烟粉虱模式识别受体βGRPs的先天免疫应答

为探索烟粉虱的β-1,3-葡聚糖识别蛋白(BtβGRP)在烟粉虱先天免疫过程中的重要作用,对烟粉虱中鉴定的3个BtβGRPs基因编码的蛋白质进行结构分析,并与其它物种的23个同源蛋白进行系统进化分析,同时通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)检测分析球孢白僵菌侵染烟粉虱不同虫态不同时间点后BtβGRPs的时间表达模式。结果显示:经鉴定3个BtβGRPs蛋白序列均含有βGRP家族特有的保守性结构域,即糖苷水解酶活性结构域;且3个BtβGRPs独自聚在进化树的一支上,可能参与烟粉虱特有的级联路径;3个BtβGRPs基因在不同虫态和不同诱导时间点与对照相比,除BtβGRP3在卵期未表达外,均有上调表达趋势,但诱导程度不同;成虫诱导24~36 h后达到峰值,比卵和若虫稍晚,可能是由于烟粉虱成虫体表有白色蜡粉等物质,延迟了球孢白僵菌对虫体的侵染。研究表明3个BtβGRPs蛋白可能参与了烟粉虱对真菌侵染的免疫反应,有可能成为烟粉虱生防的新靶标。     

芋疫霉重组聚合酶扩增结合侧流层析试纸条快速检测方法的建立及应用

为建立芋疫霉Phytophthora colocasiae快速准确的分子检测方法,基于Ypt1基因特异序列,设计芋疫霉的特异性引物与探针,建立一种快速、准确、可视化的芋疫霉重组聚合酶扩增结合侧流层析试纸条（recombinase polymerase amplification-lateral flow dipstick,LFD-RPA）检测方法,对该检测方法进行优化,评估其特异性与灵敏度,并对田间疑似样品进行检测。结果表明,优化后的芋疫霉LFD-RPA检测方法最适反应条件为39℃恒温反应30 min。LFD-RPA检测方法能够特异性地检测出芋疫霉,而对其他卵菌近缘种和常见植物病原真菌均未检出,且该检测方法对芋疫霉DNA的检测灵敏度达到1 pg/μL。对田间带病组织检测发现,LFD-RPA检测方法能够快速准确地从田间自然发病植株中检测出芋疫霉。表明本研究所建立的芋疫霉LFD-RPA快速可视化检测方法特异性好、灵敏度高、简单快捷,可用于芋疫病的田间快速诊断。     

烟草丛顶病毒RNA依赖RNA聚合酶(RdRp)介导的体外复制体系

通过重叠PCR扩增得到烟草丛顶病毒(Tobacco bushy top virus,TBTV)中国分离物RdRp的编码序列,构建以pMALC2X为基础载体的原核表达载体pMAL-TB-RdRp。0.5 mM IPTG诱导可特异性表达分子量约为120 kDa的MBP-RdRp融合蛋白。温度梯度实验显示,18℃下诱导表达的MBP-RdRp融合蛋白的可溶性比例较高,约17%;经亲和层析纯化的MBPRdRp可特异性识别TBTV正链和负链的3'末端序列,催化体外复制;对正负链的3'末端的体外复制效率存在差别,识别负链3'末端的体外复制效率明显高于正链3'末端。本研究创建的TBTV RdRp介导的体外复制体系为进一步研究TBTV基因组复制调控奠定了基础。     

重组酶聚合酶扩增技术在植物病原快速检测中的应用

植物病原的快速检测对于植物病害防控具有重要意义。重组酶聚合酶扩增(recombinase polymerase amplification,RPA)是近年建立的等温核酸扩增技术,具有灵敏度高、特异性强、适用于现场快速检测等特点,已在多个领域广泛应用。本文对重组酶聚合酶扩增技术的原理、特性、检测方法及其在植物病原体检测领域的应用进展加以综述。     

向日葵茎溃疡病菌RPA检测方法的建立

向日葵茎溃疡病菌(Diaporthe helianthi)是我国进境植物检疫性病原真菌.本研究针对D.helianthi的cal基因保守序列设计特异性引物,建立该病菌的重组酶聚合酶扩增检测技术(RPA)方法,并对其特异性、灵敏度及适用性进行评价.结果 显示,建立的RPA方法特异性强,只有3个D.helianthi样品能...     

