基于侯选基因标记的四倍体马铃薯休眠QTL关联分析

休眠期是马铃薯(SolanumtuberosumL.)重要的块茎性状之一,寻找调控马铃薯块茎休眠的关键基因,揭示其分子机制以选育具有适宜休眠期长度的马铃薯品种,对于解决当前马铃薯产业中过长或过短休眠期带来的经济损失和食品安全隐患等问题十分关键。前期研究在二倍体马铃薯连锁群体中定位了6个加性休眠QTL,本研究拟在四倍体马铃薯育种材料中验证这些休眠QTL。基于休眠QTL连锁的候选基因标记,采用混合线性模型(MLM),模型中考虑群体结构和亲缘关系(Q+K),在四倍体马铃薯自然群体St-hzau中对马铃薯块茎休眠期进行了关联分析。5号染色体上休眠QTL DorB5.3连锁的候选基因标记S199_300和GWD (根据葡聚糖水双激酶α-glucan water dikinase基因设计)与马铃薯块茎休眠期具有显著的关联(P0.05),分别解释了休眠期表型变异的7.8%和3.2%,分别能增加休眠期7.1 d和4.5 d,即在二倍体马铃薯连锁群体中定位的稳定主效休眠QTL DorB5.3在四倍体马铃薯关联群体St-hzau中也表现显著, DorB5.3的稳定性在关联分析结果中得到了验证,表明候选基因标记策略在马铃薯块茎休眠QTL关联分析中是一种有效的策略。本研究所验证的主效休眠QTL DorB5.3及相应连锁标记可以直接用于马铃薯休眠育种。据此可以推测GWD可能在控制还原糖含量和块茎休眠2个方面均发挥作用,马铃薯块茎休眠机制与还原糖含量变化机制可能存在着部分交叉。     

甜樱桃花芽不同发育时期内参基因的筛选与验证

为了筛选甜樱桃花芽不同发育时期均稳定表达的内参基因,以甜樱桃桑提娜和黔樱一号不同发育时期花芽为材料,通过qRT-PCR技术检测28S rRNA、EF1-a1、EF1-a2、UBC、RPL13、18S rRNA、RSP3、CYP40、ACT2和α-TUB3等10个常用看家基因的表达水平,并利用GeNorm、NormFinder和BestKeeper综合评价其表达稳定性。结果表明,EF-1a2和RSP3在所有样品中稳定性最好。分别以EF-1a2、RSP3及EF-1a2+RSP3作为内参基因检测不同发育时期花芽生长素运输载体AUX1基因及生长素响应因子ARF基因的表达模式,该2个基因在不同内参基因标定下表达模式相同。表明EF-1a2、RSP3及EF-1a2+RSP3可作为甜樱桃花芽不同发育时期的内参基因。     

甘肃、青海地区小麦条锈菌监测及群体遗传多样性分析

【目的】了解甘肃和青海小麦条锈菌（Puccinia striiformis f. sp. tritici）春季流行传播路线、群体遗传多样性和生殖模式,明确春季流行期两省小麦条锈菌的传播关系及菌源交流规律,进而为两省小麦条锈病的预测预报、确定越夏初始菌源来源和有效治理提供理论依据。【方法】选择条锈病常发生的地区作为调查和研究区域。甘肃省4个试验点：陇南市文县、陇东平凉市崆峒区、中部麦区定西市临洮县、临夏州临夏县;青海省2个试验点：西宁市城北区、海东市互助县。2017年秋季,在甘肃和青海省6个试验点内根据当地小麦播种适期依次种植82份变异观察圃材料。2018年4—8月,对试验点82份变异观察圃材料进行田间病害调查,并采集到551份小麦条锈菌标样,使用15对引物进行SSR分子标记分析。利用GenAlEx和POPPR v2.5.0软件对数据进行相关分析, 不显著的rbarD值表示连锁平衡,用于推断群体是否发生有性重组。【结果】82份变异观察圃材料在甘肃地区发病比青海地区严重。15对引物组合共扩增出81个位点,每对引物组合产生的多态性位点为2—12个。551份样本克隆矫正后,共鉴定出505个多位点基因型（MLG）,其中仅有32个MLG被克隆并进行了2—6次重新采样。甘肃和青海群体总的基因型多样性（G=0.917）较高,其中,甘肃平凉群体的最高,青海互助群体次之,甘肃临洮群体最低。小麦条锈菌的遗传变异主要在各群体内部个体之间。春季流行期,菌源在各群体之间交流频繁,青海东部（互助和西宁）群体与甘肃（平凉和临夏）群体之间的基因流高于青海（互助和西宁）群体与甘肃文县群体之间的基因流。最小时空网络图（MSN）和非参数主成分分析（DAPC）表明青海互助和西宁的群体与来自于甘肃平凉和临夏的群体之间菌源关系最密切,差异最小;与临洮群体遗传距离相对较远且临洮群体相对独立;文县群体则是一个完全独立的群体,与其他5个群体之间的差异最大。连锁不平衡分析表明,甘肃文县、临夏和青海西宁群体存在不显著的rbarD值表示连锁平衡,是有性生殖群体,其中文县群体（rbarD=0.0139,P=0.186）显示出明显的有性重组特征。【结论】小麦条锈病春季流行期,甘肃地区与青海东部地区的传播路线以甘肃平凉、临夏到青海的传播为主,甘肃文县到青海的传播为辅。甘肃文县、临夏和青海西宁3个群体存在有性生殖现象,对甘肃、青海地区条锈菌丰富的遗传多样性的形成具有一定作用。     

