野生甘蓝表皮毛对菜青虫的抗性机理

为明确一种全身覆盖表皮毛的野生甘蓝Brassica incana（编号C01）是否具有抗虫性,通过测定菜青虫Pieris rapae对野生甘蓝C01和无毛甘蓝B. alboglabra（编号C41）的拒食、取食和产卵行为进行抗虫性分析,同时通过测定两者的内源激素含量、表皮毛发育相关基因表达量和防御酶活性探讨野生甘蓝C01对菜青虫的抗性机理。结果显示,生长至8～10叶期,无毛甘蓝C41叶片被菜青虫啃食严重,但野生甘蓝C01叶片未被啃食;菜青虫对无毛甘蓝C41和剪除表皮毛的野生甘蓝C01叶片取食面积差异不显著,但均显著大于对野生甘蓝C01叶片的取食面积;着卵的无毛甘蓝C41植株显著多于野生甘蓝C01。野生甘蓝C01叶片中茉莉酸和茉莉酸甲酯含量都显著高于无毛甘蓝C41叶片,而两者中水杨酸和水杨酸甲酯的含量差异不显著。BolJAZ1基因在无毛甘蓝C41叶片中高表达,而BolGL3和BolGL2基因在野生甘蓝C01叶片中高表达;且野生甘蓝C01叶片中多酚氧化酶、过氧化物酶和苯丙氨酸解氨酶3种防御酶的活性均显著高于无毛甘蓝C41。表明野生甘蓝C01叶片的表皮毛会影响菜粉蝶产卵,对菜青虫表现出显著抗...     

麦二叉蚜唾液蛋白基因Sg1655时空表达谱及其抑制小麦防御反应的功能

为进一步明确麦二叉蚜Schizaphis graminum唾液蛋白Sg1655在调控小麦防御反应中的作用,基于麦二叉蚜唾液腺转录组数据,利用PCR技术克隆获取Sg1655开放阅读框,利用生物信息学软件对其序列进行分析;通过实时荧光定量PCR（real-time quantitative PCR,RT-qPCR）技术检测Sg1655在麦二叉蚜不同组织及不同龄期中的表达;利用细菌III型分泌系统将Sg1655导入到小麦叶片内,检测其瞬时表达对小麦胼胝质积累的影响。结果表明,Sg1655开放阅读框全长375 bp,编码125个氨基酸残基,N端1~21位为信号肽序列,预测蛋白分子量为14.90 kD。麦二叉蚜Sg1655氨基酸序列与玉米蚜Rhopalosiphum maidis同源序列相致性最高,为89.52%;麦二叉蚜Sg1655与玉米蚜同源序列聚为一个分支,亲缘关系最近。Sg1655在麦二叉蚜各组织中均有表达,其中在唾液腺中表达量最高,Sg1655在麦二叉蚜各个龄期均有表达,且在不同龄期之间无显著差异。小麦叶片内Sg1655瞬时表达可显著抑制小麦胼胝质积累,表明该蛋白可作为潜在效应子参与抑制小麦防御反应。     

烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析

为探讨UV-B胁迫对烟蚜Myzus persicae热激蛋白Hsp90基因表达量的影响,采用RT-PCR与RACE技术克隆了烟蚜热激蛋白Hsp90基因的全长,并对其进行生物信息学分析,利用实时荧光定量PCR技术研究了烟蚜Hsp90基因在不同时长UV-B胁迫下的表达量变化。结果表明,烟蚜Hsp90基因的cDNA全长为2 670 bp,编码728个氨基酸,编码蛋白质的相对分子量为82.6 kD,等电点为4.95,获得的氨基酸序列具有Hsp90蛋白家族的1个签名序列及C末端MEEVD基序,推测其属于胞质型热激蛋白。系统进化树结果显示,烟蚜Hsp90与其它昆虫Hsp90具有很高的相似性。实时荧光定量PCR结果表明,不同时长UV-B胁迫下烟蚜Hsp90均有表达,随着照射时间延长,Hsp90表达量表现为先上升后下降的趋势;与对照相比,照射时间为15、30、60、90和120 min时,Hsp90表达量均显著升高,且在60 min时Hsp90表达量达最大,是对照组的2.05倍。表明Hsp90基因在不同时长UV-B胁迫下差异表达,在烟蚜适应紫外胁迫的分子机制中具有重要作用。     

