that in BSMV:00 infected spikes, and the reduction persisted until at least 25 dpi. The amount of the HMW-GS 1Bx14 was also detectably decreased. The percentage of glutenin macropolymeric proteins in total proteins was significantly reduced in the grains of 1Bx14-silenced plants as compared with that in the grains of BSMV:00 infected control plants, indicating that HMW-GS 1Bx14 is one of major components participating in the formation of glutenin macropolymers in wheat grains.
This is one of the first reports of successful application of BSMV-based virus-induced-gene-silencing (VIGS) for gene knockdown in wheat spikes and grains and its application in functional analysis of the 1Bx14 gene. The established BSMV-VIGS system will be very useful in future research on functional analysis of genes contributing to grain quality and the metabolic networks in developing seeds of wheat.
基于大麦条纹花叶病毒（BSMV）的载体已被开发并用于大麦和小麦幼苗中的基因沉默，以评估其在抗病原体或抗昆虫方面的基因功能，但尚未评估其在穗和籽粒中的基因沉默条件。在这项研究中，我们探讨了使用 BSMV 在小麦穗或籽粒中进行基因沉默的可行性。
在接种后 13 天（dpi），BSMV：PDS 感染的穗在抽穗期出现明显的光漂白，持续到 30 dpi，而同时期接种 BSMV：00 的穗保持绿色。感染 BSMV：PDS 的穗的籽粒也表现出光漂白。分子分析表明，光漂白的穗或籽粒是由于内源 PDS 转录本丰度降低所致，表明 BSMV：PDS 能够诱导小麦穗和籽粒中 PDS 的沉默。从抽穗到开花期接种到小麦穗上是在小麦穗中高效沉默 PDS 的最佳方法。此外，我们使用基于 BSMV 的系统将感染 BSMV：1Bx14 的穗的籽粒中的 1Bx14 基因（编码高分子量谷蛋白亚基 1Bx14（HMW-GS 1Bx14）的转录本水平降低了 97%，与感染 BSMV：00 的穗相比，这种降低一直持续到至少 25 dpi。HMW-GS 1Bx14 的含量也可检测到降低。与感染 BSMV：00 的对照穗相比，1Bx14 沉默植株的籽粒中谷蛋白大聚合体蛋白在总蛋白中的百分比显著降低，表明 HMW-GS 1Bx14 是参与小麦籽粒中谷蛋白大聚合体形成的主要成分之一。
这是首次成功应用 BSMV 基于病毒诱导的基因沉默（VIGS）在小麦穗和籽粒中进行基因敲低及其在 1Bx14 基因功能分析中的应用的报道之一。所建立的 BSMV-VIGS 系统将在未来对影响籽粒品质的基因和小麦发育种子代谢网络的功能分析研究中非常有用。