黄瓜叶酸合成关键基因克隆与分析

【目的】分析黄瓜基因组中与叶酸合成代谢相关的基因数量、定位以及表达特征,对关键酶基因进行生物信息学分析与克隆,旨在为黄瓜叶酸合成调控研究奠定基础。【方法】根据已报道的拟南芥叶酸合成相关基因,利用黄瓜基因组数据库中9930_V3版本进行BLAST比对。利用MapChart绘制黄瓜染色体物理图谱并对基因定位。利用qRT-PCR分析这些基因在黄瓜果实发育不同时期和不同材料中的表达量。通过MEGA、WebLOGO、ExPASy等工具对关键酶基因进行生物信息学分析。通过PCR扩增对关键酶基因进行克隆,并测序分析基因的序列差异。【结果】同源比对获得19个黄瓜叶酸代谢相关基因,这些基因不均匀分布在黄瓜7条染色体上,且以Chr.4和Chr.5上分布最多。通过对其中11个调控叶酸合成的基因在测序黄瓜9930果实发育不同时期以及果实叶酸含量高低差异显著的2份材料的表达量分析,发现CsFPGS、CsHPPK/CsDHPS和CsDHNA 3个基因与果实叶酸含量变化趋势完全一致;CsADCS、CsADCL、CsDHNA、CsHPPK/CsDHFS、CsFPGS、CsDHFS等基因的表达量在2份材料中具有显著差异。通过对2个调控叶酸合成限速步骤的关键酶基因CsGCHI和CsADCS的蛋白序列及蛋白结构域分析,发现各物种中CsGCHI的同源基因均具有2个GTP_cyclohydroI结构域;CsADCS的同源基因均具有2个GATase结构域、1个Anth_synt_I_N结构域和1个Chorismate_bind结构域。它们在不同物种中高度保守,进化树分析亲缘关系近的物种聚类到一起。分别扩增黄瓜果实低叶酸含量自交系65G和高叶酸含量自交系02245中CsGCHI和CsADCS的同源基因,序列分析表明CsaV3_1G041250全长为3 012 bp,CDS序列长度为1 413 bp,3个SNP位点的突变导致了氨基酸序列的变异;CsaV3_7G026240全长为3 047 bp,CDS长度1 407 bp,序列无变异;CsaV3_5G036360全长7 941 bp,CDS序列长度为2 706 bp,序列无变异。【结论】鉴定出19个不均匀分布在7条染色体上的黄瓜叶酸代谢相关基因,基因CsFPGS、CsHPPK/CsDHPS 、CsDHNA和CsADCS是影响黄瓜果实叶酸含量变化、导致叶酸含量高低显著差异的关键基因,调控叶酸合成限速步骤的关键酶基因GCHI及ADCS功能相对保守,CsGCHI在65G、02245中有3个SNP位点的突变导致了氨基酸序列的差异。     

