烟草白粉病抗性基因型的多重PCR检测体系及其在回交育种中的应用

为提高烟草白粉病隐性抗病基因（感病基因）NtMLO1（M1）和NtMLO2（M2）在分子标记辅助选择育种中基因型的选择效率,根据已报道的M1和M2突变基因序列,设计4对单基因特异性引物,组成2个双重PCR反应体系,分别用于同时扩增M1和M2突变型或M1和M2野生型等位基因。结果表明,建立的2个双重PCR体系扩增的特异性片段与单重PCR体系扩增的片段完全吻合,突变基因纯合体只在突变基因多重PCR体系中产生191和306 bp的特异性片段,野生基因纯合体只在野生基因多重PCR体系中产生437和652 bp的特异性片段,杂合体在2个反应体系中均有特异性片段。利用该体系可经过1次或2次PCR扩增准确检测出回交转育过程中回交或自交后代M1和M2的基因型,加快抗病品种育种进程。      

晒烟品种塘蓬烟抗白粉病基因的等位性测定和序列分析

[背景和目的]广东连江县晒烟品种"塘蓬烟"是我国特有的烟草隐性遗传白粉病抗性种质资源,但到目前为止对其抗病机制未见深入报道.[方法]利用塘蓬烟花粉与抗白粉病烟草品种Kutsaga E1及感病品种K326杂交进行抗病基因的等位性检测;利用等位基因分子标记检测塘蓬烟中感白粉病基因NtMLO1/2的突变情况;利用PCR克隆技...     

烟草种质资源抗马铃薯Y病毒病基因型鉴定

  背景和目的  从作物种质资源中进行等位基因鉴定,特别是优异等位基因的发掘和应用,是使种质资源加速转变为基因资源的关键所在。  方法  采用苗期汁液摩擦接种的方法,对烟草种质资源的马铃薯Y病毒（Potato virus Y,PVY）抗病性进行鉴定;通过烟草隐性抗PVY基因eIF4E1（又称为感PVY基因eIF4E1）等位性测验、等位基因分子标记检测和RT-PCR扩增测序,对PVY抗病资源进行基因型分析。  结果  （1）从收集到的900多份来自国内外的烟草种质资源中筛选出19份高抗PVY资源。（2）根据等位性测验结果推断19份抗源所具有的PVY抗性均由eIF4E1基因所控制。（3）等位基因分子标记检测和RT-PCR扩增测序结果将19份抗源的eIF4E1基因区分为4种突变类型。  结论  本研究结果丰富了我国对抗PVY烟草种质资源的筛选和利用的内容,为后续烟草PVY抗性优异等位基因的发掘和育种利用提供了基础材料和信息支撑。      

基于tdh1基因变异的大流行副溶血性弧菌鉴别方法的建立

目的基于副溶血性弧菌tdh1 DNA序列中特异性变异位点,建立新的大流行株鉴别方法。方法通过对多种型别菌株的tdh序列进行比对,查找大流行株特异性变异位点,并基于该位点设计位点特异性PCR体系和检测方法。以GS-PCR和tdh基因联合检测为参照方法,用已知大流行株、非大流行株和2014年收集的1 067株副溶血性弧菌对新方法进行验证。结果 3株菌株的6条tdh全序列存在26个多态性位点,其中tdh1基因的第368位碱基的变异[G/A]能用于鉴别大流行株,本研究基于此位点建立了tdh1_368位点特异性PCR。该变异能将1996年后的大流行O3∶K6型菌株与1996前的进行区分,也能将大流行株其他血清型变种与非流行菌株进行区分。对2014年的1 067株菌株的检测结果显示,tdh1_368位点特异性PCR与参考方法检测结果完全一致。结论本研究以副溶血性弧菌tdh基因的多态性位点建立检测大流行菌型的试验方法,从对1 067株菌株的实测结果来看,tdh1_368位点特异性PCR与既往报道的方法具有高度的一致性,且指标更为直接。     