小麦矮腥黑粉菌g9890基因编码效应蛋白的生物信息学分析及亚细胞定位

为明确小麦矮腥黑粉菌Tilletia controversa g9890基因编码效应蛋白的生物学功能,根据小麦矮腥黑粉病菌转录组测序结果,筛选出效应蛋白g9890,通过PCR技术获得g9890基因cDNA的全长序列,并对其进行生物信息学以及亚细胞定位分析。多种生物信息学数据库分析表明,g9890基因全长为1 038 bp（包括终止密码子）,共编码345个氨基酸,相对分子质量为37 353.32,理论等电点为5.02。g9890效应蛋白不稳定系数为15.94,疏水性指数为-0.312,是一种亲水性且稳定的蛋白。将g9890基因与pbin-GFP载体重组,利用冻融法转化至根癌农杆菌Agrobacterium tumefaciens GV3101中,将其注入烟草进行瞬时表达分析,并通过共聚焦激光显微镜观测该基因的定位状况,结果显示,g9890定位在细胞膜和细胞核上。     

大丽轮枝菌效应蛋白VdSRP2的功能分析

为明确效应蛋白VdSRP2在大丽轮枝菌Verticillium dahliae中的生物学功能,从大丽轮枝菌落叶型菌株V592中克隆VdSRP2基因并进行生物信息学分析,利用酵母转化酶分泌系统对其信号肽活性进行测定,采用实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术分析VdSRP2基因在大丽轮枝菌中的表达模式,并以V592菌株为材料获得VdSRP2基因的敲除突变体和过表达体菌株,通过表型分析和致病性测定确定VdSRP2基因的生物学功能。结果显示,VdSRP2基因编码232个氨基酸,含有5个半胱氨酸残基,N-端信号肽具有分泌活性,为真菌的典型效应蛋白;VdSRP2基因主要在大丽轮枝菌菌丝和微菌核中表达,其中经棉花根系诱导培养24 h时表达量最高;与野生型菌株V592相比,VdSRP2基因敲除导致大丽轮枝菌的产孢量和孢子萌发率显著下降,不能形成微菌核,对棉花的致病力明显减弱;但VdSRP2基因敲除不影响大丽轮枝菌的穿透能力;VdSRP2基因在本氏烟和棉花叶片瞬时表达不会诱导细胞死亡。表明VdSRP2是大丽轮枝菌微菌核形成必需...     

南方根结线虫MiV901基因在拟南芥中的异源表达及其表型分析

为明确南方根结线虫Meloidogyne incognita效应蛋白MiV901在其寄生过程中的生物学功能,通过构建MiV901基因的植物表达载体,利用根癌农杆菌Agrobacterium tumefaciens介导的花序浸染法将其转化拟南芥Arabidopsis thaliana,并采用室内人工接种法测定转基因植株对灰葡萄孢 Botrytis cinerea侵染及南方根结线虫寄生的影响。结果显示：经Southern blot检测,MiV901基因以不同的拷贝数插入到转基因拟南芥株系901-6、901-8和901-12的基因组中,且qPCR检测结果证实 MiV901基因能够正常表达。3个转基因拟南芥株系901-6、901-8和901-12叶部接种灰葡萄孢3 d后,叶片上形成的病斑平均直径分别为1.00、1.06、1.05 cm,比野生型对照扩大了9.9%~16.5%。相比野生型对照,转基因拟南芥株系901-12、901-6和901-8接种南方根结线虫2龄幼虫后根系上产生了更多的雌虫和卵块,雌虫数分别显著增加了45.4%、34.4%和23.7%,卵块数分别显著增加了51.2%、 46.3%和31.7%。表明异源表达MiV901基因能够抑制植物免疫,增加拟南芥对灰葡萄孢和南方根结线虫侵染的敏感性。     

苹果中新抗旱相关基因GR- RBPs的克隆与表达分析

在构建楸子叶片SSH cDNA文库及EST分析的基础上,通过电子克隆(in silico cloning)和RT-PCR方法分别从楸子和平邑甜茶叶片中各克隆得到了1个富含甘氨酸RNA结合蛋白(GR-RBP)基因,分别命名为MpGR-RBP1( HM042682)和MhGR-RBP1 (HQ380209).它们的cDNA...     

