玉米大斑病菌黑色素合成酶基因的全基因组定位及表达模式分析

【目的】确定玉米大斑病菌（Setosphaeria turcica）黑色素合成酶基因StPKS、St3HNR、St4HNR、StSCD、StLAC1、StLAC2在基因组中的位置和基因结构,分析黑色素合成途径中6个合成酶基因在玉米大斑病菌侵染过程中从分生孢子萌发至穿透不同时期及菌丝生长时期的表达模式,明确6个基因与病菌发育和致病的关系。【方法】利用玉米大斑病菌基因组数据库,通过Blastp相似性搜索,鉴定6个基因在基因组中的定位,解析在基因组中的串联分布情况;收集玉米大斑病菌从分生孢子萌发到侵染的不同时期及菌丝生长时期的菌体材料,提取总RNA,以β-tubulin作为内参基因,根据黑色素合成6个关键酶基因序列设计引物,采用qRT-PCR技术检测基因的表达模式,对比病菌在营养生长和生殖生长时期的表达情况。【结果】在基因组数据库中,玉米大斑病菌黑色素合成酶基因StPKS、St3HNR分别位于scaffold_12的正链和负链上,St4HNR、StLAC2位于scaffold_11的负链上,StSCD位于scaffold_1正链上,StLAC1位于scaffold_7正链上,且StPKS和St3HNR在基因组中串联分布在26.9 kb范围内;6个基因的相对表达量在分生孢子诱导萌发到穿透过程的5个时期中呈上调-下调-上调或上调-下调-上调-下调两种模式;分生孢子时期,StLAC2表达量最高,明显高于其他基因,差异极显著;菌丝生长时期,St3HNR、StSCD表达量最高,明显高于其他基因,差异极显著。【结论】玉米大斑病菌6个黑色素合成酶基因中StPKS和St3HNR在基因组中位置邻近,串联在26.9 kb范围内;6个基因从分生孢子萌发至侵染的5个时期均有表达,表达量存在显著差异,但各基因的表达模式相似,说明6个基因参与了玉米大斑病菌的侵染过程,在玉米大斑病菌的致病方面具有重要作用;同时在菌丝生长时期St3HNR和StSCD发挥的作用更明显,分生孢子时期StLAC2更活跃。     

MAPKs and acetyl-CoA are associated with Curvularia lunata pathogenicity and toxin production in maize

Mitogen-activated protein kinase (MAPK) cascades play an important role in extracellular signal transduction and are involved in the pathogenicity of fungal pathogens to host plants. In Curvularia lunata, the roles of two MAPK genes, Clk1 and Clm1, have already been studied. Clk1 is involved in conidia formation and pathogenicity, and Clm1 is closely related to pathogen cell wall formation and pathogenicity to maize leaves. In this study, a third C. lunata MAPK gene, Clh1, which is homologous to hog1, was successfully cloned. We found that a Clh1 deletion mutant had lower intracellular glycerol accumulation than the wild-type stain and was unable to grow normally under osmotic stress conditions. Furthermore, the deletion mutants of three C. lunata MAPK genes (Clk1, Clm1 and Clh1) had lower levels of acetyl-CoA, which is an important intermediate product in the synthesis of melanin and furan toxin, and down-regulated expression of pathogenicity-associated genes. Furthermore, pathogenicity and the ability to produce toxin were restored after adding acetyl-CoA to the culture medium, suggesting that acetyl-CoA is closely involved in the pathogen MAPK signaling pathway.     