一种基于RPA的番茄褪绿病毒检测方法

番茄褪绿病毒Tomato chlorosis virus(ToCV)引起番茄褪绿病毒病,给番茄生产造成严重危害。开发快速准确的检测方法对该病害的防控具有重要意义。利用番茄褪绿病毒外壳蛋白(CP)基因序列,设计特异性引物,建立了ToCV的重组酶聚合酶等温扩增(recombinase polymerase amplification, RPA)检测方法,同时分析了该方法的灵敏度和特异性。结果表明,建立的ToCV-RPA方法在38℃恒温下40 min可从ToCV阳性的番茄样品中扩增出246 bp的特异性条带。扩增时间短,对设备要求低,且与番茄其他病毒无交叉反应,特异性好,灵敏度可达到PCR方法的10倍,适用于ToCV的快速检测。     

Wax matters: absence of very‐long‐chain aldehydes from the leaf cuticular wax of the glossy11 mutant of maize compromises the prepenetration processes of Blumeria graminis

Conidial germination and differentiation, the so‐called prepenetration processes, of the barley powdery mildew fungus (Blumeria graminis f.sp. hordei) are triggered in vitro by very‐long‐chain aldehydes, minor constituents of barley leaf wax. However, until now it has not been demonstrated that these cuticle‐derived molecules also play a significant role in the initiation and promotion of the fungal prepenetration processes in vivo, on the surface of a living plant leaf. In the maize (Zea mays) wax mutant glossy11, which is completely devoid of cuticular very‐long‐chain aldehydes, germination and appressorial differentiation of B. graminis were strongly impeded. Spraying the mutant leaf surface with aldehyde‐containing wild‐type wax or pure n‐hexacosanal (C₂₆‐aldehyde) fully restored fungal prepenetration, whereas maize wild‐type leaf surfaces coated with n‐docosanoic acid exhibited reduced conidial germination rates of 23%, and only 5% of the conidia differentiated infection structures. In vitro studies were performed to further corroborate the extensive prevention of fungal germination and differentiation in response to artificial surfaces coated with aldehyde‐deficient maize wax. Because of its phenotype affecting the B. graminis prepenetration processes, the glossy11 mutation of maize may become a valuable molecular target and genetic tool that could provide a means of developing basal powdery mildew resistance in the globally important crops wheat and barley.     

人心果AFLP反应体系的建立与优化

为给人心果分子生物学研究奠定基础,以人心果嫩叶为材料,采用改良CTAB法提取到高质量的总DNA,可用于AFLP实验分析。通过优化酶切—连接反应、预扩增和扩增反应过程中的关键因素,建立了适合人心果的AFLP实验体系和反应条件,得到了带型清晰、多态性丰富的AFLP指纹图谱。研究结果对人心果遗传多样性分析及其分子标记辅助育种具有重要意义。     

Evaluating the impact of moulting and chilled storage of spermatophores on the integrity of plasma membrane,acrosome and DNA of black tiger prawn (Penaeus monodon) spermatozoa

To explore the impact of moulting and short‐term chilled storage of spermatophores on the sperm quality for a commercially important penaeid prawn, spermatozoa of Penaeus monodon from early (B‐C), middle (D0‐1) and late (D2‐3) moult stages were compared for sperm quality parameters relating to the structural integrity of plasma membrane (Viab%), acrosome (AR%) and DNA (SDF%) after being stored at 4°C for 0–26 days. The three different sperm extenders used for chilled storage included artificial lobster haemolymph (AH), calcium free saline (CS) and filtered seawater (FS); two storage conditions were applied either as a free sperm suspension or retained within the intact spermatophore. Results showed that (a) the lowest natural AR% was shown for B‐C spermatozoa in CS whereas the highest levels were for B‐C, D0‐1 and D2‐3 spermatozoa in FS and D2‐3 spermatozoa in AH; (b) the calcium ionophore A23187 agent used in this study was able to increase the mean AR% by 6.22%; (c) the Viab% was significantly lower in CS than that in FS; (d) the SDF% significantly increased over the period of chilled storage for B‐C and D0‐1 spermatozoa, while the SDF% of D2‐3 spermatozoa was initially elevated and did not change significantly over time; and (e) there was no difference in sperm quality between two storage conditions. This study has successfully demonstrated the moult‐related variance in the percentages of acrosome‐reacted and DNA‐damaged spermatozoa, providing evidence of moult‐related spermatophore renewal cycle in this species.     

煤制油供应链整体优化

随着人们环保意识与政策要求的不断加强以及对石油需求的不断增加,大力发展煤制油技术对保障中国能源安全、调整优化能源结构具有重要意义。以煤制油炼厂为主体,考虑炼厂建设、采购、生产、运输、销售过程及煤炭产地供应与市场需求等相关约束条件,以煤制油炼厂收益最大为目标函数,建立煤制油供应链的混合整数线性规划优化模型,并采用基于分支定界法的大规模数学规划求解器(Gurobi)进行求解。研究成果成功应用于中国某地区煤制油炼厂,对煤制油供应链的建设具有较强的指导意义,一定程度上降低了资源消耗,提升了供应链收益,实现了整个供应链的高效运作。