多重位点特异性PCR快速检测牛肉中常见掺假动物源性成分

目的:基于动物线粒体cytb基因的多态性位点,建立一种特异性多重PCR体系检测牛肉、猪肉和鸡肉的方法。方法:提取肉类的基因组DNA,利用不同物种mtDNA cytb基因序列的SNP位点的差异,设计特异性引物。进行多重PCR扩增,利用扩增产物片段大小不同,检测牛肉中常见的掺假动物源性成分。通过灵敏性试验,确定最低检测量。结果:实验设计的引物特异性良好,在同一反应体系中,在同一退火温度52℃条件下,牛肉DNA扩增后产生149 bp的特异性条带,猪肉DNA扩增片段为261 bp,鸡肉DNA扩增片段为554 bp,未发生非特异性扩增。且检测的最低浓度达到100 pg/μL,具有高度的灵敏性和适用性。结论:根据动物线粒体cytb基因的差异性位点,开发的多重PCR体系,可一次性地同时检测牛肉、猪肉和鸡肉,可快速、灵敏、高通量地分析食品中掺假动物成分的来源。     

阪崎肠杆菌zpx基因的分子信标一实时PCR技术研究

目的建立分子信标-实时PCR技术检测婴幼儿乳粉中阪崎肠杆菌的快速方法。方法在PCR反应体系中加入分子信标探针,探针的5’端标记FAM,3’端标记TAMRA,建立阪崎肠杆菌zpx基因分子信标-实时PCR技术快速检测方法。结果检测方法特异性强,无非特异性扩增;分子信标-实时PCR反应体系DNA灵敏度为180fg/PCR反应体系,纯阪崎肠杆菌菌液的检出限为102 CFU/ml,无交叉反应;以此反应体系检测23份样品,其中2份为阳性,余未检出,与传统检测方法结果一致。结论分子信标-实时PCR检测体系快速、灵敏度高、特异性强,可用于婴幼儿乳粉中阪崎肠杆菌的快速检测。     

基于CRISPR/Cas9技术的烟草烟碱相关基因敲除及功能研究

CRISPR/Cas9是一种重要的基因组定向编辑技术,近年来在植物基因组的定向敲除和分子育种材料创制中得到了广泛应用。为了发掘可用于分子育种的低烟碱烟草,以烤烟品种K326为试验材料,利用CRISPR/Cas9基因组编辑技术对控制烟草烟碱合成和转运的5个基因（PMT1、QPT1、A622、NtNUP1和JAT1）进行定向敲除,并对T2代纯合基因型烟草植株进行烟碱含量检测。结果表明,定向敲除5个基因后获得100株成功编辑的T0代烟草植株,突变检出率为29.9%,突变类型以单碱基插入或删除为主。对定向敲除烟碱合成基因QPT1和转运基因JAT1的T1代纯合基因型烟草植株进行序列分析,发现存在4种突变类型,分别是插入一个T、C、A碱基的插入突变和一个缺失44个碱基的缺失突变,从而引发移码使得翻译的氨基酸链大幅缩短,其T2代植株上部叶烟碱含量显著低于野生型植株。综上所述,CRISPR/Cas9技术能够高效定向敲除烟碱关键基因,为低烟碱烟草分子育种提供了理论和技术支撑。     

33份晒烟种质资源SSR标记的指纹图谱构建

为分析我国晒烟种质资源的遗传多样性,并构建晒烟种质资源的指纹图谱,本研究利用25对SSR标记对33份晒烟材料进行分析。结果表明,25对SSR引物共检测到112个等位基因,平均每个标记4.31个,观测杂合度（Ho）平均值为0.0028,预期杂合度（He）平均值为0.6039。Shannon's信息指数I为1.1389,Nei's多样性指数（H）为0.5693,33份晒烟种质资源的遗传多样性相对丰富。UPGMA聚类分析表明,在相似性系数0.345处,可将供试烟草资源分为两个类群。同时,利用2 5对引物构建了33个晒烟品种的指纹图谱,为晒烟品种鉴定体系的研究奠定理论基础。本研究可为我国晒烟种质资源的鉴定与利用、优异基因的挖掘、育种亲本材料的选择等提供科学依据。      