棉花黄萎病菌VdHP1的克隆及功能分析

【目的】明确棉花黄萎病菌（大丽轮枝菌Verticillium dahliae）中一个新基因（VdHP1）的功能,为解析棉花黄萎病菌的致病机制以及棉花黄萎病的防治提供依据。【方法】以大丽轮枝菌野生型菌株V592的基因组DNA和cDNA为模板,对VdHP1全长进行克隆并测序;利用逆转录实时荧光定量PCR（RT-qPCR）分别对棉花根系诱导不同时间VdHP1的表达量及V592菌株不同组织中VdHP1的表达量进行测定;构建针对VdHP1的敲除载体、互补载体和过表达载体,通过农杆菌介导的遗传转化筛选VdHP1基因敲除突变体、互补菌株和过表达菌株;以野生型菌株V592为对照,对VdHP1基因敲除突变体及互补菌株的菌落及菌丝形态进行观察,并对微菌核量、产孢量及致病力进行测定;通过RT-qPCR测定其他致病力相关的基因在VdHP1基因敲除突变体及过表达体菌株中的表达情况。【结果】VdHP1全长为862bp,预测编码蛋白含268个氨基酸,与GenBank中已注释的基因没有任何的序列相似性。野生型菌株V592受棉花根系诱导6—12 h时VdHP1表达水平显著上调,表明VdHP1在大丽轮枝菌侵染早期发挥作用。VdHP1在分生孢子中的表达量显著高于在菌丝和微菌核中的表达量,表明VdHP1在大丽轮枝菌不同组织中的表达具有差异性。与野生型菌株V592相比,VdHP1基因敲除突变体产孢量和产孢梗显著减少,菌丝分支呈螺旋状,对棉花的致病力明显下降。与侵染钉形成相关基因（VdCrz1、VdNoxB、VdPls1）、分泌蛋白释放相关基因（VdSep5）及分生孢子产生相关基因（Vdpf、VdSge1、VGB、VdPLP、VdCYC8、VdNLP1、VdNLP2）在VdHP1基因敲除体中的相对表达量显著下调,在过表达菌株中上调;而与黑色素合成相关基因（VdCmr1、VdSho1、VdLAC、VdPKS1）在VdHP1基因敲除突变体中则显著上调,在过表达菌株中下调。【结论】VdHP1与大丽轮枝菌分生孢子和产孢梗的产生有关,参与大丽轮枝菌致病;VdHP1对与侵染钉形成、分泌蛋白释放及分生孢子产生相关基因的表达具有正调控作用,对黑色素合成相关基因的表达具有负调控作用。     

Melanin,DNA replication,and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism

Setosphaeria turcica(syn. Exserohilum turcicum) is the pathogenic fungus of maize(Zea mays) that causes northern leaf blight, which is a major maize disease worldwide. Melanized appressoria are highly specialized infection structures formed by germinated conidia of S. turcica that infect maize leaves. The appressorium penetrates the plant cuticle by generating turgor, and glycerol is known to be the main source of the turgor. Here, the infection position penetrated by the appressorium on maize l...     

Stk2, a Mitogen-Activated Protein Kinase from Setosphaeria turcica,Specifically Complements the Functions of the Fus3 and Kss1 of Saccharomyces cerevisiae in Filamentation,Invasive Growth,and Mating Behavior

Setosphaeria turcica, an essential phytopathogenic fungus, is the primary cause of serious yield losses in corn; however, its pathogenic mechanism is poorly understood. We cloned STK2, a newly discovered mitogen-activated protein kinase gene with a deduced amino acid sequence that is 96% identical to MAK2 from Phaeosphaeria nodorum, 56% identical to KSS1 and 57% identical to FUS3 from Saccharomyces cerevisiae. To deduce Stk2 function in S. turcica and to identify the genetic relationship between STK2 and KSS1/FUS3 from S. cerevisiae, a restructured vector containing the open reading frame of STK2 was transformed into a fus3/kss1 double deletion mutant of S. cerevisiae. The results show that the STK2 complementary strain clearly formed pseudohyphae and ascospores, and the strain grew on the surface of the medium after rinsing with sterile water and the characteristics of the complementary strain was the same as the wild-type strain. Moreover, STK2 complemented the function of KSS1 in filamentation and invasive growth, as well as the mating behavior of FUS3 in S. cerevisiae, however, its exact functions in S. turcica will be studied in the future research.     

MAP kinase gene STK1 is required for hyphal,conidial,and appressorial development,toxin biosynthesis,pathogenicity,and hypertonic stress response in the plant pathogenic fungus Setosphaeria turcica

The mitogen-activated protein kinase(MAPK), a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes. However, little is known of the role MAPK plays in phytopathogenic fungi. In this research, we cloned the MAPK gene STK1 from the northern corn leaf blight pathogen Setosphaeria turcica and found that the gene shared high homology with the high osmolality glycerol(HOG) MAPK gene HOG1 of Saccharomyces cerevisiae. In addition, gene knockout technology was employed to investigate the function of STK1. Gene knockout mutants(KOs) were found to have altered hyphae morphology and no conidiogenesis, though they did show similar radial growth rate compared to the wild-type strain(WT). Furthermore, microscope observations indicated that STK1 KOs did not form normal appressoria at 48 h post-inoculation on a hydrophobic surface. STK1 KOs had reduced virulence, a significantly altered Helminthosporium turcicum(HT)-toxin composition, and diminished pathogenicity on the leaves of susceptible inbred corn OH43. Mycelium morphology appeared to be significantly swollen and the radial growth rates of STK1 KOs declined in comparison with WT under high osmotic stress. These results suggested that STK1 affects the hyphae development, conidiogenesis, and pathogenicity of S. turcica by regulating appressorium development and HT-toxin biosynthesis. Moreover, the gene appears to be involved in the hypertonic stress response in S. turcica.     

