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

【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。     

Study on Anti-viral Effect of Rhubarb on Porcine Epidemic Diarrhea Virus in vitro

The inhibitory effects of rhubarb on cell lesion, which was caused by porcine epidemic diarrhea virus (PEDV), and its mechanism had been studied in this research.The maximum safe concentration of rhubarb solution applied to Vero cells was determined through morphological observation, and then the PEDV infected Vero cell model in vitro was established, by which the inhibitory effect of rhubarb on virus was studied.The experimental method had also been established based on this cell model.The result showed that the maximum safe concentration was 3.28 mg/mL.Both morphological observation and MTT assay were indicated that rhubarb could not only block the attachment of the virus to Vero cell and inhibit the synthesis and reproduction of the virus, but also could directly inactivate the virus.This research showed that rhubarb had an inhibitory effect on PEDV, and provided a theoretical basis for the prevention and treatment of clinical porcine epidemic diarrhea.     

Dense planting with less basal nitrogen fertilization might benefit rice cropping for high yield with less environmental impacts

Dense planting and less basal nitrogen (N) fertilization have been recommended to further increase rice (Oryza sativa L.) grain yield and N use efficiency (NUE), respectively. The objective of this study was to evaluate the integrative impacts of dense planting with reduced basal N application (DR) on rice yield, NUE and greenhouse gas (GHG) emissions. Field experiments with one conventional sparse planting (CK) and four treatments of dense planting (increased seedlings per hill) with less basal N application were conducted in northeast China from 2012 to 2013. In addition, a two-factor experiment was conducted to isolate the effect of planting density and basal N rate on CH₄ emission in 2013. Our results show that an increase in planting density by about 50% with a correspondingly reduction in basal N rate by about 30% (DR1 and DR2) enhanced NUE by 14.3–50.6% and rice grain yield by 0.5–7.4% over CK. Meanwhile, DR1 and DR2 reduced GWP by 6.4–12.6% and yield-scaled GWP by 7.0–17.0% over CK. According to the two-factor experiment, soil CH₄ production and oxidation and CH₄ emission were not affected by planting density. However, reduced basal N rate decreased CH₄ emission due to it significantly reduced soil CH₄ production with a smaller reduction in soil CH₄ oxidation. The above results indicate that moderate dense planting with less basal N application might be an environment friendly mode for rice cropping for high yield and NUE with less GHG emissions.     

淮安区水稻测土配方施肥配方校正与示范对比试验报告

为验证肥料配方的准确性,发挥配方肥料施用的最大效益。通过田间校验,示范对比测土配方施肥的增产效果,进一步验证并完善淮安区的肥料配方,优化测土配方施肥技术参数。     

小麦TaeEF1β基因的克隆、同源性及表达分析

真核翻译延伸因子(eukaryotic translation elongation factor,eEFs)是一种重要的多功能调控蛋白,eEF1β是eEF1的组成部分,在蛋白质生物合成过程中发挥着重要的作用。本文通过RT-PCR扩增克隆小麦(Triticum aestivum L.)的eEF1β基因,并命名为TaeEF1β。氨基酸同源性分析发现,TaeEF1β具有高度保守性,且其保守结构域位于137~226 aa处。qRT-PCR结果表明,中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)侵染小麦植株后,可以诱导TaeEF1β基因转录水平的上调表达。另外,本文也进一步分析了TaeEF1β基因在小麦根、茎、叶的表达水平和CWMV侵染不同时间点的表达情况。     

水稻OsZ314基因克隆及转录激活验证

穗是水稻(Oryza sativa L.)重要的生殖器官,其生长发育直接影响水稻的产量和品质。前期研究中,我们对水稻生殖生长时期穗发育各阶段的转录组、蛋白组数据进行联合分析,筛选到一个MYB基因家族的转录因子OsZ314基因。生物学信息分析表明,该基因位于1号染色体上,拥有典型的R2R3-MYB型转录因子结构域,且在水稻幼穗时期表达量最高;亚细胞定位结果显示,OsZ314基因定位于细胞核中;转录激活试验表明,OsZ314基因具有转录激活功能。本研究为深入探索OsZ314基因在水稻穗发育时期的分子生物学功能奠定了良好的基础。     

野生稻种质在水稻抗病育种上的研究及应用进展

野生稻是一个重要的基因资源库,包含了水稻各种病害的抗性基因。本文综述了野生稻丰富的抗性资源及其基因的挖掘,以及在水稻育种上的利用研究进展,并对目前存在的突出问题,提出了野生稻种质资源抗病育种的发展建议。     

番木瓜环斑病毒弱毒株的构建及分析

弱毒株是利用交叉保护防治植物病毒病害的关键限制因子。通过对马铃薯Y病毒属病毒(强、弱毒株)的全长及部分氨基酸序列比对分析,筛选出在亲本强毒株PRSV-LM的P1和HC-Pro基因上的8个可能与致病性相关的氨基酸突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))。采用定点突变和Gibson拼接法,分别成功构建了4种含有2个突变位点(L_(754)和N_(787))、4个突变位点(I_(309)、K_(481)、F_(753)和D_(944))、6个突变位点(I_(309)、K_(481)、F_(753)、L_(754)、N_(787)和D_(944))和8个突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))的PRSV-LM全长c DNA侵染性克隆突变体:p Gprsvm~2、p Gprsvm4、p Gprsvm6和p Gprsvm8。经人工接种、病症观察和RT-PCR检测,4种突变体克隆均能系统性侵染非转基因番木瓜植株,除克隆p Gprsvm~2的病症表现与亲本强毒株PRSV-LM一致外,其他多位点突变的克隆p Gprsvm4、p Gprsvm6和p Gprsvm8在番木瓜植株上的病症严重程度出现依次减弱,而克隆p Gprsvm8的病症最弱,且延迟2周发病。本实验初步验证了6个氨基酸突变位点(I_(309)→S、K_(481)→Q、K_(598)→E、F_(753)→L、R_(728)→I和D_(944)→N)与PRSV-LM的病症表现及致病力相关,其中K_(598)→E和R_(728)→I可能是影响致病性的关键位点。     

Identification and Molecular Characteristics Analysis of Porcine Epidemic Diarrhea Virus Variants

To ascertain a diarrhea case in a pig farm in Shandong province,the pathological changes of dead piglets were observed and nested RT-PCR test was carried out on 7 diarrhea samples for porcine epidemic diarrhea virus (PEDV).Pathological examination revealed that intestine was detected as enlargement,hyperemia and edema.After histological examination,the typical microscopic lesions of intestine were disappearance of epithelial cells,villus shrinkage and shortening.The result of nested RT-PCR showed that all of the 7 samples could amplify a specific target band of PEDV.Molecular characteristics of two field strains showed that they had an amino acid homology of 92.3% to 92.4% with vaccine CV777 and 96.6% to 98.6% with other previous field strains which sequences were downloaded from GenBank.Phylogenetic tree analysis further revealed that all of PEDV strains could be mainly divided into two clusters of G1 and G2. G2 consisted of the field strains of our study and other field strains from USA,China and so on,which had the same sequence characteristics of two insertions and one deletion,while G1 consisted of all vaccines of CV777 and several older field strains from China and Korea.These results indicated that our field strains were the dominant strains in recent epidemic diarrhea occurrence.Moreover,its molecular characteristics might be a characterization of differentiating the field and vaccine strains.