马铃薯瓢虫和茄二十八星瓢虫体表细微结构的比较研究

本研究利用扫描电镜对马铃薯瓢虫Henosepilachnavigintioctomaculata (Motschulsky)和茄二十八星瓢虫H .vigintioctopunctata (Fabricius)体表的细微结构进行了比较研究。结果表明 ,马铃薯瓢虫和茄二十八星瓢虫在细微结构上存在明显差异。第一 ,2种瓢虫鞘翅表面凹陷内 ,毛的着生位置不同 ,马铃薯瓢虫的毛着生于凹陷中心 ,茄二十八星瓢虫的毛着生于凹陷边缘 ;第二 ,鞘翅表面凹陷的深浅不同 ,马铃薯瓢虫的凹陷较深于茄二十八星瓢虫。这为 2种瓢虫的形态学研究提供了细微结构方面的依据     

RNAi介导的HvCDA1基因沉默影响茄二十八星瓢虫存活和发育

【目的】茄二十八星瓢虫Henosepilachna vigintioctopunctata是茄科(Solanaceae)植物上的重要害虫。昆虫体内几丁质脱乙酰酶1(chitin deacetylase 1, CDA1)催化N-乙酰氨基-D-葡萄糖胺脱去乙酰基,促使几丁质转化为壳聚糖,控制昆虫体内几丁质纤维有序堆积,并维持角质层结构的完整性。抑制虫体中CDA1基因的表达会抑制壳聚糖的合成,影响昆虫表皮结构的形成,使昆虫不能正常发育而亡。【方法】利用RT-qPCR方法测定HvCDA1基因在茄二十八星瓢虫不同发育阶段(卵、1-4龄幼虫和预蛹)和4龄幼虫不同组织(表皮、脂肪体、中肠和马氏管)中的表达模式。通过饲喂茄二十八星瓢虫1龄幼虫不同浓度dsHvCDA1溶液浸泡处理1 min的茄子叶片后及直接饲喂4龄幼虫不同浓度dsHvCDA1溶液,探究沉默茄二十八星瓢虫HvCDA1基因对其幼虫存活和发育以及HvCDA1基因表达量的影响。【结果】发育表达谱结果表明,HvCDA1在茄二十八星瓢虫的各发育阶段均有表达,但在1龄末和2龄末幼虫中的表达量最高。组织表达谱结果显示,在茄二十八星瓢虫4龄幼虫的表皮中H...     

七星瓢虫触角转录组及嗅觉相关基因分析

【目的】瓢虫科食性高度分化。本研究旨在通过建立七星瓢虫Coccinella septempunctata触角转录组数据库,探讨其触角嗅觉相关基因与食性分化的关系。【方法】采用Illumina HiSeq 4000高通量测序平台对七星瓢虫成虫触角转录组进行测序、组装、注释,挖掘嗅觉相关基因,并与已发表的茄二十八星瓢虫Henosepilachna viginyioctopunctata转录组进行比较。【结果】共获得七星瓢虫触角转录组31 775条unigenes,其中69.71%的序列得以注释,NR数据库中注释最多,为20 539条。据注释信息,挖掘到27个嗅觉相关基因,包括1个气味结合蛋白(odorant binding protein, OBP)基因,13个化学感受蛋白(chemosensory protein, CSP)基因,4个气味受体(odorant receptor, OR)基因,7个味觉受体(gustatory receptor, GR)基因和2个感觉神经元膜蛋白(sensory neuron membrane protein, SNMP)基因。相对应地,在植食性茄二十八星瓢虫转录组中鉴定到38个嗅觉相关基因,包括七星瓢虫中未发现的1个离子型受体(ionotropic receptor, IR)基因。在各类型嗅觉相关基因中,茄二十八星瓢虫转录组的OBP基因比例(13.16%)高于七星瓢虫触角转录组的(3.70%),而七星瓢虫触角转录组的GR基因比例(25.93%)则高于茄二十八星瓢虫转录组的(13.16%)。【结论】触角嗅觉相关基因数目不是昆虫食性分化的主因。本研究获得了七星瓢虫触角转录组学资源,初步探讨了嗅觉相关基因同瓢虫食性分化的关系,为了解瓢虫乃至昆虫食性分化的分子基础提供了信息。     

