小麦矮缩病毒引起的植株矮化与赤霉素代谢的相关性分析

Correlational Analyses Between Dwarfing of Plant Height Induced by Wheat dwarf virus(WDV) Infection and Gibberellin Metabolism

【目的】由异沙叶蝉(Psammotettix alienus)传播的小麦矮缩病毒病是近年来中国西北部麦区严重发生的小麦病毒病害之一。受侵染的小麦植株严重矮化,有效分蘖减少,产量损失严重。论文旨在明确小麦矮缩病毒(Wheat dwarf virus,WDV)侵染小麦植株后矮化症状形成与赤霉素代谢调控的关系,为该病害的防治打下基础。【方法】以小麦品种扬麦12为试验材料,以异沙叶蝉为传毒介体饲毒后转移到1叶期的健康幼苗(3头/株)上进行传毒,同时以无毒异沙叶蝉取食健康幼苗为对照。根据试验需要,不同时间取样备用。为保证试验的准确性,经PCR检测为阳性的作为处理组试验材料;采用间接酶联免疫吸附(ELISA)法,利用植物赤霉素(GA3)试剂盒测定分析侵染第21天取样的小麦叶片赤霉素含量;将带毒条沙叶蝉接种的小麦苗分为两个平行处理组,接种后第7天分别用GA3(浓度为50 mg·L-1)和H2O进行叶面喷施处理,每隔一周处理一次。以无毒叶蝉接种后长势一致的小麦苗作为对照组,根据株高统计结果分析外施赤霉素对受侵染小麦植株的表型变化;以山羊草(Aegilops tauschii)的内根-贝壳杉烯合成酶(ent-kaurene synthase-like 3,KSL3)的基因编码区序列为参考基因设计引物(KSL3-F:5′-ATGATGGTGAATCCGCCGC-3′;KSL3-R:5′-TTAATGGTTGATCTTTGTTT-3′),对扬麦12的KSL3进行克隆和序列分析;分别取接种后7、14和21 d的小麦植株叶片,提取RNA后反转录,以克隆得到的Ta KSL3基因序列设计引物(Ta KSL3-F:5′-GAGACATGTGCCATGGCGTTC-3′;Ta KSL3-R:5′-CGTGTCACTCAGATCGGTGGAG-3′),选择小麦翻译延伸因子1A(EF-1α)作为内参基因,利用荧光定量PCR方法分析赤霉素代谢相关基因的转录水平。【结果】经ELISA检测发现,接种21 d的发病植株赤霉素含量与健康植株相比降低了28.9%;通过施用浓度为50 mg·L-1的赤霉素后,发病植株的平均株高相比对照组显著增加35.9%;采用同源克隆得到了完整的小麦赤霉素合成途径关键酶KSL3的编码区序列,长度为1 827 bp,编码608个氨基酸,BLAST比对分析发现该DNA序列与山羊草KSL3编码区序列相似度为85.2%。经荧光定量检测发现受小麦矮缩病毒侵染后小麦KSL3表达量显著下降,接种14 d降低为对照组的35.7%,21 d降低为对照组的9.6%。【结论】小麦矮缩病毒的侵染导致赤霉素合成途径关键酶的表达量降低,可能使赤霉素合成受阻,赤霉素含量降低引发受赤霉素调节的细胞生物学过程异常,从而诱导矮化症状形成。研究结果为揭示小麦矮缩病毒侵染的致病机理和病害防控打下了基础。

【Objective】 In recent years, wheat dwarf virus disease transmitted by leafhopper（Psammotettix alienus） is becoming one of the severe virus disease on triticeae crops in the northwest of China. Infected plants may be severely dwarfed and reduced effective tillering so that it causes serious production loss. The objectives of this study are to clear the correlation between dwarfing of plant height induced by Wheat dwarf virus（WDV） infection and gibberellin metabolism and provide a solid basis for the control of the disease.【Method】The cultivar Yangmai 12 was used as experimental material. Leafhoppers were fed on viruliferous wheat containing WDV and then transferred to healthy seedling at single-leaf stage（3 heads/plant）. The leaves were collected as samples at different time points according to experimental requirements and the seedlings treated with non-viruliferous leafhoppers as control. To guarantee the accuracy of the experiment, all the infected samples were validated by PCR. The plant GA3 ELISA Kit was used to assay the GA3 contents of both healthy and WDV-infected wheat leaves at 21 days post-infection（dpi）. Viruliferous treatments were divided into two parallel groups, one was treated with spraying of exogenous GA3 at 50 mg·L-1 and the other treated with water at 7 dpi. The spraying of GA3 solution was carried out 4 times every 7 days. Meanwhile, the control group consisted of similar size seedlings were treated with non-viruliferous leafhoppers. The phenotype was analyzed by statistical analysis of the plant heights after application of exogenous GA3 at 50 mg·L-1. According to the coding sequence of ent-kaurene synthase-like 3（KSL3） of Aegilops tauschii for reference, the complete coding sequence of KSL3 of Yangmai 12 was cloned with the primer pair（KSL3-F： 5′-ATGATGGTGAATCCGCCGC-3′, KSL3-R： 5′-TTAATGGTTGATCTTTGTTT-3′） and its structure was analyzed with BLAST. The total RNA of infected plants at 7, 14 and 21 dpi were prepared and reversed transcription into c DNA. The primers were designed following the obtained sequence of Ta KSL3（Ta KSL3-F： 5′-GAGACATGTGCCATGGCGTTC-3′, Ta KSL3-R： 5′-CGTGTCACTC AGATCGGTGGAG-3′）. With EF-1α as the reference gene, RT-q PCR was used to analyze the expression level of gene related to GA metabolism pathway.【Result】The GA3 content of infected plants was reduced by 28.9% at 21 dpi by indirect ELISA and the height of infected plants increased by 35.9% owing to application of exogenous GA3 at 50 mg·L-1 compared with control. Using homology-based cloning, the complete coding sequence of Yangmai 12 KSL3 was obtained, which is the key enzyme in gibberellin synthesis pathway. The sequence of KSL3 has the length of 1 827 bp, encoding 608 amino acids. By BLAST analysis, its DNA sequence was found to have a similarity of 85.2% compared with KSL3 of A. tauschii. Particularly, by q RT-PCR analysis, the expression level of KSL3 compared to healthy plants significantly decreased to 35.7% at 14 dpi and 9.6% at 21 dpi, respectively.【Conclusion】 It was concluded that WDV could interfere the biosynthesis of GA leading to the decreased content of GA, which was responsible for the development of dwarf symptom that can be relieved by exogenous GA. This study will provide a solid basis for further research on pathogenesis of WDV and control of the disease.