人溶菌酶密码子优化及其在牛乳腺细胞中高效表达

【目的】分析牛乳腺中7种主要乳蛋白基因密码子使用偏好性,筛选高频密码子和低频密码子,依据其对人溶菌酶基因进行密码子局部优化和全局优化,通过牛乳腺上皮细胞等多种细胞对优化效果进行分析评价,为提高重组人溶菌酶表达量,开发新型、高效、安全的重组人溶菌酶提供理论依据。【方法】利用CodonW和EMBOSS等软件对7种主要牛乳蛋白和人溶菌酶基因密码子偏好进行生物信息学分析,筛选牛乳蛋白基因高频、低频密码子并根据牛乳蛋白基因密码子使用偏好对人溶菌酶基因翻译起始区前22位密码子（LYZop22）和全局密码子(LYZop)分别进行优化。构建人溶菌酶-荧光素酶融合表达载体（pGL3-LYZcw/op22/op）和人溶菌酶过表达载体（pcDNA-LYZcw/op22/op）,将上述载体分别转染牛乳腺上皮细胞(BMEC)、牛成纤维细胞（BFFC）和C127小鼠乳腺上皮细胞等3种细胞,通过荧光素酶、实时荧光定量PCR及Western-blot等方法检测密码子优化对溶菌酶表达的影响。【结果】牛乳蛋白基因密码子偏好以GC结尾,GC3s平均含量为0.537±0.062,而人溶菌酶GC3s含量为0.407,偏好以AT结尾;聚类结果表明,牛乳中酪蛋白与乳清蛋白类基因在密码子使用偏好性上也存在一定差异。依据筛选获得的牛主要乳蛋白基因5个高频密码子（RSCU>1.5）和7个低频密码子（RSCU<0.5）对人溶菌酶基因密码子进行优化并转染多种细胞进行表达效果分析。荧光素酶分析发现,相比野生型溶菌酶密码子（LYZcw）,翻译起始区密码子优化类型LYZop22分别在BMEC、BFFC细胞中提高1.48倍（P<0.01）和1.30倍（P>0.05）;而全局密码子优化类型LYZop分别在BMEC、BFFC中提高2.2倍（P<0.01）和2.44倍（P<0.01）,说明密码子优化能明显提高人溶菌酶在多种细胞中表达量。实时荧光定量PCR结果表明,LYZop22相比LYZcw在BMEC和BFFC细胞中分别提高了2.08倍（P<0.05）和1.5倍（P>0.05）,而LYZop则分别提高了22倍（P<0.01）和17.8倍（P<0.01）,mRNA表达水平与密码子优化后的mRNA二级结构稳定性呈正相关。Western-blot结果也进一步表明,密码子优化后的LYZop22和LYZop能明显提高重组人溶菌酶在牛乳腺上皮细胞中的表达量。上述结果表明,根据牛乳腺中主要乳蛋白基因密码子使用偏好进行密码子优化,能显著提高人溶菌酶在牛乳腺上皮细胞及成纤维细胞中的表达量,且溶菌酶基因全局密码子优化效果优于翻译起始区密码子优化效果。【结论】通过生物信息学分析获得了牛乳蛋白基因使密码子使用偏好及高低频密码子;依据牛乳蛋白密码子使用偏好性对人溶菌酶密码子优化能显著提高重组人溶菌酶mRNA水平和蛋白水平的表达量,为今后利用生物反应器高效生产重组人溶菌酶奠定基础。     

无机碳源对零价铁介导的自养脱氮序批式反应器处理高氨氮废水的影响

无机碳源作为自养微生物的能量来源,是影响自养脱氮细菌富集的重要因素。文章通过添加不同量的KHCO3作为ZVI介导的自养脱氮体系中无机碳源,研究反应体系的脱氮效果影响趋势以及适宜的无机碳源添加量。结果如下:1)KHCO3作为无机碳源可以显著提高NH^+4-N去除率,KHCO3添加量越多,其NH^+4-N去除率越高。KHCO3添加量分别为2 g,1 g,0.5 g的SBR反应器R2,R1,R0.5的NH^+4-N去除率分别为98.39%,65.18%,44.56%,相较不添加KHCO3的R0分别提高86.5%,53.29%,32.67%。ZVI的添加会降低KHCO3对NH^+4-N氧化的促进作用。2)KHCO3可明显提高TIN去除效果,促进总氮脱除的高低顺序是R1>R0.5>R2,SBR反应器R2,R1,R0.5的平均TIN去除率分别为19.01%,32.04%,27.62%,相较于空白组R0分别提高了10.60%,23.63%,19.21%。3)KHCO3可中和硝化反应产生的H^+,对反应体系具有缓冲作用,可使微生物处于较适宜的酸碱环境中,更有利于反应器的稳定运行。4)适量的无机碳源KHCO3可以促进氨氧化细菌和厌氧氨氧化细菌的活性。该研究探讨了无机碳源在零价铁脱氮体系中的影响趋势,为铁型脱氮技术提供了理论支撑。     