StKU80, a component in the NHEJ repair pathway,is involved in mycelial morphogenesis,conidiation, appressorium development,and oxidative stress reactions in Exserohilum turcicum

Homologous recombination(HR) and nonhomologous end joining(NHEJ) are considered the two main double-strand break(DSB) repair approaches in eukaryotes. Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes. However, little is known about the roles of the NHEJ pathway in foliar pathogens. Here we identified a gene designated St KU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum. Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in St Ku80. Phylogenetic analysis indicated that St Ku80 is most closely related to Ku80(XP_001802136.1) from Parastagonospora nodorum, followed by Ku80(AGF90044.1) from Monascus ruber. Furthermore, the gene knockout mutants ΔSt KU80-1 and ΔSt KU80-2 were obtained. These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type(WT) strain but similar HT-toxin activity. The mutants did not produce conidia and mature appressoria. On the other hand, the mutants were highly sensitive to H_2O_2, but not to ultraviolet radiation. In summary, the St KU80 plays devious roles in regulating the development of E. turcicum.     

土著菌根真菌和混生植物对羊草生长和磷营养的影响

植物种间相互作用直接影响植物生长、根系可塑性及养分吸收,而与植物共生的丛枝菌根真菌可以改变植物个体和种间养分资源的分配,具有协调种间竞争的潜力.以我国北方草甸草原建群种羊草(Leymus chinensis)和混生植物紫花苜蓿(Medicago sativa)及独行菜(Lepidium spetalum)为供试植物,通过模拟盆栽试验,研究了土著菌根真菌和混生植物对羊草生长、根系形态及磷营养的影响.试验结果表明,土著菌根真菌能够与羊草及紫花苜蓿形成良好共生,而独行菜根内基本未形成菌根共生结构.土著菌根真菌显著降低了羊草及独行菜的生物量,但促进了紫花苜蓿的生长;混种紫花苜蓿显著促进了羊草的生长,而混种独行菜则显著抑制了羊草的生长.土著菌根真菌对羊草根系形态的影响表现出与植株生物量类似的趋势,但不同混生植物对羊草根系生长均无显著影响.土著菌根真菌和混生植物对羊草植株磷含量均无显著影响.与混生植物相比,羊草具有较高的比根长和磷吸收能力,这也解释了其负向菌根依赖性.研究证实了菌根真菌和植物种间相互作用均是影响草原优势植物生长和根系发育的重要因素,深入研究其交互作用对于科学管理草地生态系统,维持植物群落的稳定性和生态系统生产力具有重要意义.     

Proteomics Identification of Differentially Expressed Leaf Proteins in Response to Setosphaeria turcica Infection in Resistant Maize

Northern corn leaf blight （NCLB）, caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A comparative proteomic study was conducted to explore the molecular mechanisms underlying the defense responses of the maize resistant line A619 Ht2 to S. turcica race 13. Leaf proteins were extracted from mock and S. turcica-infected leaves after inoculated for 72 h and analyzed for differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry identification. 137 proteins showed reproducible differences in abundance by more than 2-fold at least, including 50 up-regulated proteins and 87 down-regulated proteins. 48 protein spots were successfully identified by MS analysis, which included 10 unique, 6 up-regulated, 20 down-regulated and 12 disappeared protein spots. These identified proteins were classified into 9 functional groups and involved in multiple functions, particularly in energy metabolism （46%）, protein destination and storage （12%）, and disease defense （18%）. Some defense-related proteins were upregulated such as 13-glucosidase, SOD, polyamines oxidase, HSC 70 and PPIases; while the expressions of photosynthesis- and metabolism-related proteins were down-regulated, by inoculation with S. turcica. The results indicated that a complex regulatory network was functioned in interaction between the resistant line A619 Ht2 and S. turcica. The resistance processes of A619 Ht2 mainly resided on directly releasing defense proteins, modulation of primary metabolism, affecting photosyntesis and carbohydrate metabolism.     

