615 resultados para T-RFLP
Resumo:
本文应用nifD-nifK IGS及16S-23S rDNAIGSPCR-RFLP方法,系统研究了分布于云南横断山的旱冬瓜以及吉林长白山的西伯利亚赤杨、色赤杨和东北赤杨根瘤内Frankia菌的遗传多样性。该研究可加深对Frankia菌起源与进化的认识,并为保护和利用这一固氮共生资源提供科学依据。从113株赤杨的根瘤样品中共检测到48种基因型的Frankia菌株。首次确切报道了自然条件下不同基因型Frankia菌可与同一宿主植株同时共生,并且这种混乱性在横断山区普遍存在。通过对Frankia菌群体遗传结构的分析,查明云南省横断山旱冬瓜Frankia菌群体内遗传变异水平由高到低的顺序是苍山群体、高黎贡山群体、鸡足山群体、来凤山群体和无量山群体。酶切带型9为云南横断山旱冬瓜Frankia菌群体中最古老的类型;大部分(75.63%)遗传变异发生在Frankia菌的各群体内,同一气候区的Frankia菌群体有较近的亲缘关系。旱冬瓜Fran辰a菌遗传多样性特点与横断山气候特征及冰川运动历史密切相关。宿主赤杨的进化地位不同,其共生Frankia菌的群体遗传多样性水平也不同。古老的旱冬瓜共生Fran舫“菌群体内遗传分化最大,分化最晚的东北赤杨共生Frankia菌群体内遗传变异最小;属同一生活型的西伯利亚赤杨和色赤杨共生Fronkia菌间存在着较近亲缘关系;早冬瓜Fran舫。菌群体可能为其它赤杨共生Fran舫“菌提供祖先,因此认为赤杨与其共生Frankia菌间存在着协同进化。
Resumo:
自养硝化过程在自然界氮素循环和污水处理系统脱氮过程中起着关键作用。因此,了解有机碳对硝化的影响和硝化菌与异养菌之间的竞争对微生物生态学和污水处理系统设计都很重要。目前对氨氧化到硝酸盐氮过程的研究文献很多,但对亚硝酸盐氧化过程在异养菌的存在下如何受到有机碳影响的研究甚少。本文从生理生化指标、基因组学、蛋白组学三方面考察了在实验室条件下有机碳(乙酸钠)对硝化细菌和异养菌组成的混合菌群的硝化性能、菌群结构及代谢功能的变化的影响。 全文分为两大部分: 第一部分为乙酸钠对游离态硝化混合菌群的硝化性能和菌群结构的短期影响。混合菌株先在自养条件下进行连续培养,两个月后硝化速率达到20 mg N/(L·d);而后离心收集菌体进行批式实验。在批式反应器中,初始亚硝氮均为126mg N/ L,乙酸钠-C 与亚硝酸盐-N 的比分别为0,0.44,0.88,4.41,8.82。结果表明:在低C/N 比(0.44 和0.88)时,亚硝酸盐去除速率比C/N=0 下高,细菌呈现一次生长;而在高C/N 比(4.41 和8.82)时,出现连续的硝化反硝化,亚硝酸盐去除率仍比对照下高,细菌呈现二次生长。不同C/N 比下微生物群落明显不同,优势菌群从自养和寡营养细菌体系(包括亚硝酸盐氧化菌,拟杆菌门,α-变形菌纲,浮霉菌门和绿色非硫细菌下的一些菌株)过渡到异养和反硝化菌体系 (γ-变形菌纲的菌株尤其是反硝化菌Pseudomonas stutzeri 和P. nitroreducens 占主导)。 第二部分为乙酸钠对硝化混合菌群生物膜的硝化性能和菌群结构的长期影响。接种富集的硝化混合菌群于装有组合式填料的三角瓶中,于摇床中自养培养;两个月后填料上形成生物膜的硝化速率达到20 mg N/ (L·d);而后进行长期实验,每12 小时更换混合营养培养基(亚硝氮约200 mg N/ L,C/N 比同上)。结果显示:相较于C/N 比=0 时的亚硝酸盐氧化反应来说,低C/N 比出现了部分的反硝化,而高C/N 比则是几乎完全的反硝化。与对照比,C/N=0.44 时亚硝酸盐氧化速率并未受乙酸钠的影响,反而上升了,但C/N=0.88 时亚硝酸盐氧化速率有所下降。菌群结构分析表明自养对照与混合营养下微生物群落的不同;PCR-DGGE未检测出混合营养下硝化杆菌的存在,而显示异养菌尤其是反硝化菌的大量存 在。荧光定量PCR 结果表明随C/N 比上升,硝化杆菌数量从2.42 × 104 下降到1.34× 103 16S rRNA gene copies/ ng DNA,反硝化菌由0 增加至2.51 × 104 nosZgene copies/ ng DNA。SDS-PAGE 的结果表明不同C/N 比下的蛋白组较为复杂且呈现一定的差异性。 有机碳对亚硝氮氧化及微生物群落的影响很复杂,本文分别讨论了对游离态和生物膜固定态两种状态的混合菌群相应的短期和长期影响研究。研究发现,有机碳并非一定带来硝化的负影响,如果控制在适当的C/N 比范围,有机碳是有利于亚硝氮氧化的。这些发现阐明了有机碳和硝化反硝化的关系,填补了硝化微生物生态学上的空白,对污水处理系统中减少异养菌的影响并提高氮去除率有一定理论指导意义。 Nitrification plays a key role in the biological removal of nitrogen in both nature and wastewater treatment plant (WWTP). So, understanding of the effect of organic carbon on nitrification and the competition between nitrifying bacteria and heterotrophic bacteria is important for both microbial ecology and WWTP design and operation. Despite the fact that the nitrification process of ammonia to nitrate has been extensively investigated, it is not known how the process of nitrite oxidization is affected by organic carbon when heterotrophic bacteria are present. By measuring different physiological and biochemical parameters, as well as using genomic DNA and proteome analysis, we investigated the influence of organic (acetate) on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria under laboratory conditions. The dissertation involves two parts: Part one deals with the effect of organic matter on functional performance and bacterial community shift of nitrite-oxidizing and heterotrophic bacteria under suspended state. The bacteria were prepared in a continuous-flow stirred reactor under autotrophic condition; after two months, the nitrification rate of the culture reached about 20 mg N/ (L·d); then the bacteria were harvested for the next batch experiments. The initial