禾谷镰刀菌和稻瘟病菌基因组中的微卫星序列比较

利用禾谷镰刀菌Fusarium graminearum和稻瘟病菌Magnaporthe grisea基因组测序结果,对这两种植物病原真菌基因组中的微卫星(SSR)序列进行了系统地分析和比较.结果表明,在禾谷镰刀菌基因组中,共发现4679个SSR序列,总长度为96.2kb,占基因组全长的0.27%.平均7.7kb碱基中有一个大于15 bp的SSR序列.在稻瘟病菌基因组中共发现16398个SSR系列,其总长度达到330kb,约占整个基因全长的0.85%,平均2.36kb碱基中就分布有1个SSR序列.在禾谷镰刀菌基因组中,数量最多的是五碱基重复序列,其次是六碱基重复序列;稻瘟病菌基因组中数量最多的是单碱基重复序列,其次为三碱基重复序列和五碱基重复序列.两基因组中数量最少的都是二碱基重复序列.尽管这两种植物病原真菌都属子囊菌,基因组大小也十分接近,但无论是在SSR的总体数量上,还是在各类SSR的分布上,两种植物病原真菌都存在十分显著的差别.     

PWL基因家族在黑龙江省水稻稻瘟病菌中的分布与变异

根据已公布的PWL家族基因序列设计4对特异性引物, 对329个采自2016年及2017年黑龙江省各稻区的水稻稻瘟病菌单孢菌株DNA进行PCR扩增与序列分析, 研究了水稻稻瘟病菌中PWL家族基因的组成及变异特征?结果显示, PWL2与PWL4在黑龙江省各稻区稻瘟菌中均有分布且稳定存在, 出现频率分别为73.86%与73.25%; PWL3出现频率为40.73%; PWL1在329个菌株DNA中均未扩增出目的条带, 再次验证了PWL1在水稻的稻瘟病菌中显示为完全缺失?对部分菌株的PCR产物进行测序分析发现, PWL2?PWL3和PWL4基因编码区在黑龙江省水稻稻瘟病菌株中的主要变异为点突变, 且引起氨基酸的突变?     

南方稻区稻瘟病菌株中无毒基因AvrPi9的变异分析

 本研究通过PCR(Polymerase Chain Reaction)和测序技术以及稻瘟病菌致病力检测手段,分析了2020年来自安徽、重庆、福建、广西、湖南、江苏、江西、云南以及浙江等省(直辖市、自治区)的300个稻瘟病分离菌株的无毒基因AvrPi9的分布和变异情况。结果表明,298份稻瘟病菌分离菌株的AvrPi9位点能被有效扩增,扩增产物经测序分析后显示,其中8株供试菌株的AvrPi9基因位点的外显子区域253位发生单碱基变异(C碱基替换成T碱基,AvrPi9^C253T),导致转录提前终止。稻瘟病菌致病力分析表明,AvrPi9^C253T菌株对带有Pi9基因的植株TP309-Pi9产生了致病性,说明该类菌株的AvrPi9位点变异后不能被Pi9识别。上述结果提示培育和种植携带有抗稻瘟病基因Pi9的品种可以较大程度上对南方稻区稻瘟病起到防控作用。这对保障水稻生产安全具有重要意义。     

稻瘟病菌无毒蛋白AvrPiz-t半胱氨酸残基的功能分析

 稻瘟病菌无毒基因AvrPiz-t编码了一个包含108个氨基酸的未知功能的预测分泌蛋白,其与对应的水稻抗稻瘟病基因Piz-t介导了基因对基因的抗病反应。序列分析发现AvrPiz-t的预测成熟蛋白AvrPiz-t₉₀含有4个半胱氨酸残基,推测它们可能参与了AvrPiz-t蛋白分子内或分子间的相互作用。为了检测这4个半胱氨酸对AvrPiz-t功能的影响,利用定点突变技术对4个半胱氨酸残基分别进行了位点突变并构建了相应的稻瘟病菌互补载体。功能互补分析发现,上述4个突变体都丧失了AvrPiz-t无毒基因的功能。由此推断,半胱氨酸对AvrPiz-t蛋白功能的表达是非常重要的。     