烟草青枯病抗性的全基因组关联分析

为发掘并定位烟草青枯病抗性遗传位点,利用RAD简化基因组重测序技术（RAD-seq）,以219份遗传多样性较高的国内外烟草品种作为供试自然群体,利用田间病圃鉴定供试材料的青枯病抗性,通过全基因组关联分析（GWAS）,发掘抗病基因组区段,并进行抗病候选基因预测。结果表明,筛选鉴定到8个高抗青枯病的烟草品种,获得了384 904个高质量的SNP位点,对烤烟群体的基因组连锁不平衡衰减（LD）距离进行了估算,平均为256 kb。在烟草基因组内发掘到1个与烟草青枯病抗性变异显著关联的区段,峰值SNP在17号染色体的23 977 382 bp处,p=6.196×10^-7,能解释14.29%的表型变异,在该区段内预测到4个抗病候选基因。本研究为烟草青枯病抗病基因克隆及分子育种提供了较为明确的基因组定位信息。     

基于blaCARB-17和toxR基因的副溶血弧菌双重PCR检测方法的建立

建立一种针对副溶血弧菌bla_CARB-17和toxR基因的双重PCR检测方法。按照副溶血弧菌的bla_CARB-17和toxR基因序列设计并合成引物,于同一反应体系中进行PCR扩增,优化退火温度、退火时间和引物比例,确定双重PCR的特异性和灵敏性,最后用实验室分离鉴定的副溶血弧菌分离株验证。结果表明:建立的双重PCR最佳退火条件是52℃ 30 s,引物比例1:1,在同一体系中特异地扩增出副溶血弧菌的bla_CARB-17和toxR基因的303、350 bp片段,其它对照菌株均无扩增,表明该方法特异性良好。双重PCR的最低检测限达1×102 CFU/mL,对实验室鉴定的56个副溶血弧菌分离株验证均为阳性。建立的双重PCR方法操作简单、高效快速、特异性强,适用于副溶血弧菌的检测。     

烟草青枯病抗性基因的遗传分析及RAPD标记

选用高感青枯病品种"红花大金元"与高抗青枯病品种"Ti448A"配制杂交组合,采用温室苗期叶片人工注射法接种鉴定两亲本及其杂交后代F₁、F₂、BC₁P₁代的青枯病抗性表现,结果表明抗病亲本材料Ti448A的抗病性由一对显性基因控制。依据混合群体分组分析法(BSA)建立抗青枯病基因池和感青枯病基因池,通过RAPD试验,从近200个随机引物中筛选出一个在两池间具有多态性的引物S503,用双亲、F₁、F₂单株DNA进行RAPD试验证明了该引物扩增出的特异性片段S503-750是一个与抗青枯病基因连锁的RAPD标记,利用JOINMAP (version 1.4)软件计算得此标记与抗青枯病基因间的重组率为5.984%,连锁距离为6.261 cM。      

Synergized bioresmethrin as a potential grain protectant

Results of grain protectant trials comparing 4 ppm bioresmethrin + 20 ppm piperonyl butoxide with 12 ppm malathion are presented. The trials utilised commercial welded-steel silos of 5 tonne capacity as commonly used for grain storage on farms and were undertaken during 1967–69 and 1973–74. Treated wheat, in 5-tonne lots, was sampled at monthly intervals and subjected to bioassays of 7, 14 and 28 days duration, F₁ and sometimes F₂ generation counts were also made.

The best overall treatment was 4 ppm bioresmethrin plus 20 ppm piperonyl butoxide. After 12 months, the treated grain controlled Rhyzopertha dominica, 2 susceptible and 1 resistant strain; Sitophilus granarius, 1 resistant strain; S. oryzae, 2 susceptible and 1 resistant strains: Oryzaephilus surinamensis; Plodia interpunctella and Ephestia cautella. A much lower efficiency was observed in the control of adult Tribolium confusum, however, there was complete suppression of the F₁ generation. Poor adult control in the 14-day bioassays contrasted with excellent control in the 28-day bioassay and the F₁ and F₂ bioassays of T. castaneum.

In Australia R. dominica resistant to organo-phosphorus insecticides poses a severe threat to the grain handling industry. Synergised bioresmethrin should be able to restore broad spectrum control, the probable exception being Tribolium species. Bioresmethrin is less toxic to mammals than pyrethrins for which there is an established tolerance of 3 ppm and 20 ppm for piperonyl butoxide on grain.     

