Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

矮秆大豆突变体叶片和豆荚生理特性的初步分析

经NaN3诱变获得大豆矮秆突变体HK808,对其豆荚和叶片生理特性及其与原品种"东农42"的比较研究表明:HK808叶片的叶绿素含量比野生型低,但叶绿素a/b比值变化不大;豆荚的叶绿素含量较其对应叶片的含量低,且野生型的含量略高于突变体。开花结荚后突变体叶片蛋白质含量、SOD活性逐渐升高,脯氨酸含量下降,且蛋白质和脯氨酸含量都低于东农42;突变体的SOD活性高于东农42;野生型的MDA含量在鼓粒期以后增高,即结荚后期突变体叶片防御自由基伤害能力强。豆荚的蛋白质、脯氨酸含量都与叶片的变化规律基本相同,即前者不断增加,后者不断下降,且豆荚蛋白质和脯氨酸含量都低于叶片;HK808豆荚的蛋白质含量比其野生型高。豆荚的MDA含量比叶片低,且东农42豆荚的MDA含量略高于HK808,豆荚的SOD活性呈下降趋势,但比东农42叶片的活性高,表明豆荚的抗氧化能力比东农42的叶片强。     

蝴蝶兰PhaSEP3基因的克隆及其在突变体中的表达

E类MADS-box是花器官发育分子模型中必不可少的基因,通过突变体研究其表达模式将为深入理解兰科植物花器官的分子机理与完善花发育调控理论提供依据。采用RACE(rapid-amplification of cDNA ends)技术从蝴蝶兰花瓣中克隆了一个E类MADS-box基因PhaSEP3(GenBank登录号为MZ436812),并采用实时荧光定量PCR(qRT-PCR)分析了该基因在蝴蝶兰不同组织和5种突变体中的表达水平。结果表明,该基因cDNA全长为1 236 bp,具有753 bp的开放阅读框(ORF),可编码250个氨基酸,其C端具有SEPⅠ和SEPⅡ基序。系统进化分析显示,该基因编码蛋白质与蝴蝶兰属的PeSEP3和AGL9亲缘关系最近。组织特异表达分析表明,PhaSEP3基因主要在生殖器官和授粉后子房中表达;在不同突变体中,PhaSEP3基因在侧萼唇瓣化突变体的萼片和唇瓣中表达水平显著升高;在退化雄蕊瓣化突变体的侧瓣、唇瓣和子房中表达水平显著降低,在蕊柱中的表达水平显著升高;在侧瓣唇瓣化突变体的侧瓣和唇瓣中表达水平显著升高;在侧瓣退化突变体的侧瓣中表达水平显著降低,而在蕊柱和子房中表达水平显著升高;在侧瓣雄化突变体中,该基因的表达水平在萼片和侧瓣中均显著升高。分析认为,PhaSEP3基因主要调控蝴蝶兰花器官各轮组织与授粉后子房的发育,在突变体花器官中,PhaSEP3类基因可能与其他花发育基因互作参与花器官形态的发育调控。该研究结果为进一步理解兰科植物花器官多样性的调控机理提供了资料。     

黄绿叶突变体冀麦5265yg的光合生理特性分析

【目的】叶色突变体是研究叶绿素合成、叶绿体发育和光合作用的理想材料,探索小麦黄绿叶突变体的光合生理特性,旨在阐明其光合作用调控机理,为小麦黄绿叶突变体的进一步利用奠定基础。【方法】以野生型冀麦5265和突变体冀麦5265yg为试验材料,对叶色表型进行观察,采用分光光度计和试剂盒法测定色素含量和酶活性,并利用Li-6400便携式光合仪和PAM100叶绿素荧光仪进行光合气体交换参数和叶绿素荧光参数测定。【结果】表型观察和色素含量结果表明,突变体苗期叶片表现为黄绿色,抽穗后叶片逐渐转变为淡绿色。遮阴处理可以使叶片颜色部分复绿,但比野生型略浅,属于光诱导转绿型突变体。突变体叶绿素a和叶绿素b含量显著低于野生型,叶绿素a/b的比值升高,为典型的叶绿素缺乏型突变体;光响应曲线和CO₂响应曲线显示,突变体的表观量子效率（AQY）、光饱和点（LSP）、最大净光合速率（P_n-max）、光补偿点（LCP）、暗呼吸速率（Rd）、羧化效率（CE）和饱和CO₂浓度（I_-sat）显著高于野生型,说明突变体叶片的光合机构稳定,强光下光合速率更高;光合气体交换参数和叶绿素荧光动力学参数表明,突变体的净光合速率（P_n）、蒸腾速率（T_r）、气孔导度（G_s）、光化学量子效率（F_v/F_m）、实际光化学效率（ΦPSII）和光化学淬灭系数（qP）显著高于野生型,说明其具有较强的光能转化和CO₂固定能力;突变体的超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性均高于野生型,丙二醛（MDA）含量下降,可溶性糖和可溶性蛋白含量升高,说明抗氧化酶系统通过清除氧自由基降低了氧化损伤,突变体叶片细胞膜损害减轻,抗逆性增强;突变体的核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）活性显著低于野生型,磷酸烯醇式丙酮酸羧化酶（PEPC）活性显著高于野生型,推测C₄ 途径光合酶PEPC活性的升高可能是突变体具有较高净光合速率的原因之一。花后遮阴以及外源喷施抗坏血酸AsA和二硫苏糖醇DTT处理表明,突变体对光强变化更敏感、叶片内AsA含量及叶黄素循环效率更高。【结论】黄绿叶突变体冀麦5265yg叶片气孔导度明显改善、热耗散降低、C₄途径光合酶活性升高,是其光合速率提高的主要原因。该结果为小麦叶色突变体高光合特性的分子调控机制研究奠定基础。     