concentrations of nitrite were 126 ± 6 mg N/ L in all flasks, and sodium acetate (C) to nitrite (N) ratios were 0, 0.44, 0.88, 4.41, and 8.82, respectively. The results showed that at low C/N ratios (0.44 or 0.88), the nitrite removal rate was higher than that obtained under autotrophic condition and the bacteria had single growth phase, while at high C/N ratios (4.41 or 8.82), continuous aerobic nitrification and denitrification occurred besides higher nitrite removal rates, and the bacteria had double growth phases. The community structure of total bacteria strikingly varied with the different C/N ratios; the dominant populations shifted from autotrophic and oligotrophic bacteria (NOB, and some strains of Bacteroidetes, Alphaproteobacteria, Actinobacteria, and green nonsulfur bacteria) to heterotrophic and denitrifying bacteria (strains of Gammaproteobacteria, especially Pseudomonas stutzeri and P. nitroreducens). Part two describes the influence of acetate on nitrite oxidizing performance, community structure and metabolic function of nitrite-oxidizing and heterotrophic bacteria in biofilms. Bacterial enrichments was transferred into flasks with polypropylene carriers and cultured under agitated and autotrophic condition. After two month, the biofilms grown on the carriers had a nitrification rate of about 20 mg N/ (L·h); then the biofilms were refreshed with mixotrophic medium (nitrite were 200 mg N/ L in all flasks, and C/N ratios was the same as above) every 12 h. the results show: normal nitrite oxidization reactions were performed when C/N = 0, but nitrite oxidization and partial denitrification occurred with low C/N ratios (0.44 or 0.88). At high C/N ratios (4.41 or 8.82), we mainly observed denitrification. In contrast to C/N = 0, the nitrite oxidization rate was unaffected when C/N = 0.44, but decreased with C/N = 0.88. The structure of bacterial communities varied significantly between autotrophic and mixotrophic conditions. Nitrobacter was hard to detect by PCR-DGGE while heterotrophs and especially denitrifiers were in the majority under mixotrophic conditions. Real-time PCR indicated that the Nitrobacter population decreased from 2.42 × 104 to 1.34 × 103 16S rRNA gene copies/ ng DNA, while the quantity of denitrifiers obviously increased from 0 to 2.51×104 nosZ gene copies/ ng DNA with an increasing C/N ratio. SDS-PAGE indicated the complexity of and a certain difference between the proteome of nitrite-oxidizing and heterotrophic bacteria at different C/N ratios. We conclude that the influence of organic matter on nitrite oxidation and the community structure of NOB and heterotrophic bacteria is complex. In this dissertation, we focused on how sodium acetate influenced the system both under suspended state and in biofilms. We observed that acetate did not necessarily have a negative impact on nitrification. Instead, an appropriate amount of acetate benefited both nitrite oxidization and denitrification. These findings provide a greater understanding about the relationship between organics and nitrification; they fill the gaps in the field of microbial ecology of nitrifying bacteria; they also provide insight into how to minimize the negative impact of heterotrophic bacteria and maximize the benefit of nitrogen removal in biological treatment systems.
