玉米大斑病菌StSNF1的基因组定位、蛋白质结构预测及表达分析

为了明确StSNF1在玉米大斑病菌基因组中的位置,解析该基因编码蛋白的结构特征,探究该基因在侵染寄主的不同时期、分生孢子萌发侵染过程中以及不同碳源培养下的表达情况。结果表明,该基因ID号为008026214,全长3 046 bp,位于scaffold_17负链的97 793-100 838位置。StSNF1与玉米圆斑病菌SNF1同源关系较近,由877个氨基酸残基编码而成。StSNF1蛋白具有氮端的蛋白激酶结构域、氮端的碳代谢产物去阻遏蛋白激酶结构域和碳端的激酶相关结构域。在蛋白激酶结构域内,具有ATP结合位点和丝氨酸/苏氨酸活性位点。Real-time PCR结果表明,StSNF1在侵染后期高表达,72 h表达量最高;StSNF1在分生孢子萌发24 h时(即侵入丝形成时期)表达量最高;StSNF1在蔗糖为单一碳源的培养基中表达量最高,果胶培养基次之。综上所述,StSNF1与侵染寄主和碳源利用密切相关,在侵染后期发挥作用,利用寄主细胞内的非发酵型碳源进行次级侵染。     

Spatial patterns of the Citrus leprosis virus and its associated mite vector in systems without intervention

Leprosis is caused by the Citrus leprosis virus cytoplasmic type and is vectored by the mite Brevipalpus yothersi. Miticide applications, which cost $54 million annually, are based on inspection for the presence of mites. The aim of the present study was to characterize the spatial patterns of B. yothersi-infested trees and trees with leprosis symptoms for further improvement in sampling and disease control. The presence of mites and the occurrence of leprosis were assessed over two years in 1160 Valencia trees and 720 Natal trees in a commercial sweet orange grove in Sao Paulo State, Brazil. To assess the natural growth and dispersal of mites and leprosis, mite populations were not controlled during the experimental period. Maps of mite-infested trees and trees with leprosis symptoms were analysed at three different levels of spatial hierarchy using complementary methods, i.e. among adjacent trees within and across rows, within quadrats, and the strength and orientation of aggregation among quadrats. The study showed that the spatial patterns of virus-infected and mite-infested trees were different, with a strong aggregation pattern of trees with leprosis symptoms that increased over time. Conversely, the spatial pattern of B. yothersi showed randomness or weak aggregation at all three spatial hierarchical levels. Disease incidence increased steadily in plots of both cultivars, unlike in mite-infested trees where incidence fluctuated over time. These results have important implications for the development of better management strategies for leprosis. Sampling methods and action thresholds for mite control should consider primary disease inoculum in addition to the incidence of mites.     

Molecular identification of blast resistance genes in rice landraces from northeastern India

Rice blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating diseases causing huge losses worldwide. In the present study, major blast resistance genes were investigated in landraces originating from northeastern India. Based on phenotypic evaluation, 288 landraces were classified into three distinct groups: resistant (75), moderately resistant (127) and susceptible (86). The genetic frequencies of the 18 major blast resistance genes were between 6.2% and 27.4%, with only two genotypes possessing a maximum of nine blast resistance genes. The cluster and population structure analysis grouped the landraces into two groups. Through principal coordinate analysis, the scatter plots partitioned the resistant and moderately resistant landraces into different groups. Analysis of molecular variance showed maximum (96%) diversity within populations and least (4%) diversity between populations. Association analysis identified six markers, CRG4_2, RM72, tk59-2, pi21_79-3, RM1233 and RM6648, that are significantly associated with blast disease and explained a phenotypic variance of 1.1–6.5%. The associated genes could be used in marker-assisted rice breeding programmes for gene pyramiding to develop rice varietal resistance against blast disease. The present study represents a valuable blast resistance genetic resource that could be used for identification of new R genes, donors for blast resistance, and genomic studies.     