The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses especially to the duck industry. However, little is known regarding the molecular basis of its pathogenesis. The ability to acquire iron under low-iron conditions is related to the virulence of a variety of bacterial pathogens. In this study, a sip (Riean_1281) deletion mutant CH3Δsip was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Results showed that siderophore-interacting protein (SIP) was involved in iron utilization and the sip deletion significantly reduced biofilm formation and adherence to and invasion of Vero cells. In addition, the sip gene was absent in 1 of 24 (4.17%) virulent strains and 2 of 3 (66.7%) avirulent strains of R. anatipestifer, and the sip gene from six R. anatipestifer strains, which belong to serotypes 1, 2, and 10, respectively, shared 100% amino acid identities to those of R. anatipestifer strains DSM15868 and RA-GD. These results suggested that siderophore-mediated iron acquisition may be an important iron-uptake pathway in R. anatipestifer. Animal experiments indicated that the median lethal dose of the CH3Δsip mutant in ducklings was about 35-fold higher than that of the wild-type CH3 strain. Thus, our results demonstrated that R. anatipestifer SIP was involved in iron acquisition and necessary for its optimal virulence.     

大白菜突变体库的构建及M2叶片表型变异的研究

 利用不同浓度甲基磺酸乙酯（EMS）诱变大白菜自交系‘A03’种子,根据M₁代植株发芽率、成活率、育性及M₂代成活率,筛选出适宜的EMS诱变浓度为0.4%。采取EMS种子诱变1次、EMS种子诱变2次及EMS花蕾诱变结合小孢子培养的方法构建了含有4 253个家系及相应自交M₂代种子的大白菜突变体库。在该突变体库M₂群体中分别对苗期6 404株幼苗及莲座期7 756个单株的叶片性状进行了形态学调查,总的表型变异频率高达37.62%和26.33%。     

Sensitive detection of the c‐KIT c.1430G>T mutation by mutant‐specific polymerase chain reaction in feline mast cell tumours

Here, we describe the establishment of mutant‐specific polymerase chain reaction (PCR) for detection of a c‐KIT c.1430G>T mutation in feline mast cell tumours. Several mutations in feline c‐KIT have been identified, with the c.1430G>T mutation accounting for a significant portion of feline mast cell tumour mutations. The c.1430G>T mutation in c‐KIT exon 9 was detected in 15.7% (11 of 70) of samples by mutant‐specific PCR but in only 7.1% (5 of 70) by PCR–restriction fragment length polymorphism (RFLP) in the genomic DNA isolated from 70 formalin‐fixed paraffin‐embedded sections or cells collected by fine needle aspiration. Mutant‐specific PCR showed remarkably higher detection rate than did PCR–RFLP. DNA sequence analysis did not always yield identical results to those of mutant‐specific PCR, suggesting heterogeneity of tumour cells. Mutant‐specific PCR is a valid and efficient screening tool for detection of the c‐KIT c.1430G>T point mutation in feline mast cell tumours compared with PCR–RFLP and sequencing analysis.     

Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley

The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC₂F₄ families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC₂F₄ families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high β-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent.     

水稻半矮杆突变体‘双辐矮糯’的农艺性状与遗传分析

倒伏是影响水稻（Oryza sativa L.）生产的重要因素,半矮杆是协调高产和抗倒性的理想资源。前期通过辐射诱变‘湘辐糯1号’获得了半矮杆突变体‘双辐矮糯’。本研究分析了‘双辐矮糯’的茎节配置特点、基本农艺性状及其遗传规律。结果表明：‘双辐矮糯’株高较‘湘辐糯1号’（133.13 cm）降低了24.88 cm,穗、第1节和第2节长度显著缩短,降幅分别达到18.10%、23.06%和19.13%,而第3、4、5节长度缩短程度不明显,表明其株高突变基因主要控制穗和高位茎节的伸长。‘双辐矮糯’的单株有效穗为10.4个,是‘湘辐糯1号’（5.2个）的2.04倍,其强分蘖能力确保了在每穗总粒数下降20.24%和千粒重下降7.51%情况下单株产量仍表现出25.26%的增长。品质分析发现,‘双辐矮糯’的粒长、粒宽、长宽比等外观品质指标及直链淀粉含量、胶稠度等食味品质指标与‘湘辐糯1号’无显著差异,说明‘双辐矮糯’株高的下降未对外观品质与食味品质产生不利影响。遗传分析发现,48对SSR标记在‘双辐矮糯’与‘湘辐糯1号’间扩增出了完全一致的基因型,二者遗传背景相似度达到了100.00%,证实了‘双辐矮糯’是源于‘湘辐糯1号’的真实变异。利用‘双辐矮糯’与‘湘辐糯1号’构建了2个正反交F₂群体,分析发现群体中半矮杆植株与野生型植株理论分离比符合1∶3,表明半矮杆性状受一个隐性细胞核基因控制。本研究明确了‘双辐矮糯’的株高表型和遗传特点,为深入挖掘其育种价值奠定基础。