香石竹斑驳病毒( CarMV) 主要基因的分子变异分析

 香石竹斑驳病毒(Carnation mottle virus, CarMV)是侵染香石竹的主要病毒之一。本试验从12 个香石竹品种中获得CarMV 分离物,通过RT-PCR 扩增包含p7、p9、CP 3 个主要基因的片段,并对扩增产物进行克隆测序。通过序列比对发现CarMV 的p7、p9、CP 3 个基因有较高的稳定性,p7 基因核苷酸序列相似性为98. 10% ,氨基酸序列相似性为97. 81% ,其中氨基酸的第11 和14 位存在显著差异;p9 基因核苷酸序列的相似性为98. 80% ,氨基酸序列相似性为99. 13% ,氨基酸序列在第4 差异明显;CP 基因核酸序列相似性为97. 58% ,氨基酸的相似性为98. 43% ,氨基酸序列的第164 和331 位的变异存在相关性,整个CP 变异位点比较分散。证实p7 和p9 的变异位点主要集中在暴露与寄主互作相关的N 端,推测这是导致病毒变异,与寄主互作变异的重要位点。     

四纹豆象及其近缘种基于COI基因的分子检测技术研究

四纹豆象是口岸检疫中经常截获的种类,本文以四纹豆象及其近缘种为研究对象,测定分析了COI基因516 bp碱基序列。序列分析结果表明:保守位点为353个,变异位点为163个,简约信息位点为134个,自裔位点为29个。基于Kimura 2-parameter模型分析遗传距离,结果显示:种内遗传距离介于0.001~0.013之间,平均遗传距离为0.008,种间遗传距离介于0.114~0.193,平均遗传距离为0.161。采用邻接法构建的COI基因序列系统发育树显示,同一物种聚为同一小支,且分支自展值均为100%。结果表明应用COI基因片段对四纹豆象及其近缘种进行分子鉴定具有可行性。     

海南省田间稻瘟病菌中AvrPiz-t基因位点变异

为评估在水稻育种中被广泛利用的广谱抗稻瘟病基因Piz-t的有效性,对不同年份分离自海南省陵水黎族自治县和三亚市水稻的273株田间稻瘟病菌株中的AvrPiz-t位点变异及其与菌株致病性的相关性进行系统研究。结果表明,海南省田间菌株中无毒基因AvrPiz-t位点的变异频率为0～100.00%,陵水黎族自治县菌株中的变异频率远远高于三亚市菌株。在菌株中共鉴定到3种AvrPiz-t位点变异类型,分别为基因位点完全缺失、DNA重复元件MGR583在基因位点启动子区-10 bp上游和编码区218 bp下游插入。所有AvrPiz-t位点变异的菌株对携带Piz-t抗病基因的单基因水稻系IRBL-11均表现出强的致病性。陵水黎族自治县菌株中AvrPiz-t位点的变异频率呈逐年增加趋势,2021年94株菌株中AvrPiz-t位点的变异频率为100.00%,其中51.06%的菌株变异是MGR583在启动子区-10 bp上游插入,表明DNA重复元件MGR583在AvrPiz-t位点插入是AvrPiz-t从无毒到有毒进化的重要机制之一。     

烟台市富士苹果上苹果锈果类病毒分子变异分析

为明确苹果锈果类病毒(Apple scar skin viroid,ASSVd)在烟台市富士苹果上的变异情况,通过特异性引物对携带苹果锈果类病毒的富士嫩叶进行ASSVd全长扩增,利用生物学软件DNAMAN对所得变异序列进行分析并构建系统进化树。结果表明,从130个样品中筛选到36个阳性样品,36个阳性样品中共克隆获得52条329~333 nt的ASSVd变异序列,其中30个阳性样品含2条或2条以上的ASSVd序列。对所得变异序列进行序列比对发现,同一样品不同克隆间核苷酸序列相似性为94.0%~100.0%,所有变异序列核苷酸序列相似性为94.0%~99.7%,与Gen Bank中来自不同国家或地区的10条ASSVd分离物核苷酸序列相似性为88.3%~99.7%。将序列相似性低于97.0%的12条变异序列进行系统进化分析,结果显示除了333 nt变异序列ZS2-6与伊朗苹果分离物在同一分支外,其余11条变异序列位于同一分支。52条变异序列多序列比对发现,变异位点主要集中在TL区、P区和C区。研究表明烟台市富士苹果上ASSVd存在一定的分子变异。     