外源ABA和钨酸钠对干旱胁迫下白菜型冬油菜ZEP表达的影响

为研究白菜型冬油菜玉米黄质环氧酶(ZEP)基因在干旱胁迫下响应外源ABA的分子机制,本研究选择白菜型冬油菜陇油7号和天油4号为试验材料,分别喷施蒸馏水、ABA溶液、钨酸钠溶液、ABA+钨酸钠溶液200 mL后置于植物培养箱干旱处理7 d。采用RT-PCR和qPCR等技术进行ZEP基因克隆和表达分析,结果表明:该基因包含一个长度为2 013 bp的ORF,编码含670个氨基酸残基的蛋白质,理论等电点为6.16,属于亲水性蛋白。该基因与拟南芥、甘蓝型油菜的同源性分别为89.25%和99.40%。实时荧光定量分析表明,干旱胁迫可诱导白菜型冬油菜ZEP基因上调表达,外源喷施ABA后陇油7号叶片ZEP基因相对表达量较CK升高66.64%,喷施外源ABA合成抑制剂钨酸钠后陇油7号叶片ZEP基因表达量较CK降低12.74%,而同时喷施外源ABA和钨酸钠后,干旱胁迫下陇油7号、天油4号叶片ZEP基因表达量较CK分别增加了3.3倍和2.28倍,陇油7号叶片中的ZEP基因表达量较根系高21.61%,表明ZEP基因在白菜型冬油菜抗旱中可能发挥一定作用。     

A PAL1 Gene Promoter–Green Fluorescent Protein Reporter System to Analyse Defence Responses in Live Cells of Arabidopsis thaliana

Arabidopsis thaliana ecotype Columbia-0 was transformed with a green fluorescent protein (GFP) gene under control of a phenylalanine ammonia-lyase (PAL) promoter. PAL is a key enzyme of the phenylpropanoid pathway and is induced to high levels during plant stress. Constitutive expression of PAL1 promoter-controlled GFP occurred in vascular tissues within stems, leaves and roots and in developing flowers. PAL1 promoter–GFP expression was examined in leaves of transgenic plants subjected to an abiotic elicitor, mechanical wounding or to inoculation with the pathogens Pseudomonas syringae pv. tomato or Peronospora parasitica. Wounding of leaves and treatment with an abiotic elicitor and compatible interactions produced low to moderate levels of GFP. However, in incompatible interactions there were high levels of GFP produced. In incompatible interactions, the intensity of GFP fluorescence was similar to that produced in transgenic plants expressing GFP driven by the CaMV promoter. The bright green fluorescence produced in live cells and tissues was readily visualised using conventional fluorescence microscopy and was quantified using spectroflourometry. This is the first report of the use of GFP as a reporter of defence gene activation against pathogens. It has several advantages over other reporter genes including real time analysis of gene expression and visualisation of defence gene activation in a non-invasive manner.     

核盘菌自噬相关基因SsATG5和SsATG8的功能分析

为探究自噬在核盘菌Sclerotinia sclerotiorum致病过程中的作用,利用酵母Saccharomyces自噬相关基因（autophagy-related gene,ATG）编码的蛋白序列比对核盘菌基因组,获得核盘菌假定ATG,并以核盘菌1980菌株为出发菌株,基于同源重组的原理对假定ATG进行敲除和回补,并测定不同突变体的生长表型和致病能力。结果表明,从核盘菌基因组中比对到2个ATG,分别命名为SsATG5和SsATG8,两者在核盘菌致病过程中均上调表达。SsATG5和SsATG8敲除突变体在菌丝生长、产草酸和侵染垫形成方面与野生型菌株无明显差异,但SsATG5敲除突变体在离体拟南芥Arabidopsis thaliana叶片上的致病力显著下降了约40%,在活体拟南芥植株上的致病力显著下降了约80%,同时SsATG5回补突变体恢复了正常的致病力。表明SsATG5参与了核盘菌的致病过程,证实自噬在核盘菌致病过程中发挥着重要作用。     

Artificial infection of Vaccinium vitis-idaea L. and defence responses to Exobasidium species