Resumo:
应用重离子加速器的 50MeV/u12C6+重离子对胡萝卜素生产菌-红酵母(Rhodotorula RYStrain )进行辐照处理,经酵母发酵实验,发现 50MeV/u12C6+重离子对胡萝卜素生产菌-红酵母具有诱变作用。初步筛选到了胡萝卜素产量有变化的辐照变异菌株,并对这些辐照变异菌株进行了 RFLP(限制性片段长度多态性)和 RAPD(随机扩增 DNA 多态性)分析,这些工作为工业上利用重离子对胡萝卜素生产菌进行诱变育种展现了新的前景。
Resumo:
从DNA/DNA杂交、RFLP分析、DNA的限制酶图谱和核苷酸序列分析、PCR技术、DNA指纹技术、RAPD分析等六个方面详细描述DNA分析技术在植物学研究中的应用 ,并讨论了DNA分析技术与植物系统学的关系。
Resumo:
生物标记物能在细胞或分子水平上指示暴露-效应关系,是进行污染土壤生态毒理诊断的主要技术手段之一。随着分子生物学技术的飞速发展,出现了一系列以聚合酶链式反应为基础的、在分子水平上检测污染物质导致的生物体DNA损伤的DNA指纹技术。DNA指纹技术的主要类型有:随机扩增多态性DNA(RAPD)、聚合酶链式反应-单链构象多态性(PCR-SSCP)、扩增片段长度多态性(AFLP)、任意引物聚合酶链式反应(AP-PCR)、差异显示反转录聚合酶链式反应(DDRT)、短DNA重复序列(SSR)及限制片段长度多态性(RFLP)等。这些技术与检测基因突变、染色体畸变和损伤为主的一系列经典研究方法如彗星分析、微核实验等相比具有简便、快速、灵敏等优点。本文着重介绍了随机扩增多态性DNA、聚合酶链式反应-单链构象多态性、扩增片段长度多态性3种重要的DNA指纹技术在污染土壤诊断中的应用。
Resumo:
土壤中的微生物多样性是十分丰富的,传统培养方法对土壤微生物多样性的研究有很大局限性。近年来,各种基于16S rDNA基因的指纹图谱分析技术取得了长足的进步,并广泛应用于土壤微生物多样性的研究。这些技术主要有变性梯度凝胶电泳(DGGE)/温度梯度凝胶电泳(TGGE)、单链构象多态性(SSCP)、随机引物扩增多态性DNA(RAPD)、限制性片段长度多态性(RFLP)和扩增核糖体DNA限制性分析(ARDRA)等。对这些技术近年来在土壤微生物多样性研究领域的应用予以简短综述,并初步探讨未来几年土壤微生物分子生态学发展的方向。
Resumo:
RAPD(随机放大多态性DNA)是1种新的DNA分子标记技术。与RFLp、AFLP及ARDRA相比,RAPD具有可在一次试验中同时观察到大量的DNA多态性片段,方法更具简单、敏感、花费少等优点。阐述了RAPD的原理方法,及目前在微生物分类鉴定研究中的应用,并分析了RAPD技术在共生固氮放线菌Frankia分类鉴定及系统发育研究中的应用前景。
Resumo:
采集长白山自然保护区北坡东北赤杨、西伯利亚赤杨、色赤杨根瘤样品 2 1个 ,对根瘤内FrankiaDNA的 16S -2 3SrDNA和nifD -nifK两个基因间隔区段 (IGS)进行PCR -RFLP分析 ,研究其基因多样性。结果表明 :与赤杨共生的Frankia菌存在丰富的基因多样性 ,基因类型与宿主种型关系密切。东北赤杨对Frankia的特异性较高 ,与西伯利亚赤杨和色赤杨共生的Frankia菌有较近的亲缘关系。上述结果说明Frankia菌与其宿主赤杨间存在着协同进化
Resumo:
Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.
