FgLEU1在禾谷镰刀菌亮氨酸生物合成及产毒致病中起关键作用

为挖掘新型药剂的潜在靶标,利用靶向基因敲除和互补技术研究赤霉病病原菌禾谷镰刀菌Fusarium graminearum中必需氨基酸亮氨酸合成酶编码基因FgLEU1的功能,并测定禾谷镰刀菌的生物学表型。结果表明,FgLEU1编码亮氨酸合成途径中的3-异丙基苹果酸脱水酶,其敲除突变体表现亮氨酸营养缺陷。生物学表型测定结果显示,与野生型菌株相比,FgLEU1敲除突变体的产孢量和孢子萌发率显著下降,产孢量仅为野生型菌株的20.96%,培养4 h后孢子萌发率下降了49.45%,且合成脱氧雪腐镰刀烯醇（呕吐毒素）能力丧失,在麦穗上的致病力下降,仅能侵染接种小穗,赤霉病症状不能扩展。外源添加一定量的亮氨酸、FgLeu1催化产物或导入含启动子的全长FgLEU1基因可以恢复敲除突变体表型缺陷。表明FgLEU1基因在禾谷镰刀菌亮氨酸合成、菌丝孢子形成及产毒致病过程中发挥着重要作用,可作为新型安全杀菌剂的潜在研发靶标,用于持续有效控制麦类赤霉病和镰刀菌毒素。     

稻瘟病菌MoCOS1基因调控MoCMR1基因表达的研究

 以往研究证明MoCOS1可能是一个新型的转录因子,其突变体不产生分生孢子,黑色素合成明显减少。经过RNA-Seq分析,发现受MoCOS1调控的基因达到442个,其中MoCMR1的表达水平受到MoCOS1的下调。本研究中通过实时定量PCR进一步证实MoCOS1基因突变导致MoCMR1基因表达水平下降。基因MoCMR1突变后,黑色素产量略有减少,但分生孢子形态及产量和MoCOS1的表达量没有发生变化。     

Mating type gene MAT1-2-1 is common among Japanese isolates of Verticillium dahliae

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence's presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium.     

Functional analysis of the 3′ end of avrBs3/pthA genes from two Xanthomonas species

The phytopathogens Xanthomonas oryzae pathovar (pv.) oryzae and Xanthomonas axonopodis pv. citri each contain several avrBs3/pthA family genes. Structural features of these genes important for avirulence and/or virulence functions include a central region of multiple direct repeats and three nuclear localization signals (NLSs) and an acidic activation domain (AAD) at the 3′ end. To identify other regions critical to function in the 3′ ends of these genes, we constructed several chimeras using apl1 and apl2 from X. axonopodis pv. citri and avrXa10 and avrXa7 from X. oryzae pv. oryzae and evaluated their functions by inoculation to citrus and rice. The apl1 and avrXa7 genes are major virulence determinants in citrus and rice, respectively, while the contributions of apl2 and avrXa10 to virulence are negligible or not measurable. Constructs that contained a 417 bp HincII-SphI fragment from the 3′ end of apl1 in combination with the repeats from avrXa7, avrXa10, and apl1 caused a canker phenotype on citrus. Interchange of the HincII-SphI fragment between avrXa7 and avrXa10 abolishes avrXa7 avirulence function and reduces its virulence but it does not affect avrXa10 avirulence function in rice. avrXa7 caused a hypersensitive response (HR) in citrus and replacement of it's 3′ end with that of apl1 resulted in loss of canker and induction of HR. Thus, the HincII-SphI fragment of the avrBs3/pthA gene family is important for avirulence and virulence functions in two different plant species, Oryza sativa and Citrus natsudaidai HAYATA.     

苹果树腐烂病菌VmMFS1~VmMFS3基因的功能分析

为绿色持久防控苹果树腐烂病,该研究分析苹果树腐烂病菌Valsa mali的3个主要协同转运蛋白超家族（major facilitator superfamily,MFS）编码基因的氨基酸序列特征,利用实时荧光定量PCR （quantitative real-time PCR,RT-qPCR）技术分析这3个基因在苹果树腐烂病菌侵染阶段的表达水平,通过构建这3个基因的缺失突变体和回补菌株分析其在病原菌营养生长、致病力和非生物胁迫应答等方面的功能。结果表明,这3个基因的氨基酸序列均具有MFS保守结构域,将其命名为VmMFS1~VmMFS3; VmMFS1和VmMFS2的进化距离较近,均与VmMFS3的进化距离较远;在苹果树腐烂病菌侵染过程中VmMFS1~VmMFS3基因表达均显著上调;与野生型03-8菌株相比,VmMFS1~VmMFS3基因缺失突变体的菌落形态无明显差异,但生长速度下降; VmMFS1~VmMFS3基因缺失突变体的致病力均显著降低; VmMFS1~VmMFS3基因缺失突变体对H₂O₂胁迫的敏感性无明显变化,但对NaCl胁迫更敏感;基因回补后基因缺失突变体的表型缺陷能恢复到野生型菌株的水平。     

