大豆GmTTG1-like基因的克隆与分析及植物表达载体构建

在大豆(Glycine max L.Merr.)中克隆了一个1008bp的表皮毛相关基因GmTTG1-like(Glycine max Transparent Testa Glabra 1-like);生物信息学分析表明,GmTTG1-like蛋白在结构上具有4个WD40重复(WD40-repeat)结构域,与烟草中NtTTG2(Nicotiana tabacum Testa Glabra 2)具有较高同源性。推测GmTTG1-like与大豆表皮毛的生物学功能有关,并可能作用于大豆发育和防卫反应过程。采用RTPCR和q-RT-PCR两种方法检测GmTTG1-like在植物根、茎、叶、花和种子中的表达。构建GmTTG1-like植物表达载体并获得可以侵染的农杆菌阳性克隆。

The BEL1-like transcription factor GhBLH5-A05 participates in cotton response to drought stress

Drought stress impairs crop growth and development.BEL1-like family transcription factors may be involved in plant response to drought stress,but little is known of the molecular mechanism by which these proteins regulate plant response and defense to drought stress.Here we show that the BEL1-like transcription factor GhBLH5-A05 functions in cotton(Gossypium hirsutum)response and defense to drought stress.Expression of GhBLH5-A05 in cotton was induced by drought stress.Overexpression of GhBLH5-A05 in both Arabidopsis and cotton increased drought tolerance,whereas silencing GhBLH5-A05 in cotton resulted in elevated sensitivity to drought stress.GhBLH5-A05 binds to cis elements in the promoters of GhRD20-A09 and GhDREB2C-D05 to activate the expression of these genes.GhBLH5-A05 interacted with the KNOX transcription factor GhKNAT6-A03.Co-expression of GhBLH5-A05 and GhKNAT6-A03 increased the transcription of GhRD20-A09 and GhDREB2C-D05.We conclude that GhBLH5-A05 acts as a regulatory factor with GhKNAT6-A03 functioning in cotton response to drought stress by activating the expression of the drought-responsive genes GhRD20-A09 and GhDREB2C-D05.

Location-based prediction model for Crohn’s disease regarding a novel serological marker,anti-chitinase 3-like 1 autoantibodies

BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.

Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase the production of reactive oxygen species(ROS),thereby promoting anoikis.Recently,we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs)from H 2 O 2-induced cell apoptosis by inducing autophagy and reducing ROS production.However,the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.AIM To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.METHODS Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA(shRNA)adenovirus transfection.Integrin(ITGs)were tested by flow cytometry.Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA,respe-ctively.The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays.The levels of the anoikis-related proteins ITGα5,ITGβ1,and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I,Beclin1 and p62 were detected by Western blotting.RESULTS In isolated mBMSCs,Mst1 expression was upregulated,and Mst1 inhibition significantly reduced cell apoptosis,induced autophagy and decreased ROS levels.Mechanistically,we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4,ITGαv,or ITGβ3 expression.Moreover,autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.CONCLUSION Mst1 inhibition ameliorated autophagy formation,increased ITGα5β1 expression,and decreased the excessive production of ROS,thereby reducing cell apoptosis in isolated mBMSCs.Based on these results,Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.

PKHD1L1 blocks the malignant behavior of lung adenocarcinoma cells and restricts tumor growth by regulating CBX7