黏虫体内两种微管蛋白基因cDNA序列的克隆与序列分析

以黏虫4龄幼虫为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到该虫的α和β微管蛋白基因的cDNA序列各1条。其中α微管蛋白基因的cDNA序列1 443个碱基,包括一个1 353个碱基的开放阅读框,编码一个含450个氨基酸的蛋白,分子量约为50.0ku。氨基酸的142~148位存在一个微管蛋白标志信号片段GGGTGSG,在氨基酸序列的C-端有一个酪氨酸残基,N-端存在一个对转录后调控非常重要的保守区MRECI序列,以上特点与其他昆虫α微管蛋白氨基酸序列相同。黏虫β微管蛋白基因cDNA序列含1 906个碱基,开放读码框1 344个碱基,编码氨基酸447个,分子量约为50.2ku,等电点4.75。1~4个氨基酸MREI为β微管蛋白转录后调控信号,140~146GGGTGSG位同样存在一个微管蛋白标志信号片段。序列比对表明,克隆的α和β微管蛋白基因与其他昆虫的α和β微管蛋白基因在核苷酸和氨基酸水平上都是高度同源的,黏虫与家蚕(Bombyx mori)α微管蛋白的氨基酸序列同源性达到99.3%,与其他3种夜蛾科昆虫α微管蛋白的氨基酸序列同源性更是达到100%。黏虫与家蚕β微管蛋白的氨基酸序列同源性达到98.7%,与烟草天蛾β微管蛋白的氨基酸序列同源性达到99.6%。两个基因的cDNA序列已经登录GenBank并获得登录号分别为EU100016和EU234504。     

烟粉虱内共生菌传毒相关groEL基因的克隆及其原核表达

 烟粉虱内共生菌groEL基因编码一种63kD的GroEL蛋白,利用一对特异性引物,通过PCR对长期培养在黄瓜上的烟粉虱体内groEL基因进行了扩增,序列测定表明其长度为1668bp,编码555个氨基酸,与GenBank中烟粉虱内共生菌groEL(AF130421)的核苷酸同源性为99.58%,仅7个核苷酸发生了变异,二者氨基酸序列同源性为99.28%,仅4个氨基酸有别。构建了一个原核表达载体,表达得到了76kD的融合蛋白     

禾谷镰孢菌γ-微管蛋白基因克隆及其与多菌灵抗药性关系分析

根据禾谷镰孢菌参考菌株NRRL31084(PH-1)的γ-微管蛋白基因核苷酸序列设计5对引物,采用PCR方法从禾谷镰孢菌(Fusarium graminearum)对多菌灵(MBC)的敏感菌株、室内诱导及田间多菌灵抗药性菌株中分段扩增测序,获得了γ-微管蛋白基因全序列.该基因全长1 868 bp,含有5个内元,编码一含493aa的多肽;与PH-1的γ-微管蛋白基因核苷酸序列同源性99%,存在10个差异核苷酸,与所编码的氨基酸序列同源性100%;与其它7种真菌的γ-微管蛋白基因所编码的氨基酸序列同源性为31%～72%.中国的2个敏感菌株和4个抗药菌株的γ-微管蛋白基因序列完全相同,认为多菌灵抗药性与该微管蛋白变异无关.     

泡桐丛枝植原体Sec分泌蛋白转运系统3个亚基基因的克隆及蛋白特性分析

采用PCR方法扩增Sec分泌蛋白转运系统3个亚基基因,并进行了生物信息学分析。通过SWISS MODEL同源建模与3D PSSM折叠子识别法构建蛋白质的三维结构,并通过PROCHECK程序验证构建的三维结构的可靠性。首次从泡桐丛枝(PaWB)植原体基因组中分离出Sec分泌蛋白转运系统3个亚基基因,各基因全长依次为2 508、1 242 bp和411 bp,分别编码835、204个及136个氨基酸的蛋白。SecA、SecY和SecE均为脂溶性稳定蛋白,SecA无明显跨膜区,SecY和SecE分别含有10个和3个明显的疏水跨膜区。二级结构主要为螺旋,其次为折叠和无规则卷曲,没有转角。构建的三维结构符合立体化学原则。泡桐丛枝(PaWB)植原体中存在的Sec分泌蛋白转运系统作为最主要的运输途径,可能直接转运菌体蛋白如毒素等直接到寄主细胞质中或介体昆虫细胞中,引起寄主病症。研究植原体的Sec蛋白转运系统对于了解植原体的致病机理及预防这类病害的发生提供了理论依据。     