舞毒蛾DnaJ1基因克隆分析及对甲萘威胁迫响应

Dna J蛋白是Dna K/Hsp70的辅助分子伴侣,通过调节Hsp70的ATPase活性来影响蛋白复合体的合成与组装。为明确舞毒蛾Lymantria dispar的LdDnaJ1基因特性及对杀虫剂甲萘威的胁迫响应,通过克隆LdDnaJ1全长基因并运用实时荧光定量RT-PCR技术测定了甲萘威对其LdDnaJ1基因表达量的影响。结果表明,舞毒蛾Ld Dna J1全长基因开放阅读框为1 062 bp,编码353个氨基酸,分子质量为39.91 kD,理论等电点为5.65;舞毒蛾Dna J与柑橘凤蝶Papilio xuthus Dna J亲缘关系较近。甲萘威对舞毒蛾2龄幼虫24 h和48 h的致死中浓度LC50分别为74.04 mg/L和31.48mg/L。低剂量(LC_5、LC_(10)和LC_(30))甲萘威胁迫下,舞毒蛾2龄幼虫Ld Dna J1基因表达量均下调,LC_(30)甲萘威处理后72 h时LdDnaJ1基因表达量最低,仅为对照的15.70%。表明甲萘威可抑制舞毒蛾LdDnaJ1基因的表达,且呈现明显的时间和剂量效应。     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis

Fusarium graminearum, Fusarium culmorum and Fusarium cerealis are major causal agents of Fusarium Head Blight (scab) which is a disease of global significance in all cereal growing areas. These fungi produce trichothecene mycotoxins, principally nivalenol (NIV) and deoxynivalenol (DON). Genes Tri13 and Tri7 from the trichothecene biosynthetic gene cluster convert DON to NIV (Tri13) and NIV to 4-acetyl-NIV (Tri7). We have developed positive–negative PCR assays based on these two genes, which accurately indicate a DON or NIV chemotype in F. graminearum, F. culmorum and F. cerealis. These assays are useful in assessing the risk of trichothecene contamination, and can be informative in epidemiological studies. All NIV chemotype isolates studied have functional copies of both Tri13 and Tri7, and all DON-producing isolates have both genes disrupted or deleted. We have identified several mutations in these genes, which are conserved across F. graminearum lineage, RAPD and SCAR groupings and between the three species. There appears to be evidence of inter-species hybridisation within the trichothecene biosynthetic gene cluster.     

二点螟几丁质酶1基因CiChi1的克隆和功能分析

为明确几丁质酶1(chitinase 1,Chi1)编码基因在二点螟Chilo infuscatellus中的潜在功能，对Chi1基因进行全长序列克隆和生物信息学分析，使用实时荧光定量PCR技术检测该基因的时空表达模式，利用RNA干扰技术探究其在二点螟生长发育方面的功能。结果表明，二化螟Chi1基因序列全长为1 788 bp，开放阅读框为1 653 bp，编码550个氨基酸，并命名为CiChi1;CiChi1基因在二点螟不同发育阶段及不同组织中均有表达，其中在成虫期的相对表达量最高，在6龄幼虫期的相对表达量次之，且在6龄幼虫表皮中的相对表达量最高；在RNA干扰试验中，注射ds CiChi1的二点螟3龄幼虫试验组在第14天的最终死亡率为62.0%，显著高于注射ds EGFP的阴性对照组（27.8%）和注射蒸馏水的空白对照组（33.9%）；此时试验组的平均虫重为0.024 g，显著低于阴性对照组（0.039 g）和空白对照组（0.040 g），且试验组试虫还伴随有外表皮急剧变黑、消解软化的致死表型；实时荧光定量PCR检测结果表明，试验组试虫体内CiChi1基因表达量较阴性对照组和空白对照组...     

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。     

番茄枯萎病菌和颈腐根腐病菌KASP-SNP检测技术的建立

为快速、准确地对番茄枯萎病菌Fusarium oxysporum f. sp. lycopersici(FOL)和番茄颈腐根腐病菌F. oxysporum f. sp. radicis-lycopersici(FORL)进行检测,基于尖孢镰刀菌F. oxysporum多聚半乳糖醛酸外切酶基因pgx4的单核苷酸多态性（single nucleotide polymorphism,SNP）位点,设计FORL、FOL生理小种1(FOL-R1)、2(FOL-R2)和3(FOL-R3)的竞争性等位基因特异性PCR-SNP(kompetitive allele specific PCR-SNP,KASP-SNP)引物,建立番茄颈腐根腐病菌和番茄枯萎病菌KASP-SNP检测技术,并通过与常规PCR比对及ITS与pgx4序列分析对该检测技术的可靠性进行验证。结果显示,在FORL、FOL-R1、FOL-R2和FOL-R3中存在35个变异SNP位点,设计出18对KASP-SNP引物,筛选出FORL＿KASP、FOLrace1＿KASP、FOLrace2＿KASP和FOLrace3＿KASP共4对分型清晰的...     