福建省烟粉虱不同地理种群遗传结构特征

为明确福建省烟粉虱种群遗传结构特征,基于福建省烟粉虱不同地理种群中40个代表性的线粒体COI基因序列,分析了种群遗传多样性、遗传分化及分子变异情况,并构建了单倍型系统发育树与网络图。结果显示:在590 bp长度的mt COI基因序列中有效位点558个,其中187个核苷酸位点存在变异;序列核苷酸中A、T、C、G含量分别为42.32%、24.36%、20.25%、13.06%,其中A+T的含量为66.68%,表现出明显的A+T偏向性;共检测出11个单倍型,其中Hap3、4、7、9、11为特殊单倍型;种群多样性指数为0.838,核苷酸多样性指数为0.093,表明遗传多样性水平较高;AMOVA分析表明种群遗传变异主要来自种群内,总种群遗传分化系数仅为0.027,种群遗传分化较低。表明福建烟粉虱种群基因交流未受地理距离明显影响,种群遗传分化不显著。     

甘蔗线条花叶病毒HC-Pro基因的分子变异分析

为了解析甘蔗线条花叶病毒Sugarcane streak mosaic virus(SCSMV)不同分离物HC-Pro基因的分子变异规律,本研究利用RT-PCR法扩增获得SCSMV HC-Pro基因的序列,通过生物信息学分析,分别从重组、系统发生、选择压力等方面研究SCSMV HC-Pro基因的分子变异特征。共测定了44条SCSMV HC-Pro基因序列,相似性最低值为70%;HC-Pro基因重组频率较低,仅发现3个重组位点,其中一个系首次报道;与先前报道相比,部分新测定云南蔗区的SCSMV分离物在HC-Pro基因上形成一个新组-第Ⅲ组;HC-Pro基因处于很强的负选择压力作用,未发现正向选择作用位点。本研究结果进一步证明SCSMV HC-Pro基因具有高度的遗传多样性。     

侵染白附子的芋花叶病毒分子变异研究

为明确侵染白附子的芋花叶病毒(dasheen mosaic virus, DsMV)的分子变异情况,对51个DsMV白附子分离物(DsMV-BF)的外壳蛋白(Coat Protein,CP)基因和3个分离物的近全长基因组序列进行了克隆和测定,DsMV-BF的CP基因大小有855个和942个核苷酸两种类型,51个白附子分离物之间CP基因的核苷酸和氨基酸一致率分别为88.3%～100%和91.9%～100%,BF8、BF30和BF38分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为82.9%～95.9%和90.7%～95.9%,与GenBank中其他分离物之间多聚蛋白的核苷酸和氨基酸序列一致率分别为76.9%～99.4%和85.6%～99.0%;P1基因的分子变异较大,P1基因大小有987个和990个核苷酸两种类型;CP基因核苷酸序列系统进化树分析结果表明,侵染白附子的DsMV分离物可分为两个亚组;重组分析结果表明BF8和BF30分离物各检测到1个重组事件,BF38检测到2个重组事件。     

trnH-psbA序列作为DNA条形码在苍耳属杂草鉴定中的应用

以trn H-psb A序列作为DNA条形码,对从国外截获的9种苍耳属杂草及1种国内苍耳进行物种鉴定研究。采用DNeasy Plant Mini Kit试剂盒进行总DNA的提取,应用通用引物对其trn H-psb A基因进行PCR扩增,测序得到10种苍耳属杂草的trn H-psb A序列,并利用MEGA 7.0软件进行比对分析构建系统进化树。结果显示:苍耳属杂草trn H-psb A基因序列共有543个位点,其中有53个变异位点,31个变异信息位点,22个单突变位点,30个碱基缺失;种间遗传距离为0~0.093,种内无遗传差异;进化树显示10个苍耳物种均处于独立分支,能够利用该条形码对苍耳属杂草进行区分鉴定。     