Resumo:
从砷污染土壤中富集砷抗性细菌,在厌氧环境中进行培养,观察其对砷的还原能力。结果表明:在21h之内,As(V)就被完全还原为As(Ⅲ);培养72h后,培养基中出现黄色沉淀,采用X射线衍射分析(XRD)和扫描电镜-能谱分析(SEM-EDS)技术对沉淀进行分析表明,沉淀主要是以3种晶型存在的硫化砷(AsS);培养150h后,大约有65%的As以上述沉淀的方式从溶液中移除。此外,本文还采用了构建16SrDNA文库的方式对该体系中的微生物种群进行分析,利用RFLP技术对16SrDNA片段进行分型,共得到72个操作单元类型(OTU),其中6个OTU占了库容的51%,从这6个OTU中各选取1个克隆进行测序,结果表明,富集到的砷还原细菌属于喜热菌属(Caloramator)、梭菌属(Clostridium)和杆菌属(Bacillus)。
Resumo:
树木外生菌根真菌在森林生态系统中发挥着重要的作用,其多样性研究能反映外生菌根真菌的种群结构。随着分子生物学技术在多样性研究中的应用,打破了传统方法诸如大多数微生物处于不可培养状态的局限性,提高了人们对外生菌根真菌群落结构的认识。本文主要介绍了几种外生菌根真菌多样性研究常用的方法,综述了它们在菌根研究中的应用状况,为森林生态系统生物多样性研究提供参考。
Resumo:
Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.
Resumo:
Genetic differentiation of the shrimp Penaeus chinensis in the Yellow Sea and Bohai Sea was investigated using the mitochondrial control region (CR). RFLP of a partial CR segment (613 bp) shows that 106 out of 122 (86.9%) individuals from six sampling localities along the coast of northern China and the west coast of the Korean Peninsula share the same haplotype, and the haplotype frequencies among localities are not significantly different. The findings are further confirmed by sequencing the complete CR. Divergence of the complete CR (992 bp) is less than 1.6% in 14 individuals from the six localities. F-statistics based on RFLP data and the TCS network of sequencing data suggest little genetic differentiation of P. chinensis in the Yellow Sea and Bohai Sea. Mismatch analysis suggests a rapid expansion of P. chinensis population to the Yellow Sea and the Bohai Sea, which probably occurred with the rapid rise in sea level after the last glacial maximum. Despite the lack of genetic heterogeneity, we propose that P. chinensis populations in this region should be treated as separate management units, as fishery management programs have to be applied on a local basis by different governments.