The effects of various plant protection methods on the development of Zymoseptoria tritici and Cephalosporium gramineum,grain yield and protein profile

Septoria leaf blotch progresses rapidly, leading to the development of Zymoseptoria titici forms resistant to fungicides. Cephalosporium stripe is caused by Cephalosporium gramineum. The aim of this study was to evaluate the effectiveness of selected pesticides in limiting the symptoms of both diseases on winter wheat leaves, and to determine their influence on grain yield and the content and composition of protein fractions in wheat kernels. Propiconazoles were most effective in inhibiting the development of Septoria leaf blotch (symptoms were reduced from 54.7% to 78.6%). Strobilurins were less effective due to the presence of isolates with the G143A mutation. Symptoms of Cephalosporium stripe were rarely observed, and protective treatments did not reduce their severity. The highest content of grain protein (14.81%) was found in plants most intensely protected with the fungicides containing fenpropimorph, pyraclostrobin and epoxiconazole. The principal component analysis revealed that the plant protection method influenced the grain protein profile. The accumulation of HMW glutenins and α/β gliadins was mutually interrelated and higher in high-input treatments; control grain was characterized by close relationships between ω-gliadins, LMW glutenins, albumins and globulins, whereas low-input treatments influenced mostly γ-gliadins.     

Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.     

番茄匍柄霉叶斑病拮抗细菌的筛选与鉴定

应用平板对峙法从连作多年的番茄根际土样中筛选得到对番茄匍柄霉叶斑病具有较强拮抗活性的细菌菌株ZF161，该菌株对番茄匍柄霉的平板抑制率达到70.51%。离体叶片试验显示，菌株ZF161对番茄匍柄霉叶斑病防治效果达到69.83%。通过菌落形态观察、生理生化特性、Biolog测定和多基因系统发育树综合分析，鉴定菌株ZF161为枯草芽孢杆菌（Bacillus subtilis）。番茄盆栽试验结果表明，菌株ZF161对番茄匍柄霉叶斑病的防治效果达到63.27%。进一步平板对峙抑菌谱试验结果显示，菌株ZF161对其他7种病原真菌也具有较好的抑制效果。上述结果说明，菌株ZF161对番茄匍柄霉叶斑病具有良好的防治效果，且对多种病原真菌也具有抑制作用，具有开发应用的潜力。     

Soluble Klotho protein suppresses THP-1-derived foam cell formation

AIMTo investigate the role of soluble Klotho protein in THP-1-derived foam cell formation. METHODSTHP-1 monocytes were induced into macrophages by treatment with 160 nmol/L phorbol myristate acetate for 48 h, and then were divided into 6 groups: negative control group (THP-1-derived macrophages), positive control group [THP-1-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL) for 48 h], and 25, 50, 100 and 200 μg/L soluble Klotho protein groups (THP-1-derived macrophages pretreated with soluble Klotho protein at the indicat?ed concentraions for 2 h and then induced by ox-LDL for 48 h). Lipid droplets in cytoplasm were observed by oil red O staining. The cholesterol outflow rate was detected by scintillation counting technique. The content of intracellular total cholesterol, free cholesterol and cholesterol ester was detected by enzyme fluorescence analysis. The expression of acyl-coenzyme A:cholesterol acyltransferase 1 （ACAT1） and ATP-binding cassette transport?er A1 (ABCA1) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTSOil red O staining and lipid mass quantification showed that THP-1-derived foam cell formation was dose-dependently suppressed by soluble Klotho protein. The cholesterol efflux rate of THP-1-derived foam cells was increased by soluble Klotho protein in a dose-dependent manner (P<0.05). In addition, soluble Klotho protein decreased the expression of ACAT1 and increased the expression of ABCA1 in a dose-dependent manner (P<0.05). CONCLUSION The soluble Klotho protein inhibits THP-1-derived foam cell formation in a dose-dependent manner by down-regulating the expression of ACAT1 and up-regulating the expression of ABCA1.