Objective:To explore the role of polycystic kidney and hepatic disease 1-like 1(PKHD1L1)in lung adenocarcinoma(LUAD).Methods:Bioinformatics tools were utilized to examine the clinical profile of PKHD1L1 and chromobox protein homolog 7(CBX7)in LUAD.The Cell Counting Kit-8,colony formation,terminal deoxynucleotidyl transferase dUTP nick end labeling,Transwell,and wound-healing assays were carried out to assess the proliferative,apoptotic,invasive,and migrative capacities of the cells.Furthermore,the interrelation between PKHD1L1 and CBX7 was validated using a co-immunoprecipitation assay.A LUAD mice model was constructed by subcutaneous injection of A549 cells.Finally,immunohistochemical staining was performed to evaluate CBX7 and Ki67 expression.Results:PKHD1L1 was downregulated in LUAD and predicted dismal outcomes in patients with LUAD.PKHD1L1 upregulation repressed the proliferative,invasive,and migrative capabilities of A549 cells and exacerbated the apoptotic rate.Additionally,PKHD1L1 may bind to CBX7 and positively modulate CBX7 expression.CBX7 deletion partly abrogated the effects of PKHD1L1 upregulation on the cellular biological activities in A549 cells.Furthermore,the PKHD1L1/CBX7 axis regulates the Hippo signaling pathway in A549 cells.PKHD1L1 restricted tumor growth in LUAD xenograft mice;this was partly abolished by CBX7 knockdown.Conclusion:PKHD1L1 can hinder LUAD progression by regulating CBX7-mediated Hippo signaling.

梅花PmSOC1-like基因的克隆与表达分析

SOC1/TM3(Suppressor of overexpression of constans 1/Tomato MADS-box gene 3)是MADS-box家族一员,它能够整合多条开花途径的开花信号,调节植物由营养生长向生殖生长的转变。为了解梅花成花转变过程的分子机理,采用RT-PCR的方法从梅花长蕊绿萼中克隆到3个SOC1的同源基因,分别命名为Pm SOC1-1、Pm SOC1-2和PmSOC1-3,并采用荧光定量PCR对3个基因的表达模式进行了分析。序列分析表明,3个基因的编码区长度分别为645 bp(Pm SOC1-1)、654 bp(Pm SOC1-2)和660 bp(Pm SOC1-3);编码的氨基酸长度分别为214,217,219 aa。系统进化结果显示,Pm SOC1-1、Pm SOC1-2和Pm SOC1-3分别与拟南芥SOC1/TM3亚家族中的SOC1、AGL42/71/72和AGL14/19聚为一组。实时荧光定量分析结果表明,3个Pm SOC1-like基因均在茎、叶等营养器官中表达量较高,在花、果实和种子等生殖器官中表达量较低;在梅花花芽分化过程中,3个Pm SOC1-like基因的表达量整体呈下降趋势,推测PmSOC1-like基因可能在诱导梅花由营养生长向生殖生长转变过程中起重要作用。

菜蛾盘绒茧蜂多分DNA病毒EP-1-like基因克隆、原核表达与多克隆抗体制备方法

本实验提取被菜蛾盘绒茧蜂寄生后24h的小菜蛾幼虫体内总RNA,反转录合成cDNA,以此为模板PCR扩增出菜蛾盘绒茧蜂多分DNA病毒(Cotesia vestalis polydnavirus,CvBV)EP-1-like基因,将其分别克隆到表达载体pET30a、pET28a中,转化宿主菌BL21(DE3),获得单克隆重组质粒pET-30a-EP1L和pET-28a-EP1L。经IPTG诱导,pET-28a-EP1L成功表达了约34.8kDa的包涵体蛋白。将诱导条件优化以后,采用割胶回收的方法纯化包涵体,将纯化后的表达蛋白免疫新西兰大耳兔制备多克隆抗体。ELISA分析表明制备的抗体效价达1:128000,Western blot检测证明抗体具有良好的免疫反应特异性。

萼脊兰SeAP1-like基因的克隆与表达载体构建

根据NCBI网站上萼脊兰AP1-like全长基因序列设计引物,采用RT-PCR法从萼脊兰花瓣中扩增Se AP1-like基因,对扩增产物进行克隆和测序。结果表明,获得的萼脊兰Se AP1-like基因为743 bp,编码247个氨基酸。将Se AP1-like基因插入p CAMBIA1304载体中,经PCR、双酶切鉴定,证实重组表达质粒中含有目的片段,表明成功构建了高效植物表达载体p CAMBIA1304-Se AP1。对Se AP1-like基因的结构域和所表达蛋白质的亲水性进行了分析,并预测了该基因所表达蛋白的二维结构。结果显示,该基因包含MADS-box和K-box保守域,属于MADS-box基因家族,该蛋白分子属于亲水性蛋白。二级结构分析表明,其包含57.49%的α螺旋、6.07%的延伸链、36.44%的不规则折叠。