假眼小绿叶蝉与其天敌的空间关系及最佳样方确定

为了分析在聚块大小不同时天敌对假眼小绿叶蝉Empoasca vitis Gothe空间上跟随关系的密切程度、聚集原因和聚集范围,为评价假眼小绿叶蝉的天敌优势种提供科学依据,用聚块样方方差分析法、灰色关联度法、空间聚集强度指数、种群聚集均数法和ρ指数法对安徽省合肥市‘乌牛早’和‘白毫早’茶园不同大小聚块条件下的假眼小绿叶蝉及其7种蜘蛛类的天敌空间关系进行分析。假眼小绿叶蝉与其7种蜘蛛类天敌均方差峰值时的聚块样方数的关联度分析结果表明:‘乌牛早’茶园中与假眼小绿叶蝉空间上跟随关系密切的前四种天敌依次是茶色新圆蛛Neoscona theisi(0.753 5)、八斑球腹蛛Theridion octomaculatum(0.720 1)、锥腹肖蛸Tetragnatha maxillosa(0.681 3)和草间小黑蛛Erigonidium graminicolum(0.644 2)。‘白毫早’茶园中与假眼小绿叶蝉空间上跟随关系密切的前四种天敌依次是粽管巢蛛Clubiona japonicola(0.823 5)、鳞纹肖蛸Tetragnatha squamata(0.800 9)、锥腹肖蛸(0.794 2)和茶色新圆蛛(0.794 2)。两种茶园前四种天敌中相同的天敌是茶色新圆蛛和锥腹肖蛸。假眼小绿叶蝉在聚块内基本样方数k为1、2、4、8时,随着聚块内基本样方数的增多,聚集分布格局时的扩散系数C不断增大,均匀和随机格局时扩散系数不断减小。聚块内基本样方数k为2、4、8时与k为1时的假眼小绿叶蝉的空间分布聚集程度差异均不显著。假眼小绿叶蝉的种群聚集均数λ多数情况均大于2,其聚集是该虫本身原因引起的,假眼小绿叶蝉在种群聚集均数λ为正值时,随着聚块内基本样方数的增加,则种群聚集均数λ不断增大。用假眼小绿叶蝉不同大小聚块的ρ指数判断个体群聚集时的最小范围是聚块中有1个基本样方,即本文的4 m~2。该研究为该虫抽样时确定样方大小提供了科学依据。     

稻瘟病菌F-box蛋白MoFbr7生物学功能分析

F-box蛋白在泛素-蛋白酶体途径(ubiquitin-proteasome pathway,UPP)中参与调控胞内蛋白降解、受体识别、信号传导等生物学过程。本研究从稻瘟病菌中克隆了F-box基因MoFbr7,序列分析表明,该基因编码产物具有1个F-box结构域(N-端)和8个连续的WD40重复序列(C-端),在丝状真菌中高度保守。利用基因敲除方法,获得4个MoFbr7基因敲除突变体,同时构建了回补菌株。表型分析结果显示,MoFbr7基因缺失突变体在产孢量、附着胞形态、原生质体释放、致病性等方面均无异常。突变体在MM、RDC培养基上生长速率下降;对细胞壁胁迫因子CFW(Calcofluor white)、刚果红敏感。以上结果表明MoFbr7参与稻瘟病菌的营养生长与细胞壁完整性,为进一步揭示其生物学功能奠定基础。     

温度胁迫下沙葱萤叶甲小热激蛋白基因GdHsp20.6的克隆及表达分析

为明确小热激蛋白在沙葱萤叶甲Galeruca daurica应对温度胁迫中的作用,采用PCR方法克隆沙葱萤叶甲小热激蛋白基因Hsp20（GdHsp20.6）完整的开放阅读框（open reading frame,ORF）序列,应用在线软件对GdHsp20.6基因进行生物信息学分析,通过原核表达技术诱导表达及纯化其编码蛋白,并通过实时荧光定量PCR（real-time quantitative PCR,RT-qPCR）技术分析不同温度胁迫下GdHsp20.6基因的表达量。结果显示,GdHsp20.6基因ORF序列长度为543 bp,编码180个氨基酸,预测分子量为20.6 kD,无跨膜区和信号肽。GdHsp20.6氨基酸序列有高度保守的α-结构域。GdHsp20.6氨基酸序列与其它鞘翅目昆虫的Hsp20氨基酸序列有较高的一致性,其中与花绒寄甲Dastarcus helophoroides的Hsp20.99氨基酸序列的一致性最高,为63%。GdHsp20.6基因在大肠杆菌Escherichia coli BL21（DE3）细胞系中成功表达,经异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-d-thiogalactoside,IPTG）诱导后GdHsp20.6蛋白成功表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）和Western-blot分析表明融合蛋白大小与预测大小一致,并纯化获得了纯度较高的目的蛋白GdHsp20.6。低温（-10~5℃）和高温（35~40℃）处理1 h以及处理后25℃恢复30 min均能诱导GdHsp20.6基因表达上调,并且0℃处理30~120 min也能诱导GdHsp20.6基因表达上调。表明GdHsp20.6基因在沙葱萤叶甲应对低温和高温胁迫中可能起着重要作用。     