普通烟草长叶柄突变性状的遗传分析

叶柄是烟草叶片的重要组成部分,烟草长叶柄性状遗传分析可用于研究烟草叶柄和叶片的发育。利用甲基磺酸乙酯（EMS）诱变普通烟草烤烟品种中烟100和红花大金元各获得一个（极）长叶柄突变体（M48和M50）,将两个突变体分别与各自的野生型中烟100和红花大金元及普通烟草烤烟品种革新三号杂交获得F₁,F₁自交获得F₂群体,F₁与其相应的野生型亲本回交获得BC₁F₁群体。对各类型材料进行表型调查和统计分析结果显示:F₁均表现为（极）长叶柄突变表型,在F₂群体和BC₁F₁群体中,经x2检验,（极）长叶柄突变表型与野生型表型的理论分离比均符合3:1和1:1,表明（极）长叶柄突变性状由染色体上一对显性单基因控制。上述结果为进一步定位和克隆（极）长叶柄突变基因,揭示其在烟草叶柄和叶片发育中的作用奠定基础。      

烤烟几个重要植物学性状的遗传分析

利用“主基因+多基因”混合遗传模型的6个世代联合分离分析方法, 分析烤烟组合丸叶×Coker319几个重要植物学性状的遗传效应。结果表明, 烤烟的株高、叶数、叶面积和鲜叶重受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制, 株高与叶数遗传以加性效应及显性×显性上位性效应为主, 叶面积和鲜叶重各遗传效应相差不多, 其上位性效应>加性效应>显性效应, F₂世代的主基因遗传率分别为57.53%、42.63%、30.32%和44.26%。移栽至中心花开放天数受2对加性-显性-上位性主基因+加性-显性多基因控制, 以加性×加性上位性效应、加性效应及显性×显性上位性效应为主, 主基因遗传率为64.79%。茎围和比叶重均受1对完全显性主基因+加性-显性多基因控制, 茎围遗传以多基因为主, 其多基因加性效应和显性效应大小相当, 比叶重遗传主基因、多基因的加性效应和显性效应大致相当, 主基因遗传率分别为2.48%和38.71%。叶形指数受1对加性-显性主基因+加性-显性-上位性多基因控制, 主基因加性效应与显性效应基本相当, 主基因遗传率为49.64%。叶长、叶宽、节距和蒴果重受加性-显性-上位性多基因控制, 多基因遗传率分别为60.75%、62.14%、75.08%和82.34%。     

戴瑞羊、小尾寒羊及其杂交后代乳成分比较分析

为培育我国自主知识产权的新品种奶绵羊,本研究对戴瑞羊(DM)、小尾寒羊(STH)、戴寒杂交F1(F1)和戴寒杂交F2(F2)乳中常规成分、脂肪酸、氨基酸、矿物质和维生素进行了比较分析.结果表明,F1乳的脂肪含量最高,其蛋白质、酪蛋白、固形物和非乳脂固形物以及必需氨基酸含量仅次于STH乳,显著高于DM和F2乳(P<0.0...     

Effects of gamma radiation on the progeny of irradiated Ephestia cautella (Walker) (Lepidoptera: Pyralidae) males

One-day-old P₁ males of Ephestia cautella, Baghdad strain, were treated with 0.15, 0.20 and 0.25 kGy of γ radiation and mated in groups (15 pairs) with untreated virgin females. Radiation effects were assayed by determining percentage of egg hatch, mating ability and F₁ sex ratio. Our results showed the greater the initial dose, the greater the reduction in the percent egg hatch, and the greater the male-to-female ratio. Radiation effects on F₁ males were greater than the effect on the P₁ males. Furthermore, the results showed that the effects on F₁ males were greater than the effects on F₁ females. These results were confirmed cytologically by examination of the developing meiotic germ cells of F₁ males which showed that the meiotic nuclei carried multiple translocations. Such chromosomal aberrations explain the sterility observed in the F₁ male generation.     

Efficacy of the insect growth regulators methoprene, fenoxycarb and diflubenzuron against Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) on maize and paddy rice

The activity of fresh deposits of methoprene, fenoxycarb and diflubenzuron against F₁ progeny of Rhyzopertha dominica on maize and paddy was compared with that on wheat, at two equilibrium relative humidities. There were differences between slopes of log concentration-probit (lc-p) lines for different compounds, and for the same compound on different grains. Judging by values of the IC_99.9, i.e. the concentration which inhibited progeny production by 99.9%, the order of activity against F₁ progeny on different grains was: methoprene, wheat and paddy > maize; fenoxycarb, wheat > paddy > maize; diflubenzuron, wheat and maize > paddy. Equilibrium relative humidity (e.r.h.) had no consistent effect on activity—at 90% e.r.h., the IC₅₀ of fenoxycarb on wheat was reduced and the IC₅₀ of diflubenzuron on maize was increased compared with 70% e.r.h., and other treatments were unaffected.