水稻条纹病毒山东济宁分离物RSV-SD-JN2分子变异分析

为从分子水平探讨强致病性水稻条纹病毒山东济宁分离物RSV-SD-JN2的变异及其致病性,采用RT-PCR技术,克隆RSV-SD-JN2的RNA3、RNA4区段cDNA。结果显示,RSV-SD-JN2的RNA3和RNA4核苷酸序列长度分别为2487、2157bp;RNA3中,NS3基因长为636bp,基因间隔区(IR)为725bp,CP基因为969bp;RNA4中,SP基因长为537bp,基因间隔区(IR)为654bp,NSvc4基因为861bp。不同时期、不同区域和不同致病性的RSV分离物的序列比较发现,RNA3、RNA4序列5′和3′末端非编码区具有高度保守性,仅存在个别碱基的差异;基因编码区保守性较高,核苷酸和氨基酸序列同源性分别在93%和97%以上,且大部分碱基变异为无意义变异;基因间隔区(IR)易于变异。RSV的分子变异与其地理分布具有密切的关系。RNA4的IR的变异导致RNA二级结构——发夹结构的稳定性提高是病毒致病性增强的重要原因。     

甘薯脉花叶病毒外壳蛋白基因在大肠杆菌中的表达及其抗血清的制备

 根据已报道的甘薯脉花叶病毒(Sweet potato vein mosaic virus,SPVMV)外壳蛋白（CP）基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPVMV河南分离物（SPVMV-HN）基因组3′端1.8 kb的基因片段,包括部分NIb 基因序列和完整的CP基因及3′端非编码区序列(3′UTR)。序列分析表明,SPVMV-HN的CP基因由996个核苷酸组成（GenBank登录号为FJ687211）,编码332个氨基酸残基。与已发表的SPVMV其他分离物相比,其推导的氨基酸序列一致性为95.2%～98.5%,与 SPVMV广东分离物的氨基酸序列一致性为97.9%。将CP基因克隆到原核表达载体pET-28a（+）上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3) pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPVMV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。利用SPVMV的抗血清,对采自全国14个省（市）的田间甘薯样品以及嫁接的巴西牵牛样品进行了检测,结果表明,SPVMV在我国甘薯上普遍存在。     

稻瘟菌无毒基因AVR-Pikm的定位

 无毒基因是病原物中决定寄主抗病性表达与否的功能基因,其功能的丧失导致毒性小种的产生。在先前的研究中,本研究小组从稻瘟病菌中分离了与无毒基因AVR-Pik^m连锁的2个SCAR标记SCO12₉₄₆和SCE12₁₄₀₆。在本研究中,作者首先通过TAC克隆末端核苷酸序列的测定及其与70-15全基因组序列的比较,将这2个标记定位到稻瘟菌第1号染色体上;然后,利用稻瘟菌70-15全基因组草图序列和SSR技术,又分离了与无毒基因AVR-Pik^m连锁的4个SSR标记:SSR47T34、SSR50CA24、SSR52TAGG18和SSR56A28。进一步分析表明:上述4个SSR标记位于与SCO12₉₄₆和SCE12₁₄₀₆相反的一侧,与AVR-Pik^m位点的遗传距离分别为4.90、7.01、19.12和21.94cM,无毒基因AVR-Pik^m位于SCE12₁₄₀₆和SSRA7T34之间。本研究获得的稻瘟菌无毒基因AVR-Pik^m的精细定位为通过染色体步移克隆该基因奠定了基础。     

Assessing new SSR markers for utility and informativeness in genetic studies of brown rust fungi on wheat,triticale, and rye

The aim of the present study was to validate new simple-sequence repeat (SSR) markers and use them to assess genetic variability among 24 isolates of Puccinia triticina collected from wheat (Pt-wheat) and triticale (Pt-triticale), and 15 isolates of P. recondita f. sp. secalis (Prs) collected from rye. The Pt and Prs isolates were tested for virulence on a set of 35 Thatcher wheat near-isogenic lines, eight rye lines with known resistance genes, and 53 triticale cultivars with uncharacterized leaf rust resistance. Molecular genotypes were determined using a newly developed set of 34 SSR microsatellite primer pairs. All SSR markers tested on Pt isolates successfully amplified fragments of appropriate size. When tested on the Prs isolates, 21 out of the 34 Pt SSRs amplified expected fragments. Sixteen of these 21 SSRs were polymorphic, providing for the first time microsatellite markers to study genetic variation in Prs. Based on virulence data, variation among Prs isolates was low, probably due to the small number of rye differential lines available. Much higher variation for virulence was observed within the collection of Pt isolates from wheat and triticale, and two separate groups were established with mixed host origin. Substantial genetic variation was detected among the isolates studied with the SSR markers, assuming two different models of SSR evolution (infinite alleles model and stepwise mutation model). The newly developed set of SSR markers proved their effectiveness in detecting genetic variation and should be useful in further population genetics investigations of the two pathogens.     