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。     

7种长蠹科昆虫的线粒体DNA ND4基因序列比较分析

长蠹科昆虫严重危害林木和仓贮物品。该文应用非损伤性DNA测序技术测定了来自不同国家的长蠹科害虫的线粒体DNA ND4基因的部分序列。在获得的204bp的序列中，7种昆虫的序列变异丰富，多数变异发生在密码子的第3位点上。将实验结果与形态学特征比较分析，探讨7个种的形态差异与基因序列的差异；结果表明，7种昆虫不仅在外形特征上存在差异，而且在ND4基因序列上的差异程度也很显著，平均为21．94％。这为分类鉴定提供了充分的证据。     

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schr?t, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.     

Response of stomatal conductance of two tree species to vapor pressure deficit in three climate zones

Stomatal behavior is a central topic of plant ecophysiological research under global environmental change. However, the physiological mechanism controlling the response of stomata to vapor pressure deficit(VPD) or relative humidity(RH) has been inadequately understood till now. In this study, responses of stomatal conductance(gs) to VPD in two species of trees(Fraxinus chinensis Roxb., Populus alba L. var. pyramidalis Bge.) in three different climate zones(Jinan with typical warm humid/semi-humid climate, Urumqi with temperate continental arid climate and Turpan with extreme arid desert climate) were measured. Levels of two phytohormones(abscisic acid, ABA; indole-3-acetic acid, IAA) in the leaves of the two tree species at these three sites were also measured by high performance liquid chromatography. The results showed that the responses of gs to an increasing VPD in these two tree species at the three sites had peak curves which could be fitted with a Log Normal Model(gs=a·exp(–0.5(ln(D/c)/b)2). The VPD/RH values corresponding to the maximum gs can be calculated using the fitting models for the two tree species in the three sites. We found that the calculated gs-max-VPD correlated negatively with relative air humidity in the three sites during the plant growth period(April to October 2010), which showed the values of gs-max-VPD were related to the climate conditions. The prevailing empirical stomatal model(Leuning model) and optimal stomatal behavior model could not properly simulate our measured data. The water use efficiency in the two tree species did not show obvious differences under three very different climatic conditions, but the highest gs, photosynthetic and transpiration rates occurred in P. alba var. of Turpan. The sensitivity in response of gs to VPD in leaves of the two trees showed positive correlations with the concentration of ABA, which implied that ABA level could be used as an indicator of the sensitivity of stomatal response to VPD. Our results confirmed that the prediction of the response of gs to VPD might be incomplete in the two current popular models. Therefore, an improved gs model which is able to integrate the results is needed. Also, the stomatal response mechanism of single peak curves of gs to VPD should be considered.     

基于线粒体COⅡ基因的乳白蚁序列分析和系统发育研究

本研究克隆测序了我国口岸截获和国内采集的7种乳白蚁的线粒体细胞色素氧化酶Ⅱ(COⅡ)基因,并进行序列分析,构建了系统发育树,进行系统发育研究.研究结果表明:乳白蚁COⅡ基因长度为684 bp,AT含量为61.2％,明显高于GC含量.乳白蚁属内各种之间的同源性为87.5％?96％,同种的不同地理种群之间的同源性均高于98...     