“一带一路”沿线分区用水结构与产业结构的分析比较

“一带一路”沿线用水结构与产业结构的分析,对支撑沿线区域发展意义重大。基于“一带一路”主体水资源区分区及用水研究,采用赫芬达尔-赫希曼指数（HHI指数）分析了“一带一路”产业用水量和GDP的地理集中度,采取用水结构指数和产业结构指数分析了分区用水结构及产业结构的差异,应用洛伦茨曲线和基尼系数对“一带一路”全区用水量与GDP的匹配度进行研究,计算了“一带一路”分区基于数列的用水量与GDP匹配度。结果表明：① “一带一路”第一产业用水量和GDP产值的集中度在三大产业中均为最高,主要集中在东亚、西亚、南亚和东南亚;② 不同分区分产业用水量和GDP产值占各分区总量的比值,在空间分布差异上从大到小依次为第一、第三、第二产业;③ “一带一路”全区第三产业用水结构与GDP产值匹配度最差,南亚是第一产业和第三产业匹配度最差的分区。分析成果有助于系统了解“一带一路”区域用水和产业结构的基本情况,为深入分析分区水资源与经济社会关系提供指导。     

甜瓜黄斑病毒在海南黄瓜上的发生分布及序列分析

 应用特异性引物MYSV-L-F和MYSV-L-R对海南5个市县采集的150份疑似感染病毒病的黄瓜样品进行RT-PCR检测,在三亚、乐东和东方等3个市县的18份样品中检测到甜瓜黄斑病毒(Melon yellow spot virus, MYSV)。选取采自三亚的分离物C29(MYSV-Hainan)进行MYSV全基因组克隆及序列分析,结果表明:该分离物的L RNA、M RNA和S RNA基因组全长分别为8 918 nt、4 815 nt和3 257 nt,与已知MYSV分离物的核苷酸相似性分别为97.4%~97.7%、96.2%~97.9%和94.8%~99.8%,系统进化关系分析与相似性分析一致,序列相似值越高,系统进化关系越近。     

Argonaute1敲除前后尖孢镰刀菌转录组分析比较

 Argonaute蛋白(AGO)介导的沉默复合体在RNA干扰(RNAi)中起着至关重要的作用。为了探究AGO1在尖孢镰刀菌RNAi中的作用机制,本文以粘团专化型尖孢镰刀菌生理1号小种FOX-A8野生型和其AGO1缺失突变体(FOX-A8-△Ago1)的菌丝和孢子为材料,分别进行了RNA提取、Illumina HiSeq 2000高通量转录组测序、差异表达基因(DEGs)的显著富集分析;选择菌丝和孢子中的DEGs 进行实时荧光定量PCR(Quantitative real-time PCR, qRT-PCR)验证。GO通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中的醇脱氢酶(NADP⁺)、孢子中的CAMK / CAMKL / CHK 1蛋白激酶均显著上调;KEGG通路注释结果显示,相对于野生型菌株,AGO1缺失突变体菌丝中与 MAPK信号通路相关的基因、孢子中与PLD信号通路相关的基因均显著下调;另外,相对于野生型菌株,编码AGO2的基因下调,但是下调不显著。qRT-PCR检测DEGs的表达模式与RNA-Seq分析结果一致,证实了RNA-Seq结果的可靠性。