松墨天牛dynamin-1-like protein基因的鉴定及表达分析

为了探讨GTP酶超基因家族中dynamin-1-like protein基因在昆虫中的表达特性,以松墨天牛为研究对象,从已构建的cDNA文库中筛选到松墨天牛dynamin-1-like protein基因,命名为Ma DLP1(Gen Bank:KU245763)。该序列长为2 133 bp,编码710个氨基酸。由此预测的蛋白质二级结构主要由α螺旋与无规则卷曲组成,其次是β片层与β转角;Ma DLP1基因编码蛋白质,定位于细胞核。通过DNAMAN软件比对发现Ma DLP1与赤拟谷盗的DLP1同源性最高,为82%,且存在3个保守酶域;用Clustal X和MEGA4.0构建系统发育树,显示松墨天牛与赤拟谷盗处在同一分支。RT-qPCR分析结果显示,Ma DLP1在各虫态不间断表达,幼虫期在5龄幼虫中表达量最高,化蛹期间表达量先上升后下降,羽化期间表达量表现为先上升后下降,并在初羽化的成虫中表达量达到最大值;Ma DLP1在幼虫和成虫头部表达量较高,且在幼虫脂肪体中表达最高;成虫的足、翅、触角和卵巢中也都有表达。说明Ma DLP1的表达与松墨天牛的完全变态发育相关。

梅花花器官发育相关基因PmSOC1-like的功能初探

SOC1/TM3(SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/Tomato MADS-box gene 3)是MADS-box家族一员,它能够整合多条开花途径的开花信号,调节植物由营养生长向生殖生长的转变。在从梅花长蕊绿萼品种中克隆到3个SOC1同源基因PmSOC1-1、PmSOC1-2和PmSOC1-3的基础上,构建由CaMV35S启动子驱动的植物表达载体,并在拟南芥中过量表达,以研究其功能。通过对转基因植株表型观察发现,3个PmSOC1-like基因都具有使野生型拟南芥提前开花的作用。其中,PmSOC1-2作用最强,PmSOC1-1居中,PmSOC1-3作用最弱。对转基因拟南芥内源成花基因的qRT-PCR检测说明,PmSOC1-like基因主要是通过上调其下游花分生组织特性基因(AGL24、LFY、AP1和FUL)来促进植物开花;此外,PmSOC1-1和PmSOC1-2还导致了花瓣细丝状、花萼叶片化、花瓣和花萼宿存等花器官形态的改变,说明它们可能还具有影响花器官发育的功能。研究结果将有助于阐明梅花由营养生长向生殖生长转变的分子机制。