草鱼种质相关SRAP及SCAR的分子标记

采用相关序列扩增多态性(Sequence-realted Amplified Polymorphism, SRAP)技术分析野生草鱼和家养草鱼,筛选与草鱼种质退化相关的分子遗传标记.共进行88对引物组合的检测, 产生标记数目共计905个.依据标记在群体中出现的频率和变化规律,共筛选出2 1个可能与种质相关的特异性标记,对这些特异性标记进行测序并将测序结果进行BLAST分析 .发现测得片段中有8个片段在GenBank中找到同源性较高的序列,而其他片段与数据库中序列的相似性较低.根据序列信息分别设计了3对引物.用这3对引物分别对草鱼三个群体进行 PCR扩增,分别产生了SCAR1(308 bp)、SCAR2(66 bp)、SCAR3(114 bp)3个扩增带.采用大样本对这3个标记进行验证,发现其中SCAR1在家养群体中呈现阳性,在野生群体中为阴性,可区分出这两种群体.以SCAR3为引物在174条家养群体中得到目的片段,在26个家养群体没有扩增出条带,分布频率为87%;在100个野生群体中有6个个体检测到该条带,分布频率为6%.以SCAR2为引物在野生群体中完全扩增出目的条带,淡水中心群体中有7条扩增到条带,前洲群体中没有扩增出条带,标记在家养种群中的分布频率为96.50%.因此SCAR1可作为草鱼家养群体的一个重要的分子遗传特征指标,为进一步进行分子标记辅助育种奠定了基础 [动物学报 54(3):475-481,2008].     

蠋蝽对茄二十八星瓢虫的捕食功能反应

【目的】明确蠋蝽对茄二十八星瓢虫的捕食能力。【方法】在实验室条件下测定蠋蝽5龄若虫对茄二十八星瓢虫2和4龄幼虫的捕食功能反应及搜寻效应。【结果】蠋蝽5龄若虫对茄二十八星瓢虫2和4龄幼虫的捕食行为均符合Ⅱ型功能反应;随着茄二十八星瓢虫密度的增加,蠋蝽的搜寻效应逐渐降低,蠋蝽5龄若虫对茄二十八星瓢虫2龄幼虫的搜寻效应明显大于4龄幼虫;蠋蝽5龄若虫对茄二十八星瓢虫2和4龄幼虫的实际最大捕食量分别为15和10头。【结论】蠋蝽5龄若虫对茄二十八星瓢虫幼虫具有较好的捕食作用。     

茄二十八星瓢虫生活史观察

<正> 茄二十八星瓢虫Henosepilachna viginti-octopunctata(Fabricius),属鞘翅目瓢虫科,俗名茄虱。该虫以成、幼虫为害马铃薯、茄子、大豆、豇豆、番茄等,喜群集于叶背、取食下表皮、叶肉,残剩表皮上有许多透明的凹纹,致使叶片变褐枯萎。近几年来由于耕作制度的变化,马铃薯、大豆等食料终年不断,该瓢虫为害明显加重。我们对该虫的生活史进行了观察,现整理如下:     

RNAi介导的HvIAP1基因沉默对茄二十八星瓢虫存活和发育的影响

RNAi抗虫技术,是一种新型绿色环保的害虫防控方法,具有广阔的应用前景.茄二十八星瓢虫Henosepilachna vigintioctopunctata是茄科植物上的重要害虫,对作物生产造成了严重的经济损失.本研究用饲喂法RNAi探究了沉默凋亡抑制蛋白1(inhibitor of apoptosis protein 1,IAP1)表达对茄二十八星瓢虫生长发育的影响.结果显示,HvIAP1在茄二十八星瓢虫的每个发育阶段均表达,1龄幼虫和3龄幼虫的表达量最高,成虫的表达量最低.取食200 ng/μL dsHvIAP1浸泡处理的叶片2 d后,可导致92％的茄二十八星瓢虫1龄幼虫死亡,同时在取食dsHvIAP124 h后,HvIAP1基因的表达量下降了2.40倍;另外,单头取食200 ng的dsHvIAP1可导致70％的4龄幼虫死亡,同时在取食48 h后,HvIAP1基因的表达量下降了2.58倍.并且RNAi处理后1龄幼虫和4龄幼虫大部分个体在48 h内出现急性取食障碍现象,基本不取食直至死亡.上述结果表明,HvIAP1基因在茄二十八星瓢虫的生长发育过程中起重要作用,当该基因表达受阻,会直接影响茄二十八星瓢虫的取食、抑制其生长发育而死亡.这一研究表明HvIAP1基因可作为潜在的防治茄二十八星瓢虫的RNAi靶标基因.     