小菜蛾鱼尼丁受体基因克隆及序列分析

利用RT-PCR和RACE技术克隆了小菜蛾(Plutella xylostella)鱼尼丁受体(Px-RyR)基因,其核苷酸序列全长15 748bp,5′非编码区267bp,3′端非编码区109bp,开放阅读区全长为15 372bp(GenBank登录号:JF927788),编码5 123个氨基酸残基。估测其蛋白分子量为579.39ku,等电点为5.45。该基因编码氨基酸序列和其他鳞翅目昆虫RyR氨基酸序列比对相似性较高(92%),与哺乳动物3种亚型RyRs的相似性为45%～47%。此外,二级结构预测,其C-末端存在6个跨膜区域;且Px-RyR中存在一个出现4次重复,长度为89～95个氨基酸的RyR结构域,平均相似性为33%。     

来源于砂梨和桃的苹果褪绿叶斑病毒分离物部分CP基因和3′端非翻译区的序列分析

 苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus, ACLSV）是引起果树病害的一种重要病毒。ACLSV寄主范围广、发生较普遍,可侵染苹果、梨等仁果类果树和桃、扁桃、李、樱桃、杏等核果类果树,据报道我国梨产区感染ACLSV达80%以上。ACLSV引起植物症状的类型与寄主种类、病毒株系有关。ACLSV为线形病毒科（Betaflexiviridae）、纤毛病毒属（Trichovirus）的代表成员^［1］。ACLSV的CP相对比较保守,研究表明不同的ACLSV分离物的CP基因具有序列多样性,存在分子变异^［2～5］,CP基因分子特性的研究可为ACLSV株系划分提供依据。来源于欧洲、亚洲和北美的桃、李等核果类果树,以及苹果寄主上的ACLSV分离物的分子变异报道较多^［2,3,5］,来源于梨寄主上的ACLSV分子变异研究较少^［4,5］。     

Analysis of Variable Short-sequence DNA Repeats on the 29 kb Plasmid of Erwinia amylovora Strains

The fire blight pathogen Erwinia amylovora has been specifically and sensitively detected by PCR assays with primers derived from plasmid pEa29. The amplified fragment of approximately 1kb can vary in length for individual strains, easily seen in a digest with restriction enzymes Sau3A or HpaII. DNA fragments from this variable region were cloned and DNA sequence analysis revealed short-sequence DNA repeat (SSR) motifs which were reiterated to various extents. The SSR units consisted of eight nucleotides (ATTACAGA), and terminated with ATTA which is part of an SSR. The shortest repetition consisted of four units and the longest one in Austrian E. amylovora strains was 15 units. The number of SSR units was remarkably stable during propagation of strains, but was occasionally changed when a strain was stressed by exposure to antibiotics, copper sulphate or storage at low temperature. Changes in the SSR number could be due to adjustment in bacterial fitness to environmental pressure. We designed oligonucleotide PCR primers from DNA sequences adjacent to the SSR region of pEA29 for rapid analysis of SSR length variations. With this PCR assay, more than 130 strains were classified into at least 11 types based on the number of repeats. E. amylovora strains isolated in Germany carried mostly six repeats in pEA29, which never changed under laboratory conditions. E. amylovora strains from Hungary and the Netherlands were quite divergent for the SSRs and further changes were sometimes observed after plating on agar medium. Homology search of nucleotide sequence data libraries revealed similarities of the SSR motif to partition functions of low copy number plasmids. Amino acid homology searches showed similarity of the deduced amino acid sequence in the ORF adjacent to the SSR motif to replication proteins of plasmids. The SSR may play a role in regulation of plasmid replication and partition as assumed for iterons.     