An inoculation method for Exobasidium splendidum and Exobasidium vaccinii was developed on the dwarf shrub Vaccinium vitis-idaea. Using inoculated ramets, we investigated whether there are differences between V. vitis-idaea populations in the susceptibility to Exobasidium infections and whether the defence reaction of V. vitis-idaea is visible at a molecular level. Sixteen V. vitis-idaea clones from four populations were propagated in tissue cultures and the ramets were inoculated with E. splendidum or E. vaccinii fungi. The expression of three flavonoid biosynthetic genes (chalcone synthase, dihydroflavonol 4-reductase and anthocyanidin synthase) and the accumulation of flavonoids and hydroxycinnamic acids were determined in response to E. splendidum infection. A pathogenesis-related (PR 4) gene was isolated and its expression was studied in host ramet leaves. To our knowledge, this was the first successful artificial infection reported with E. splendidum. Disease frequencies of the inoculated ramets were between 32% and 47% for E. splendidum and 33% for E. vaccinii, but below 10% in uninoculated control ramets. There were no differences in disease frequencies between V. vitis-idaea populations. Both symptomatic leaves and healthy leaves of diseased ramets showed activation of flavonoid biosynthesis at the gene level, whereas expression of PR 4 was observed only in symptomatic leaves. The increase of flavonoid biosynthesis in healthy leaves of diseased ramets may represent a general response to stress or a role in defence against the pathogen E. splendidium. Ability of V. vitis-idaea to defend chemically against Exobasidium fungi and the heterogeneity of genotypes, age, size, and growth rates in host plant populations might be reasons for the low infection incidence of Exobasidia in nature.     

玉米抵御玉蜀黍尾孢菌侵入的生理机制

为深入探讨玉米抗灰斑病的机制,以抗/感玉米灰斑病自交系78599-1、OH43Ht N和掖478、K12为材料,采用比色皿法和RT-PCR法相结合研究了接种玉蜀黍尾孢菌毒素后玉米叶片中防御酶活性、丙二醛(MDA)含量及防御酶合成相关基因SOD、CAT、APX和GR的表达量。结果显示,接种该病菌毒素后,抗、感材料叶片中防御酶活性均升高,多数在接种7 d时达到峰值,且抗病材料防御酶活性峰值高于感病材料;供试材料叶片中MDA含量均降低,且抗病材料低于感病材料;供试材料叶片中SOD、CAT和GR的表达量均上升,其中CAT的表达量在78599-1、掖478和K12接种5d时达到峰值,灰度值分别为228.67、161.33和178.00,与其酶活性变化趋势一致;SOD和GR的变化规律与其酶活性变化不一致;APX的表达量仅在OH43Ht N中上升,接种7 d后达到峰值。表明抗病材料调控防御酶活性的能力强,防御酶基因的表达与其酶活性变化存在关联性。     

甜菜夜蛾中肠碱性磷酸酶alp2基因的克隆、表达及功能分析

为探究甜菜夜蛾Spodoptera exigua中肠碱性磷酸酶(alkaline phosphatase protein 2,ALP2)是否为Cry1Ac杀虫蛋白的受体,采用同源克隆和RACE技术克隆了编码alp2基因的完整c DNA序列,利用荧光定量PCR比较了甜菜夜蛾幼虫中肠不同龄期ALP2表达量的差异,利用Ligand blot方法检测了中肠ALP2与Cry1Ac杀虫蛋白的结合。结果表明,alp2基因序列全长1 629 bp(Gen Bank序列号为KP420013),编码542个氨基酸,预测在氨基酸序列N端包含1个由21个氨基酸组成的信号肽,在C端存在1个GPI修饰的锚定位点,且在整个氨基酸序列中存在多个糖基化修饰位点。在整个甜菜夜蛾幼虫期均有ALP2表达,但不同龄期的表达量差异显著,1龄幼虫期表达量最低,4龄幼虫期最高。Ligand blot方法检测结果表明原核表达的ALP2片段与活化的Cry1Ac杀虫蛋白可以结合。研究表明,甜菜夜蛾中肠的ALP2可能是Cry1Ac的受体之一。     

Inhibitors of N-linked glycosylation induce systemic acquired resistance in cucumber