The efficacy of these compounds on maize and paddy against F₁ and F₂ progeny was evaluated during 48 weeks storage at 30°C, 70% r.h. The resolved S isomer of methoprene was also included. Slopes of lc-p lines were greater against the F₂ than against the F₁, particularly using diflubenzuron on paddy, with corresponding smaller values of the IC_99.9. Equally effective concentrations did not decline systematically over 48 weeks. Minimum effective application rates were judged as the concentrations that prevented living F₂ progeny in at least 2 of 3 replicates. Estimates for 48 weeks protection on maize were: methoprene, 2 mg kg⁻¹; S-methoprene, 1 mg kg⁻¹; fenoxycarb, 10 mg kg⁻¹; and diflubenzuron, 5 mg kg⁻¹. Corresponding estimates on paddy were 0.15 mg kg⁻¹, 0.05 mg kg⁻¹, 5 mg kg⁻¹, and 5 mg kg⁻¹.     

雪茄外包皮烟叶片重要性状的遗传分析

为研究雪茄外包皮烟叶重要性状的遗传规律,应用“主基因+多基因”混合遗传模型的4个世代联合分离分析方法,对雪茄烟品种Beinhart1000-1×鹤峰把把烟组合3个不同生育时期的叶面褶皱程度、叶片厚薄和主侧脉夹角3个叶片重要性状进行了分析。结果表明,控制3个性状的遗传模型和基因互作效应在各个时期存在着差异。叶面褶皱度的遗传率在团棵期和现蕾期分别为42.70%、57.71%,叶片厚薄在现蕾期和中心花开放期的遗传率分别为64.44%、50.62%,主侧脉夹角在现蕾期主基因遗传率达90.93%,在中心花开放期多基因遗传率为42.55%。可以看出,同一性状在不同生育时期的遗传效应存在差异,会因环境影响而表现出不同,所以雪茄烟品种培育过程中应注意基因效应与外界环境的相互作用。     

Control of Ephestia cautella (Walker) (Lepidoptera: Phycitidae) by synthetic sex pheromones in the laboratory and store

The reduction in mating of Ephestia cautella caused by permeation of the atmosphere with synthetic sex pheromone components was investigated in the laboratory and store. In the laboratory the three main components of the pheromone complex were tested either singly or in combination, and (Z, E)-9,12-tetradecadienyl acetate (ZETA) was found to be the most effective. Field trials were carried out in simulated cocoa stores with four different initial application rates of ZETA and two moth population densities. The concentration of airborne pheromone was measured, and the emergence of the F₁ generation was used to assess the degree of reproductive control. Decreased numbers of copulating pairs were observed in the stores treated with pheromone, and F₁ emergences were reduced by 91 to over 99% when population densities of moths of 0.1–0.3 moths/m² surface area were exposed to pheromone concentrations of 4–10 μg/m³. The results provide a basis for investigation of the effects of factors such as method of pheromone application and store conditions on the effectiveness of this control technique.     

Analysis of the role of the Listeria monocytogenes F0F1 -AtPase operon in the acid tolerance response

As little is known about the genes involved in the induction of an acid tolerance response in Listeria monocytogenes, the role of the F₀F₁-ATPase was analyzed as a consequence of its role in the acid tolerance of a number of other bacteria and its conserved nature. It was found that acid adapted cells treated with N,N′-dicyclohexylcarbodiimide (DCCD) exhibited greatly enhanced sensitivity to low pH stress. Degenerate primers were designed to amplify and sequence a portion of the atpD gene. Subsequently, a PCR product from atpA to atpD was identified. While we were unable to create a deletion in the atpA gene, the plasmid pORI19 was inserted in a region between atpA and atpG to reduce, rather than eliminate, expression of the downstream genes. As expected this mutant displayed enhanced resistance to neomycin and exhibited slower growth than the wild type strain. This mutant could still induce an acid tolerance response and remained susceptible to DCCD treatment, but its relative acid sensitivity was difficult to assess as a consequence of its slow growth.