Genetic relationships among Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) genotypes based on the SSRs at the quantitative trait Loci for resistance to Ascochyta Blight

Breeding for resistance to ascochyta blight in chickpea has been challenged by several factors including the limited sources of good resistance. Characterization of a set of genotypes that may contain different genes for resistance may help breeders to develop better and more durable resistance compared to current cultivars. The objective of this study was to evaluate the genetic relationships of 37 chickpea germplasm accessions differing in reaction to ascochyta blight using Simple Sequence Repeat (SSR) markers linked to Quantitative Trait Loci (QTL) for resistance. The results demonstrated that ILC72 and ILC3279, landraces from the former Soviet Union, had SSR alleles that were common among the kabuli breeding lines and cultivars. A lower SSR allele diversity was found on LG4 than on other regions. No correlation was found between the dendrogram derived using SSRs at the QTL regions and the SSRs derived from other parts of the genome. The clustering based on 127 alleles of 17 SSRs associated with the QTL for ascochyta blight resistance enabled us to differentiate three major groups within the current germplasm accessions. The first group was the desi germplasm originating from India and cultivars derived from it. The second group was a mix of desi genotypes originating from India and Greece, and kabuli breeding lines from ICARDA and the University of Saskatchewan. The third and largest group consisted of landraces originating mostly from the former Soviet Union and breeding lines/cultivars of the kabuli type. Several moderately resistance genotypes that are distantly related were identified. Disease evaluation on three test populations suggested that it is possible to enhance the level of resistance by crossing moderately resistant parents with distinct genetic backgrounds at the QTL for resistance to ascochyta blight.     

西瓜花叶病毒2号新疆昌吉分离物外壳蛋白基因核苷酸序列分析

 利用RT-PCR从新疆昌吉地区表现花叶、疱斑、扭曲等症状的南瓜病株上检测到西瓜花叶病毒2号新疆昌吉分离物(简称WMV-2-XJ-CJ),并测定了该分离物外壳蛋白(CP)基因序列。序列分析表明,新疆昌吉分离物CP基因全长850个核苷酸,编码197个氨基酸。与国内外报道的12个WMV-2CP基因相比,其核苷酸序列同源性为92.6%~98.3%,由此推导的氨基酸序列同源性为94.7%~99.3%。新疆昌吉分离物在CP N'端可变区明显不同于国内外报道的核苷酸序列。WMV-2新疆昌吉分离物与日本和郑州分离物较其它国家和地区的分离物多出6个核苷酸,但其核苷酸及其推导的氨基酸序列差异较大。新疆昌吉分离物外壳蛋白有2个氨基酸残基明显不同于其它分离物,其中蚜传株系的特征结构域DAG突变为DAE。     

应用转录组测序高通量发掘东方粘虫SSR标记

为高通量发掘粘虫Mythimna separata(Walker)的SSR标记,利用MISA软件对第2代转录组测序获得的108 494条粘虫unigenes进行SSR检测。结果显示,共发现12 483个SSR位点,出现频率为11.505%,分布于10 685条unigenes中,发生频率为9.848%,平均每6 126 bp含有1个SSR位点。单核苷酸和三核苷酸是主要重复类型,分别占SSR总数的75.206%和15.325%;六核苷酸重复所占比例最小,仅为0.008%。粘虫转录组SSR中共发现42种重复基元,单核苷酸重复基元A/T出现频率最高,占总SSR的74.141%;其次是AC/GT和ATC/ATG,分别占4.390%和4.094%。10次重复的SSR数量最多,有6 557个,占总SSR的52.527%;11次重复的为1 872个,占14.996%;12次以上重复所占比例均不足5.000%。表明粘虫转录组中SSR位点数量多、出现频率高、基元类型丰富。     

Development of simple sequence repeat markers for the classification of the clubroot pathogen <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious diseases in cruciferous crops. To classify isolates, we developed simple sequence repeat (SSR) markers for P. brassicae. Twenty-four Japanese isolates were used in this study: 12 isolates of an unknown pathotype from the Kyoto Prefecture, as well as 12 isolates of known pathotypes, including three single-spore lines. From the 12 isolates from Kyoto Prefecture, 11 were classified into either pathotype 2 (three isolates) or 4 (eight isolates). We designed 23 SSR markers based on the P. brassicae genome, of which 11 markers from intergenic regions showed polymorphisms in the 24 isolates. Many haploid isolates belonging to pathotypes 2 and 4 were monomorphic, and typical alleles were detected in some isolates not belonging to pathotype 4. Two bands were detected for eight SSR loci in five isolates, indicating that different genotypes were mixed in these isolates. We constructed a phylogram based on the 11 polymorphic SSRs. Pathotypes 2 and 4 formed a cluster, from which pathotypes 3 and 1 were successively placed. These results strongly suggest a close genetic relationship between isolates in pathotypes 2 and 4, consistent with our finding that isolates in these two pathotypes were found at one collection site. In combination with pathotype classification and other marker systems, the SSR markers can be used for more detailed analyses to improve the control of clubroot disease.