Resumo:
本文利用不同的分子标记方法,分别对牙鲆及大黄鱼不同养殖群体的生长、抗病等经济性状的候选基因进行了序列多态性研究,检测到了几个SNP位点和微卫星的多态性位点,并分析了它们与经济性状之间的相关性;同时,利用微卫星的多态性位点对牙鲆2个养殖群体的遗传变异进行了分析,这些均为海水鱼类遗传育种及标记辅助选育工作提供了基础数据。 在牙鲆胶南养殖群体中,以100个个体为实验材料,根据其生长激素(GH)基因的6个外显子序列设计引物,通过SSCP分析技术显示该群体GH基因的第4外显子存在多态性,检测到2种基因型,AA型和AB型。DNA测序结果表明,AB型在第1763位发生碱基突变,c→t,与AA型同源性达到99%。连锁分析结果表明:这2种基因型的个体在体重和头长上表现出显著的差异,AB型个体的体重和头长都明显大于AA型个体(P<0.05),由此推测等位基因B是一个对牙鲆体重和头长都有利的等位基因;这2种基因型个体之间在其体型性状上也存在显著差异(P<0.05);同时,该多态位点的Hardy-Weinberg平衡性检验结果表明,该群体处于Hardy-Weinberg平衡状态。在牙鲆GH基因第1外显子区域还发现了一个微卫星位点,对该位点进行多态性分析,检测到5种基因型、3种等位基因,one-way ANOVA统计结果显示,基因型AC个体的体重、头长和体高明显大于其它基因型个体(P<0.05),C是一个对体重、头长和体高有利的等位基因。 对2个大黄鱼养殖群体的GH基因进行SSCP分析后发现,浙江群体大黄鱼GH基因在第196位存在1个SNP(g→a)位点,检测到2种基因型,AA和AB。t检验结果表明,AA型个体的体高比AB型个体的高(P≤0.05),但AB型个体在体长/体高上占优势(P≤0.05),提示该突变位点可以作为大黄鱼体型性状的候选标记。福建群体大黄鱼GH基因在第692位有1个SNP位点(t→c),共检测到2种基因型,CC型和CD型,其中,CD基因型个体的体重和全长显著大于CC基因型个体(P≤0.01),提示该位点可以作为大黄鱼体重和头长性状的候选标记。 在牙鲆胶南和日照2个养殖群体中,采用牙鲆GHR基因5’端Promoter区的一个微卫星标记,进行了群体遗传变异的研究,并探索了该基因多态性位点与牙鲆生长性状之间的相关性。结果表明,2个群体在该位点检测到的等位基因数为12和9个,有效等位基因数为6.26和5.04个。两个群体该位点的Hardy-Weinberg遗传偏离指数均为正值,并没有显示出杂合子缺失,但各基因型分布频率都在一定程度上偏离Hardy-Weinberg平衡(P<0.01)。连锁分析发现,在胶南群体中,IM基因型对应的个体在全重、全长、体长、头长、体高和眼径形态学数据中均是最大的,但仅在体重上极其显著的大于全部其它基因型个体;在日照群体中,BC基因型对应的个体在全重、全长、体高、尾柄高、尾柄长和眼径数据中均是最大的;而CJ基因型对应的个体在体长和头长这两组数据中是最大的。由此认为,该位点IM基因型可以作为牙鲆体重性状的潜在标记。 在进行牙鲆抗病性状标记的筛选时,利用迟缓爱德华氏菌(Edwardsiella tarda)LSE40对牙鲆鱼进行攻毒感染实验,得到死亡群体和未死亡群体。选择Toll样受体基因中的TLR2、TLR3和TLR9基因作为候选基因,分别对这3个基因中的部分序列共设计7对引物进行扩增,同时对扩增产物进行RFLP多态性分析,目前只在TLR3基因内检测到一个EcoRI的酶切多态性位点,测序后发现,这是由于在TLR3基因第3806位的EcoRI酶切位点在某些个体中缺失所致。酶切产物共呈现出3种基因型,分别定义为AA,AB和BB。χ2检验证明该多态性位点与牙鲆抗迟缓爱德华氏菌LSE40的能力有一定关系。利用多因素非条件Logistic回归分析对死亡组和存活组牙鲆的各种形态学数据以及不同基因型之间进行了分析,发现体长、头长和体高均具有显著的相关性(P<0.05),而这几个因素与体重的相关性不显著(P>0.05)。多因素非条件Logistic分析后发现:AA基因型对死亡率具有显著的影响(P<0.05),是主要的危险因素,而AB基因型的作用不显著(P>0.05);头长是主要的保护因素(P<0.05),体重对死亡率的影响很小。χ2检验证明,等位基因A是对死亡的主要危险等位基因,B是对存活有利的主要等位基因。推测该位点可以作为牙鲆抗迟缓爱德华氏菌的潜在标记。
Resumo:
栉孔扇贝(Chlamys farreri)是我国北方地区主要的养殖贝类之一,曾为沿海各省带来巨大的经济效益。但自1997年以来,陆续爆发的病害问题给扇贝养殖业造成了巨大的经济损失,严重影响了该产业的健康发展。目前认为培育抗病性强的扇贝优良品种是解决病害问题的根本途径。由于传统的育种方法费时费力,无法满足对良种的迫切需求,因此有必要通过分子手段加快抗病品种的培育步伐。标记辅助育种(marker assisted selection,MAS)是成功应用于动物育种中的分子手段之一,但由于缺乏与抗病性状相关的标记,MAS目前还无法在软体动物中得到应用。因此,寻找与抗病性状相关的分子标记是在软体动物中发展MAS的关键。 本研究利用鳗弧菌(Listonella anguillarum)对栉孔扇贝进行攻毒感染实验,初步得到敏感群体和抗病群体后采用PCR、PCR-RFLP、Bi-PASA PCR等方法研究了CfLysG、CfC1qDC和CfLITAF基因多态性及其与栉孔扇贝对鳗弧菌抗性的关系。 研究发现,栉孔扇贝CfLysG的基因序列中共有104个单核苷酸多态性(SNP)位点和29个插入/缺失(I/D)多态性位点。有17个多态性位点位于启动子区域,选择其中的-753 I/D、-391A/G和-284I/D多态性进行检测,发现这三个位点的基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡(P>0.05)。其中-753 ID基因型和-284 ID基因在抗病群体中的频率高于在敏感群体中的频率,但两者之间无显著性差异(P>0.05)。-391 AG基因型在抗病群体中的频率显著高于敏感群体(P=0.007),表明-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。为验证这一相关性,对-391位点不同基因型的扇贝进行攻毒感染实验。统计发现,具有-391 AA基因型的扇贝累计死亡率显著高于具有-391 AG基因型的扇贝(P=0.001),进一步证实了CfLysG基因-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因的外显子共有3处SNP,其中仅第三外显子上的+3473 A/C为非同义突变。统计分析表明,+3473位点不同基因型在敏感群体中的分布频率符合Hardy-Weinberg平衡(P>0.05),而在抗病群体中则偏离Hardy-Weinberg平衡(P<0.01)。+3473 AA基因型在抗病群体中的频率显著高于在敏感群体中的频率(P=0.022),表明+3473 AA基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因第1内含子存在+96 I/D和+487 I/D两处大片段的I/D多态性。统计发现,这两个位点的基因型在敏感群体和抗病群体中的分布频率均符合Hardy-Weinberg 平衡(P>0.05)。其中+96 DD基因型和+487 ID基因型在抗病群体中的频率均略高于在敏感群体中的频率,但两者之间无显著性差异(P>0.05)。表明这两个位点的多态性与栉孔扇贝对鳗弧菌的抗性无显著相关性。对CfLysG基因各多态性位点的统计分析表明,各位点之间存在不同程度的连锁不平衡,提示有单体型的存在。对19种频率>1%的单体型在敏感群体及抗病群体中的频率进行分析,发现-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型在抗病群体中的频率显著高于敏感群体(P=0.044),表明该单体型与栉孔扇贝对鳗弧菌的抗性显著相关。 在栉孔扇贝CfC1qDC基因cDNA序列上共发现14处SNP。对+423 T/C多态性与栉孔扇贝对鳗弧菌抗性的关系进行了分析。统计发现,+423位点各基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡(P>0.05)。+423 TT基因型在抗病群体中的频率显著高于在敏感群体中的频率(P=0.005),表明+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关。 在栉孔扇贝CfLITAF基因cDNA序列中共发现3处SNP及1处I/D多态性。对+145 I/D多态性进行研究,发现所有敏感个体及抗病个体中均同时存在+145 位点所有等位基因,表明+145位点多态性与栉孔扇贝对鳗弧菌的抗性不相关。 以上研究表明,栉孔扇贝CfLysG基因-391 AG基因型、+3473 AA基因型、-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型以及CfC1qDC基因+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关,提示它们可作为与栉孔扇贝抗病相关的候选分子标记应用于贝类抗病育种中,为贝类的标记辅助育种提供参考。此外,抗病相关分子标记的发现还有利于加深对扇贝发病机理的理解,并有助于发掘预防及治疗贝类疾病的新方法。