MoFbc1调控稻瘟病菌生长与致病机制初探

真核生物体内,F-box蛋白作为泛素连接酶复合物(Skp1-Cullin1-F-box,SCF)的调节亚基,参与降解底物的特异性识别。为探究F-box家族基因MoFbc1在稻瘟病菌生长发育及侵染致病中的功能,采用生物信息学方法分析Mo Fbc1蛋白结构域并构建进化树。利用同源重组方法获得MoFbc1基因敲除及其回补菌株并进行表型分析。结果表明,MoFbc1敲除菌株在产孢、附着胞形成及致病性等方面与野生型菌株均无显著差异。但其在MM和RDC培养基上生长速率下降,表明MoFbc1基因可能参与调控稻瘟病菌对部分营养物质的利用;突变体对细胞壁胁迫因子CFW、CR敏感,提示其细胞壁结构可能发生改变。结果为进一步揭示基因MoFbc1调控稻瘟病菌生长发育机制奠定基础。     

Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5

Cadmium (Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food. Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains. OsNRAMP5 is the major transporter for Cd and manganese (Mn) uptake in rice. Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield. In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties. We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth. Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn. Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants. In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield. The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation. Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.     

苹果树腐烂病菌根皮苷水解酶基因Vmlph1的功能

为明确根皮苷降解酶在苹果树腐烂病菌侵染过程中的作用,从苹果树腐烂病菌基因组序列中鉴定出候选根皮苷降解酶基因Vmlph1,利用Double-joint PCR和PEG介导的原生质体转化技术构建Vmlph1敲除突变体,观察突变体营养生长状况、产孢情况以及致病力的变化;利用HPLC方法检测基因敲除对根皮苷降解速率的影响。结果发现,经PCR及Southern blot验证后获得Vmlph1基因的敲除突变体;与野生型菌株相比,突变体菌落颜色变白,生长速率和产孢能力显著降低;接种到叶片和枝条上,突变体致病力显著降低;突变体根皮苷降解能力与野生型没有显著差异;在根皮苷诱导下,野生型菌株黑色素合成调控转录因子Cmr1基因的表达量显著上调,但突变体Cmr1基因的上调倍数显著低于野生型菌株。表明,根皮苷降解酶基因Vmlph1与苹果树腐烂病菌营养生长、致病力、分生孢子的产生及黑色素合成有关。     

玉米大斑病菌小柱孢酮脱水酶StSCD家族鉴定及其功能分析

【目的】小柱孢酮脱水酶（scytalone dehydratase,SCD）是黑色素合成过程中的关键酶。本文旨在鉴定玉米大斑病菌（Setosphaeria turcica）小柱孢酮脱水酶基因（StSCD）家族,并分析玉米大斑病菌附着胞发育过程中StSCD基因家族表达量差异及SCD抑制剂对黑色素合成的影响,为进一步研究StSCD基因家族在黑色素合成和附着胞发育中的作用打下基础。【方法】利用玉米大斑病菌野生型菌株01-23的全基因组数据,获得StSCD基因家族的全序列,并与玉米小斑病菌（Cochlibolus heterostrophus）、稻瘟病菌（Pyricularia oryzae）、瓜类炭疽病菌（Colletotrichum lagenaria）等真菌的SCD进行序列比对;收集玉米大斑病菌附着胞不同发育时期的材料进行实时荧光定量PCR（qRT-PCR）分析,获得不同时期、不同StSCD的表达量,从而确定与病菌侵染和附着胞黑色素化密切相关的脱水酶基因;使用SCD抑制剂环丙酰菌胺处理玉米大斑病菌,测定菌落生长速度、黑色素合成量、附着胞膨压等,确定StSCD在附着胞发育过程中的作用。【结果】玉米大斑病菌全基因组中共鉴定到4个StSCD,其编码蛋白具有SCD保守结构域及保守的催化及底物结合氨基酸残基。StSCD3与灰葡萄孢（Botrytis cinerea）中功能冗余的SCD2蛋白具有较高同源性,StSCD4与多主棒孢（Corynespora cassiicola）中参与黑色素合成的SCD蛋白具有较高同源性。通过分析玉米大斑病菌生长发育过程中不同时期StSCD的表达量发现,在附着胞时期4个StSCD表达量均上调,其中StSCD3、StSCD4表达量上调尤为显著,附着胞再生菌丝时期StSCD3、StSCD4的表达量均显著下降,并且在附着胞诱导整个时期StSCD4的表达量较高。环丙酰菌胺处理玉米大斑病菌后,黑色素合成受阻,附着胞膨压显著降低。【结论】玉米大斑病菌含有4个StSCD,推测StSCD4参与DHN（1,8-间苯二酚）黑色素的合成,并进而影响附着胞膨压积累。     