不同温度下茄二十八星瓢虫的实验种群生命表

近年来,茄二十八星瓢虫Henosepilachna vigintioctopunctata在江汉平原地区危害严重,本研究旨在探明温度对该地区茄二十八星瓢虫生长发育和繁殖的影响,组建茄二十八星瓢虫的实验种群生命表,为该虫防治提供一定的理论依据。本研究采用室内恒温饲养方法,分别设置19℃、22℃、25℃、28℃、31℃和34℃等6个温度,测定各温度下用马铃薯饲养不同虫态的生长发育历期。结果表明,茄二十八星瓢虫在不同恒温条件下各虫态的发育历期随着温度的升高而逐渐缩短,19℃恒温下发育历期最长为3219 d,31℃恒温下仅为1315 d。幼虫期和蛹期的发育起点温度分别为1093和1213℃,日有效积温为18604和5706 d·℃。在19-31℃5个温区下幼虫和蛹的存活率都很高,并依据该温区内的发育起点温度和有效积温建立了发育历期预测式与Logistic模型。在25℃时单雌平均产卵量达到最大值107450粒,世代存活率和种群趋势指数最高,分别为9333%和50147,高温和低温均不利于其种群数量增加。     

茄二十八星瓢虫成虫天花板爬行行为研究

【目的】探明茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)成虫的天花板爬行行为,为气流控制技术防治农业害虫提供技术参数。【方法】本研究通过采用录像观察法对茄二十八星瓢虫雌雄成虫的天花板爬行行为进行观测,明确其成虫的运动行为参数,并分析了其步速分别与步距、步频和负荷因数的关系。【结果】茄二十八星瓢虫雌雄成虫天花板爬行时的速度为3.77~19.32 mm/s,步距为2.63~4.51 mm,步频范围为1.32~4.74 Hz。步距和步频均与速度之间存在极显著线性关系,速度的增加同时依靠步距和步频的增加,但步距的变化相对步频的变化小;前足、中足和后足的负荷因数均随着速度的提高而降低,且负荷因数均与速度存在显著相关性;一个步态周期内,足的摆动时间均远小于支撑时间。【结论】茄二十八星瓢虫成虫天花板爬行时速度的增加主要是依靠步频的提高来实现。     

SCAR标记在黑木耳栽培菌株分类鉴定中的应用

应用序列特异性扩增区域（SCAR）标记技术分析50株黑木耳栽培菌株的遗传多样性。在SRAP标记分析过程中发现1条1 200 bp的特异性条带,经回收克隆测序转为SCAR标记。根据序列设计出1对特异性引物,经PCR可以扩增出1 000 bp大小的片段,说明成功构建出了＂黑931＂的指纹图谱。     

SCAR标记在茶树种质资源鉴定中的应用

SCAR标记是一种在RAPD技术的基础上发展起来的新型分子标记技术,提高了分子标记辅助选择育种的效率,在茶树种质资源的合理开发与利用中具有广阔的应用前景.运用优化后的RAPD反应体系对10个茶树品种的基因组DNA进行遗传差异分析,随机引物S89、S4分别在白毫早和福云6号中扩增得到长度为498 bp、1 622 bp的差异片段,命名为BHZ498、FY1622.根据它们的测序结果分别设计了一对特异引物,BHZ498的特异引物为SB1/SB2;FY1622的特异引物为SC1/SC2,用这两对特异引物对10个茶树品种的基因组DNA进行扩增.引物SB1/SB2和SC1/SC2分别在白毫早和福云6号中扩增出唯一的一条扩增带,而这两对引物在其他供试茶树材料中均无相应的扩增带,结果表明已将BHZ498、FY1622标记成功转化成SCAR标记.     