Niemann-Pick C1-Like 1表达缺陷缓解肝X受体激动剂诱导的小鼠肝脏脂肪性变

目的探讨Niemann-Pick C1-Like 1在肝X受体激动剂诱导的肝脏脂肪性变中的作用。方法给C57BL/6小鼠和Niemann-Pick C1-Like 1基因敲除小鼠喂食0.015%胆固醇饮食21天,胃饲溶剂或肝X受体激动剂T0901317一周后,收集小鼠肝脏,称重;抽提肝脏脂质并用酶法检测脂质含量;用实时定量聚合酶链反应法检测肝脏表达与脂肪合成相关基因固醇调节元件结合蛋白1c、脂肪酸合成酶和硬脂酰CoA去饱和酶1的mRNA水平。结果在胃饲T0901317一周后,C57BL/6小鼠肝脏由1.1±0.1g增大到2.8±0.3g,肝脏甘油三酯含量由34.2±18.1mg/g增至232.2±67.9mg/g,固醇调节元件结合蛋白1c、脂肪酸合成酶和硬脂酰CoA去饱和酶1mRNA水平在肝脏的表达也显著增高;而Niemann-Pick C1-Like 1基因敲除小鼠肝脏由0.9±0.1g增大到1.5±0.1g,肝脏甘油三酯含量由43.7±26.5mg/g增至104.9±62.1mg/g,脂肪酸合成酶和硬脂酰CoA去饱和酶1mRNA水平在肝脏的表达也显著增高。但在T0901317诱导下,Niemann-Pick C1-Like 1基因敲除小鼠肝脏脂肪酸合成酶mRNA水平仍比C57BL/6小鼠低63%。结论Niemann-Pick C1-Like 1表达缺陷降低肝X受体激动剂诱导的肝脏固醇调节元件结合蛋白1c和脂肪酸合成酶的表达,缓解小鼠肝脏脂肪性变。

池蝶蚌组织蛋白酶L1-like基因的克隆及表达特征分析

为了解淡水贝类是否存在组织蛋白酶L的亚型及其亚型的免疫相关作用,本实验利用已构建的池蝶蚌血细胞全长c DNA文库,筛选获得与之同源的EST序列,结合RACE技术进一步克隆了池蝶蚌一个新的组织蛋白酶L基因的c DNA全长,命名为Hs Cts L1-like基因(Gen Bank登录号为KF015273)。该序列全长为1280 bp,5′-非翻译区(5′UTR)为31 bp,3′-非翻译区(3′UTR)为256 bp,开放阅读框区(ORF)为993 bp,编码330个氨基酸,预测蛋白相对分子量为36.86 ku,理论等电点为6.23。序列分析结果显示,Hs Cts L1-like与其他软体动物相对应序列具有共同结构特征,包含信号肽、前肽抑制域和成熟肽三部分,在其他物种中已鉴定的Cts L签名序列标签(ERF/WNIN、GNFD、GCXGG和QCHN等)在Hs Cts L1-like中均可找到。其氨基酸序列同缢蛏Cts L1(AGL33704.1)同源性最高,达67%;与报道的三角帆蚌Cts L(ADV03094)和池蝶蚌中另一个Cts L(AEX88474)仅均为52.91%;系统进化分析表明,Hs Cts L1-like与缢蛏、长牡蛎和合浦珠母贝的Cts L1聚为一分支,推测Hs Cts L1-like属于Cts L家族中的亚型1。实时荧光定量PCR(q RT-PCR)检测显示,Hs Cts L1-like m RNA在肝脏中表达量最高,其次是卵巢和精巢。注射鳗弧菌后,血细胞和肝脏Hs Cts L1-like m RNA转录水平显著升高,暗示其是一个免疫有关的基因,参与了池蝶蚌的先天免疫应答反应。