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance (R) genes. In Arabidopsis, some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR-1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR1 and EDS1.     

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。     

棉铃虫YTHDF1基因的表达模式及在核型多角体病毒侵染中的作用

为明确N6-甲基腺苷（N6-methyladenosine,m⁶A）修饰在棉铃虫核型多角体病毒（Helicoverpa armigera nucleopolyhedrovirus,HearNPV）侵染棉铃虫中的作用,根据基因组和转录组数据,对棉铃虫m⁶A结合蛋白基因YTHDF1（YTH domain-containing family protein 1）进行鉴定和分析,利用实时荧光定量PCR技术检测棉铃虫YTHDF1基因的时空表达模式、HearNPV处理后的表达变化情况以及RNA干扰效率,并调查该基因下调后对HearNPV复制及感染HearNPV棉铃虫死亡情况的影响。结果显示,棉铃虫YTHDF1基因开放阅读框全长为2 019 bp,编码672个氨基酸,含有1个保守的YTH结构域,其氨基酸序列与斜纹夜蛾Spodoptera litura同源物序列一致性达87.52%。YTHDF1基因在棉铃虫不同发育阶段均有表达,其中在进入5龄期24 h幼虫中的表达量最高,在2龄取食期幼虫中的表达量最低。YTHDF1基因的空间表达谱呈现一定的组织特异性,在幼虫血细胞和成虫头部的表达量最高。HearNPV侵染棉铃虫后YTHDF1基因上调表达,经RNA干扰使该基因下调后显著抑制了HearNPV多角体蛋白基因polyhedrin的表达并延迟了感染HearNPV棉铃虫的死亡时间。表明YTHDF1基因在HearNPV侵染棉铃虫的过程中发挥着重要作用。     

珠江三角洲地区黄毛鼠抗药性的发生趋势及其Vkorc1基因多态性

为阐明珠江三角洲地区黄毛鼠Rattus losea对第1代抗凝血灭鼠剂的抗性发生趋势及其遗传机制,以杀鼠灵为标准药物,采用致死期食毒法对2017—2021年在广东省江门市捕获的165只黄毛鼠进行生理抗性检测,并测定每只试鼠的维生素K环氧化物还原酶复合物亚单位1(vitamin K epoxide reductase complex subunit 1,Vkorc1)的编码基因序列,分析其突变情况。结果显示,江门市黄毛鼠对第1代抗凝血灭鼠剂杀鼠灵的抗性率为27.03%～50.00%,在黄毛鼠Vkorc1基因中检测到6个不同的突变位点,包括2个错义突变位点Arg58Gly及Tyr139Cys和4个沉默突变位点Ala41Ala、Cys96Cys、Arg98Arg及Ala143Ala,突变率分别为87.27%、0.61%、1.21%、0.61%、1.21%和0.61%,其中Ala143Ala是在黄毛鼠中新发现的沉默突变位点。表明珠江三角洲地区黄毛鼠已对第1代抗凝血灭鼠剂产生了群体抗性并呈上升趋势,第58位的精氨酸突变成甘氨酸(Arg58Gly)是黄毛鼠抗性基因Vkorc1的主要突变位点。     

苹果树腐烂病菌染色质重塑因子Vmles4的功能研究

染色质重塑因子INO80是一类由多亚基构成的遗传学调控因子，调控多种DNA代谢，在基因的表达中发挥着重要的调控功能。但其在苹果树腐烂病菌（Valsa mali）中是否存在及其生物学功能并不清楚，为此，本研究从病菌基因组中分析鉴定到INO80的一个亚基基因Vmles4，利用qRT-PCR技术分析了Vmles4在病菌侵染过程中的表达模式，利用Double-joint PCR技术和PEG介导的原生质体转化方法进行了基因敲除，然后对3个突变体的营养生长及致病力进行测定和分析，并对病菌的非核糖体肽合成酶基因VmNRPS12及VmNRPS14在突变体中的表达进行定量分析。结果表明：Vmles4在侵染初期的表达显著上调，接种后6 h上调表达6.2倍。敲除突变体的生长速率平均降低28%且菌丝生长稀疏，在富士苹果品种（Malus domestic cv. Fuji）叶片和枝条上的致病力分别降低到22.5%和27.5%，VmNRPS12及VmNRPS14基因在Vmles4突变体侵染苹果枝条24 h后的表达量分别下调86.5%和50%；综上所述，Vmles4正调控腐烂病菌的营养生长、致病力以及次级代谢合成酶基因VmNRPS12和VmNRPS14的表达。     

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。     

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。     

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.