烟草SCAR标记技术的建立

目的:通过烟草随机扩增多态性DNA(RAPD)标记技术建立烟草特征序列扩增区域(SCAR)标记技术,用于烟草品种鉴定。方法:对12个烟草品种的复烤叶片DNA进行RAPD分析,得到2个RAPD特异片段S1和S2,通过切胶回收,连接pUCm-T载体克隆转化,片段测序,设计特异性引物S1-1/S1-2和S2-1/S2-2,对SCAR-PCR扩增退火温度进行优化。结果:2个RAPD标记成功地转化为稳定快捷的SCAR标记,可将红花大金元和NC102等2个品种从12个烟草品种中快捷准确地鉴别出来。结论:SCAR标记可作为准确稳定的DNA水平的烟草品种鉴定方法,可对种植、复烤和配方品种的烟叶或叶片进行鉴别。     

Development of a SCAR marker and a strain‐specific genomic marker for the detection of the biocontrol agent strain CPA‐8 Bacillus amyloliquefaciens (formerly B. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA‐8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA‐8 that was sequenced and used to design SCAR (sequence‐characterised amplified regions) primer pairs. The SCAR‐4 marker amplified a semi‐specific fragment of 665 bp not only for the strain CPA‐8 but also for other 12 strains whose morphology was completely different from CPA‐8. Another approach was developed to obtain a strain‐specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA‐8. Our results revealed that these two molecular markers, SCAR‐4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA‐8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA‐8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphicDNA)分子标记技术,寻找谭清苏铁(Cycas tanqingii)中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465(CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域(SCAR)分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

水稻抗褐飞虱基因SCAR标记的获得

采用282个随机引物对药用野生稻1665和栽培稻桂99远缘杂交的抗褐飞虱近等基因系B3F4分离群体的不抗池DNA和抗池DNA进行了特异性RAPD标记筛选,从中筛选到一个具有明显的特异性扩增带谱的RAPD标记S1159,序列分析表明,S1159序列长度为1408bp,与基因库中已报道的水稻第四号染色体的BAC克隆(编号OSJNBa0070O11)序列(67114-69100)有51.86%的同源性。为了提高所找到的RAPD标记S1159在应用上的稳定性,将RAPD标记转化为SCAR标记检测近等基因系群体,结果表明与RAPD标记结果一致,说明该研究得到的RAPD标记具有较好的稳定性和重复性,为进一步的研究打下了良好的基础。     

Recombinant bovine interferon alpha causes only a temporary prolongation of the lifespan of the corpus luteum

Abstract

A method is described for developing a sheep‐ vs. goat‐specific DNA marker using sequence characterized amplified regions (SCARs) derived from a random amplified polymorphic DNA (RAPD) marker from sheep DNA samples. A sheep 645 bp DNA fragment that was absent in goat DNA was identified by analyzing pools of sheep and goat DNA with RAPD primers. This fragment was cloned and partially sequenced to design extended, strand‐specific 24‐mer oligonucleotide primers. Each primer contained the original 10 bases of the RAPD primer and the following 14 internal bases. The pair of primers resulted in the amplification of a single band of 645 bp when used to amplify sheep DNA, and in no amplification when used to amplify goat DNA. These SCAR primers successfully amplified the equivalent of DNA from one nucleated sheep cell in a sample of 5000 nucleated goat cells. This level of sensitivity is especially desirable for research involving the detection of interspecific chimerism.     

与苹果Co基因紧密连锁的RAPD标记的筛选及其SCAR标记转换

以短枝富士(Spur Fuji)X舞姿(Telamon)的105株F1群体为试材,利用RAPD技术,结合集群分类分析法(BSA)进行了苹果柱型基因(Co)分子标记的研究。通过对300条随机引物的筛选,获得一个与Co基因紧密连锁的RAPD标记S1142682,连锁距离为2．86cM。对该标记片段进行序列测定,然后根据序列特点设计了4条特异引物(其中正向引物与反向引物各两条)。PCR结果显示,这4条引物的4种组合都可以扩增出柱型性状的特征带。选其中之一进行群体上的分析,结果表明该SCAR标记特征带与柱型性状的共分离行为与原RAPD标记表现一致。可见,此组合的引物可以作为该SCAR标记的特异引物。通过对S1142682标记片段序列分析发现,在 45～ 251区域含有一个可编码68个氨基酸残基的ORF。