Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene
Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene
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DOI：
10.1111/pbi.12270
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发表时间：
2014-05-01
影响因子：
13.8
通讯作者：
Xu, Huijun
中科院分区：
文献类型：
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作者：
Chen, Ming;Sun, Liying;Xu, Huijun
核因子Y（NF-Y）是一种普遍存在的转录因子，由三个不同的亚基组成，即NF-YA、NF-YB和NF-YC。本研究从三倍体狗牙根（Cynodon dactylonxCynodon transvaalensis）中分离得到NF-YC（Cdt-NF-YC1）胁迫响应cDNA，并探讨其在非生物胁迫耐受中的作用。在所有营养组织中均检测到 Cdt-NF-YC1 转录物，其中在根中观察到较高水平。叶子中 Cdt-NF-YC1 的转录是由脱水、盐度和脱落酸 (ABA)、过氧化氢 (H2O2) 或一氧化氮 (NO) 处理诱导的，但不会因寒冷而改变。 Cdt-NF-YC1 脱水或盐诱导的转录被 ABA 合成抑制剂和 H2O2 或 NO 清除剂阻断，表明 ABA、H2O2 和 NO 参与了 Cdt-NF-YC1 脱水和盐诱导的转录。 Cdt-NF-YC1 的过度表达导致转基因水稻对干旱和盐胁迫的耐受性提高，并且对 ABA 的敏感性增加。转基因水稻中胁迫/ABA 响应基因（OsLEA3、OsRAB16A、OsLIP9 和 OsP5CS1）、ABA 合成和信号基因（OsNCED3 和 OsABI2）以及 ABA 独立基因（OsDREB1A、OsDREB1B 和 OsDREB2A）的转录水平显着高于野生型植物。结果表明，Cdt-NF-YC1是一个很好的候选基因，可以通过调节ABA依赖性和ABA非依赖性途径的基因调控来提高转基因水稻的耐旱性和耐盐性。小麦黄花叶病毒（WYMV）在中国主要小麦产区迅速蔓延，造成严重的减产。由于它是由在土壤中长期存活的类真菌生物禾本科多粘菌传播的，因此很难通过传统的作物管理或杀菌剂来消除。商业品种的抗性也很有限。在本研究中，通过反义NIb8基因（WYMV的NIb复制酶）和选择性基因bar的共转化获得了14个独立的转基因事件。四个原始转基因品系（N12、N13、N14 和 N15）和一个后代品系（N12-1）在田间表现出对 WYMV 的高且持久的抗性。后代的PCR和除草剂抗性测试表明，4个抗性品系已分离，仅含有NIb8（不含bar）。 N12-1品系对中国不同地点的WYMV分离株表现出广谱抗性。在受侵染的土壤中生长后，转基因小麦的组织印迹和蛋白质印迹分析无法检测到 WYMV。转基因小麦的籽粒产量比野生型敏感对照高出约10%。 Northern印迹和小RNA深度测序分析表明，转基因小麦植株中没有针对NIb8基因的小干扰RNA的积累，这表明转基因RNA沉默（病毒源性抗病的常见机制）并不参与WYMV抗性过程。转基因小麦对 WYMV 的持久和广谱抗性将有助于减轻 WYMV 造成的损害。
Nuclear factor Y (NF-Y) is a ubiquitous transcription factor formed by three distinct subunits, namely NF-YA, NF-YB and NF-YC. A stress-responsive cDNA of NF-YC (Cdt-NF-YC1) was isolated from triploid bermudagrass (Cynodon dactylonxCynodon transvaalensis), and its role in abiotic stress tolerance was investigated in this study. Cdt-NF-YC1 transcript was detected in all vegetative tissues with higher levels being observed in roots. Transcription of Cdt-NF-YC1 in leaves was induced by dehydration, salinity, and treatments with abscisic acid (ABA), hydrogen peroxide (H2O2) or nitric oxide (NO), but not altered by cold. The dehydration- or salt-induced transcription of Cdt-NF-YC1 was blocked by inhibitor of ABA synthesis and scavenger of H2O2 or NO, indicating that ABA, H2O2 and NO were involved in the dehydration- and salt-induced transcription of Cdt-NF-YC1. Overexpression of Cdt-NF-YC1 resulted in elevated tolerance to drought and salt stress and increased sensitivity to ABA in transgenic rice. Transcript levels of stress/ABA responsive genes (OsLEA3, OsRAB16A, OsLIP9 and OsP5CS1), ABA synthesis and signalling genes (OsNCED3 and OsABI2), and ABA-independent genes (OsDREB1A, OsDREB1B and OsDREB2A) were substantially higher in transgenic rice than in wild-type plants. The results suggested that that Cdt-NF-YC1 is a good candidate gene to increase drought and salinity tolerance in transgenic rice through modulating gene regulation in both ABA-dependent and ABA-independent pathways.Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat-growing areas in China. Because it is vectored by the fungus-like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co-transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12-1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12-1 showed broad-spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild-type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus-derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad-spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.