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)

Laccase(EC 1.10.3.2)is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2(PxLac2),from the third instar larvae using polymerase chain reaction(PCR)and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR(qPCR).The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant(K_m)of 0.97 mmol L~(-1),maximal reaction speed(V_m)of 56.82 U mL~(-1),and activation energy(E_a)of 17.36 kJ mol~(-1) to oxidize2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt).Type II copper enhanced laccase activity below 0.8mmol L~(-1) and reduced enzyme activity above 0.8 mmol L~(-1) with an IC_(50) concentration of 1.26 mmol L~(-1).This study provides insights into the biological function of laccase.     

基因编辑酪氨酸酶(TYR)基因不同功能区对鱼类体色的影响

酪氨酸酶(Tyr)基因在动体的黑色素合成过程中起关键作用,其突变会导致人类和其他物种出现白化现象。本研究以AB系斑马鱼(Danio rerio)为研究对象,应用CRISPR/Cas9基因编辑技术,对纯合亲鱼受精卵进行显微注射,通过编辑损坏Tyr基因的CDS区第二外显子和3′-UTR非poly-A加尾信号区,使基因发生突变,检测基因不同功能区经编辑后对鱼类体色的影响。结果显示：Tyr基因在野生型斑马鱼的胚胎各个发育时期均能正常表达,眼睛出现黑色素时的表达量最高;编辑损坏Tyr基因的CDS区后,突变型斑马鱼的胚胎和仔鱼均未出现黑色素细胞,表现为体表白化,而成鱼会再度出现黑色素细胞,表现为体表黑色条纹断裂。编辑损坏Tyr基因的3′-UTR非poly-A加尾信号区后,突变型胚胎、仔鱼和成鱼均未出现黑色素减褪,黑色素合成未受影响。本研究表明,编辑损坏基因的CDS区会对生物表型产生显著影响,而编辑损坏3′-UTR非poly-A加尾信号区对生物表型的影响有限。     

稻瘟病菌Mofbox基因缺失突变体构建及生物学表型分析

采用啤酒酵母菌(Saccharomyces cerevisiae)F-box蛋白的氨基酸序列对稻瘟病菌（Magnaporthe oryzae）全基因组数据库进行Blastp分析,找到一个与之同源的、具有典型F-box结构域的蛋白编码基因,命名为Mofbox。采用同源重组原理,将构建的基因敲除载体转化稻瘟病菌,获得该基因缺失突变体2个。研究表明,在稻瘟病菌中,Mofbox缺失后可导致稻瘟病菌生长速率显著下降,分生孢子产孢量和附着胞形成率显著降低,对外源过氧化氢敏感,改变细胞壁完整性,同时降低对大麦和水稻敏感品种CO-39的致病性。     

深圳近海海域产纤维素酶、脂肪酶和漆酶 真菌的筛选及酶学性质分析

现有自深圳海域分离出的真菌31株,其中包括22株丝状真菌和9株酵母菌.对各菌株进行纤维素酶、脂肪酶和漆酶等胞外酶的定性筛选后,进行胞外酶定量分析.结果表明,在定性初筛的培养基生长5d后,大多数菌株呈现出2～3种胞外酶活力的溶解圈,而9个酵母菌菌株都没有检出漆酶活力.在定性测量的结果基础上,挑选出产酶潜力大的2株丝状真菌(PKU F16、PKU F18)和2株酵母菌(PKU Y5、PKU Y8),进行3种胞外酶的定量分析.在各菌株培养4、6、9d时,分别对3种胞外酶活进行测定,结果发现各菌株生产3种胞外酶的定量分析结果与定性分析结果基本吻合,且随着培养时间的增长,菌株的胞外酶活力呈现先增后减的趋势.在第6天,F18和Y8产纤维素酶活力分别高达1.13、1.31 U/min·mL.Y8的脂肪酶活力产量高达21.94 U/min·mL.F18和F16产漆酶活力高达14.61、10.50 U/min· mL.