河南省地黄病毒病初步鉴定

 利用血清学、RT-PCR并结合核苷酸序列测定等方法,对河南省地黄病毒病进行了初步鉴定。结果表明,烟草花叶病毒(TMV)为侵染地黄的主要病毒;对TMV地黄分离物(TMV-RH) CP基因的序列分析结果表明,TMV-RH与TMV-U1株系CP基因的核苷酸同源性为86.5%,氨基酸同源性为94.3%;与已发表的TMV其它株系CP基因的核苷酸同源性在76.3%~88.5%之间,氨基酸同源性在79.3%~95.0%之间,同源性较低。根据不同株系CP的氨基酸序列进化树分析,推测该分离物可能为TMV的一个新株系。     

Genetic variation and evolutionary forces shaping Cucumber vein yellowing virus populations: risk of emergence of virulent isolates in Europe

The genetic variation and evolutionary mechanisms shaping Cucumber vein yellowing virus (CVYV) populations were investigated by analysis of nucleotide sequences coding for P1b, P1b/P3 and coat proteins (CP) from isolates collected in different countries. The complete genome sequence of isolate ISM from Israel was also determined and compared to those of isolates Jor from Jordan and ALM32 from Spain. This isolate had overall nucleotide identities of 94·23 and 94·96% with ALM32 and Jor, respectively. Nucleotide variation among isolates was not homogeneously distributed, with the 5′ half of the genome being more variable than the 3′ half. A Bayesian phylogenetic tree of the CP showed that CVYV isolates clustered into two main clades: isolates from the Middle East region (Lebanon, Israel and Jordan) clustered in both clades whereas the isolate from Tunisia clustered in clade I and the European isolates clustered as a homogeneous phylogroup in Clade II. A similar topology was observed for P1b but with incongruences with respect to the CP, suggesting genetic exchange among virus isolates, which were confirmed with recombination algorithms. The low genetic diversity within the European phylogroup with respect to the other isolates, neutralist tests and genetic differentiation analyses suggest that the Middle East region is the cradle of CVYV and that a unique virus introduction event occurred in Europe, where the virus spread rapidly. Taken together, these findings indicate a risk of emergence of virulent CVYV isolates in Europe either through migration from the Middle East or by genetic changes of the European isolates.     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

来源于湖北省枣疯病植原体分离物16S rDNA和rp基因的序列分析

以田间采集的来源于我国湖北省枣树产业主产区随州市随县种植的表现为"枣疯病"症状的枣树分离株为试材,对其16S rDNA和核糖体蛋白(ribosomal protein,rp)基因采用Nested-PCR进行扩增以及序列分析。结果表明,湖北JWB-Hubei植原体分离物16S rDNA基因的核苷酸序列与我国山东、河南等地的分离株一致率均为99%以上,在进化树中位于同一亚组的不同进化分支;虚拟RFLP图谱分析表明,JWB-Hubei属于16SrV-B亚组一个成员,与其进化树分组结果一致。JWBHubei分离株rp基因的核苷酸序列也与我国山东、陕西等地区的分离株一致率均为99%以上,在进化树中聚为同一亚组,与报道的基于RFLP分类属于rpV-C亚组的中国枣疯病分离物(JWB)聚集于同一亚组不同分支。该研究结果明确了湖北省枣疯病植原体的分类地位以及与来源于我国不同地区枣疯病分离株之间的遗传进化关系,为进一步研究植原体的株系划分、基因遗传变异研究提供了理论基础。     

葡萄浆果内坏死病毒变种类型1分离物全长基因组序列分析

正近年来,随着高通量测序技术的运用,国内陆续发现和报道了一些新的葡萄病毒~([1～4]),但由于尚缺乏相关的基础性研究及防治措施,给葡萄产业的健康发展带来一定影响。其中,葡萄浆果内坏死病毒(Grapevine berry inner necrosis virus,GINV)为2016年国内新报道的葡萄病毒,可引起一些葡萄砧木和品种产生褪绿斑驳和环斑症状,造成严重