Localized treatment of cucumber (Cucumis sativus L. cv. Wisonsin) cotyledons with inhibitors of N-glycosylation such as tunicamycin or amphomycin resulted in systemic acquired resistance in the first leaf to the fungal pathogen Colletotrichum lagenarium. Resistance was maximal as early as 2 days after application and best results were observed when the inhibitor was used at 100 μ . The same treatment also induced salicylic acid accumulation as well as the expression of chitinase and a PR1-like protein. The systemic effect is not caused by the transport of tunicamycin, since tunicamycin was not detected in the leaves. Within 2 h after application tunicamycin inhibited N-glycosylation, but not protein synthesis as indicated by labelling experiments. The amount of large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase decreased after tunicamycin treatment and after pathogen inoculation and the expression of BiP, a protein localized in the endoplasmic reticulum was enhanced. The activation of defense reactions seems to be dependent and sensitive to N-linked glycosylation.     

禾谷缢管蚜水通道蛋白基因RpAQP1的克隆、分子特性和表达分析

为探究禾谷缢管蚜Rhopalosiphum padi (Linnaeus)水通道蛋白基因RpAQP1的序列特征及其在不同龄期的表达,利用RT-PCR和RACE技术克隆了RpAQP1基因的cDNA全序列,采用生物信息学软件分析了RpAQP1编码蛋白的特性,利用qRT-PCR分析了RpAQP1在不同龄期的表达量。结果显示:禾谷缢管蚜水通道蛋白基因RpAQP1的cDNA全长为1 216 bp,其750 bp的开放阅读框编码250个氨基酸,蛋白分子量为27.36 kD。RpAQP1属于水通道蛋白亚家族DRIP(果蝇内嵌蛋白Drosophila integral protein)的一员,具有6个跨膜区,2个保守的NPA结构单元和1个压缩区域Ar/R;qRT-PCR结果显示,RpAQP1在整个发育历期均有表达,其在2龄若蚜中表达水平最高,是成蚜表达量的1.432倍;在4龄若蚜中表达量最低,为成蚜表达量的0.444倍,显著低于其它龄期,而其余各龄期RpAQP1表达量无显著差异。     

基于转录组测序解析干旱胁迫对祁连山黄参基因表达的调控

以栽培2个月的黄参为试材,设置对照（土壤相对含水量70%~80%）和适度干旱胁迫（土壤相对含水量55%~60%）处理,利用高通量转录组测序BGISEQ-500平台,对测序结果进行基因功能注释、差异表达基因(DEGs, differentially expressed genes)筛选。结果表明：(1)获得的68193条Unigene中,分别有34230(50.20%)、34170(50.11%）、31727(46.53%)、27701(40.62%)、27092(39.73%)和22793(33.42%)个Unigene分别被分配到NCBI非冗余蛋白(NR)、eggNOG(基因的进化谱系, Evolutionary genealogy of genes: Non\|supervised Orthologous Groups)、基因本体(Gene ontology,GO)、Pfam (Protein family)、SwissProt (Reviewed protein sequence database)和KEGG (Kyoto encyelopedia of genes and genomes)六大功能数据库。(2)DEGs分析显示,黄参块状根和叶中分别有10674个和13402个DEGs;GO富集结果表明,根和叶中的DEGs功能部位中的分布基本一致,主要富集在生物过程、DNA的复制和翻译调控、氧化还原过程、蛋白质磷酸化、防御响应等;KEGG富集分析表明,根中DEGs显著富集在苯丙烷类生物合成、半乳糖代谢、半胱氨酸和甲硫氨酸代谢、淀粉和蔗糖代谢、植物-病原菌相互作用、植物激素信号转导等途径,叶中DEGs则主要富集在半乳糖代谢、淀粉和蔗糖代谢、苯丙烷类生物合成、戊糖、葡萄糖醛酸转换、植物激素信号转导等途径,说明淀粉和蔗糖代谢、半乳糖代谢、苯丙烷类生物合成途径、植物激素信号转导途径在黄参应对干旱胁迫中起重要作用。干旱胁迫影响黄参不同器官中差异基因的表达,为解析黄参耐受干旱的生物学途径、黄参药效成分的生物合成和分子机制提供了理论依据。