ORM1-like3基因单核苷酸多态性与客家人群哮喘发病的关系

目的 探讨ORM1-like 3基因的单核苷酸多态性（SNPs）与客家人群哮喘发病的相关性.方法 选取确诊的客家人群哮喘患者194例（哮喘组）和同期健康查体者200例（对照组）为研究对象,利用MassARRAY-IPLEX技术和基质辅助激光解吸电离飞行时间质谱平台对ORM1-like 3基因的6个SNPs位点进行基因分型.结果 哮喘组与对照组的性别（χ^2＝0.013,P=0.909）和年龄（t=0.728,P=0.259）差异均无统计学意义.与对照组相比,哮喘组嗜酸性粒细胞计数（Z=-5.670,P=0.000）和IgE（Z=-9.158,P=0.000）明显升高,CRP水平（Z=-1.716,P=0.086）差异无统计学意义,吸入性过敏原（χ^2＝55.452,P=0.000）和食物过敏原（χ^2＝12.853,P=0.000）检出率明显升高.单纯哮喘组与哮喘合并过敏性鼻炎组比较,除rs3859192差异有统计学意义（χ^2＝6.659,P=0.036）外,其余5个位点差异均无统计学意义（P〉0.05）.总的基因型分型成功率为97.34%.与对照组相比,哮喘组rs7216389的等位基因分布（χ^2=7.103,P=0.008）和基因型分布（χ^2=7.695,P=0.021）差异均有统计学意义,但进行Bonferroni校正后基因型分布差异无统计学意义（Pc=0.126）.与对照组比较,哮喘组rs12603332、rs8069176和rs3859192的等位基因分布（χ^2=2.119,P=0.145;χ^2=2.167,P=0.141;χ^2=0.026,P=0.871）和基因型分布（χ^2=2.197,P=0.333;χ^2=5.686,P=0.058;χ^2=5.052,P=0.080）差异均无统计学意义.与对照组比较,哮喘组rs2305480和rs4795400的等位基因分布（χ^2=14.722,P=0.000;χ^2=10.417,P=0.001）和基因型分布（χ^2=12.351,P=0.002;χ^2=12.271,P=0.002）差异均有统计学意义,进行Bonferroni校正后两组间差异仍均有统计学意义（Pc=0.012;Pc=0.012）.与CC基因型相比,rs2305480的CT、TT基因型显著增加哮喘发生的危险性（P=0.033,P=0.003）,OR值（95% CI）分别为1.763（1.044～2.978）和5.681（1.600～20.171）,CT＋TT基因型增加哮喘的发生,OR值（95% CI）为2.130（1.306～3.475）.与TT基因型相比,rs4795400的CT基因型不增加哮喘发生的危险性（P=0.059）,而CC基因型显著增加哮喘发生的危险性（P=0.002）,OR值（95% CI）为4.440（1.600～12.316）.在82例初次确诊的哮喘患者中,与CC基因型相比,rs2305480的CT＋TT基因型的FVC占预计值百分比和FEV1占预计值百分比下降明显（均P〈0.05）.与TT＋CT基因型相比,rs4795400的CC基因型的FVC占预计值百分比和FEV1占预计值百分比下降亦明显（均P〈0.05）;rs2305480的基因型分布在初诊哮喘的病情严重程度不同分级之间差异无统计学意义（χ^2=2.130,P=0.144）,rs4795400差异有统计学意义（χ^2=9.522,P=0.002）.结论 ORM1-like 3基因的rs2305480和rs4795400位点与客家人群哮喘的发病有关,它们的基因型分布可能影响初诊哮喘患者的肺功能,而且rs4795400可能与病情严重程度有关.

油茶转录因子基因CoSOC1-like的克隆和表达分析

[目的]克隆油茶的SOC1同源基因(CoSOC1-like),分析其序列特征和表达模式及其蛋白进化。[方法]以3年生油茶嫩叶为材料提取RNA,利用RT-PCR和RACE方法克隆油茶的CoSOC1-like基因,用生物信息学工具分析其序列特征,用荧光定量PCR分析其表达模式,用基因瞬时表达法分析其蛋白的亚细胞定位。[结果]油茶CoSOC1-like基因的CDS全长为654 bp,推测蛋白质由127个氨基酸组成,蛋白分子量为24.958 kD,等电点(pI)为6.8,基因库的登录号为MT036382。CoSOC1-like具有植物Ⅱ型MADSbox基因的蛋白结构,且C末端含有MOTIF结构域,是一个MADS-box家族转录因子。CoSOC1-like蛋白有31个磷酸化位点,建立在二级结构基础上的CoSOC1-like蛋白的三级结构具有明显活性部位;CoSOC1-like基因瞬时表达分析表明,油茶CoSOC1-like蛋白定位于细胞核,符合转录因子的细胞核定位特征。系统进化分析表明:油茶CoSOC1-like与茶树SOC1-like蛋白聚类在同一个进化支上。荧光定量PCR分析表明:CoSOC1-like基因存在于油茶所有的器官中,尤其在花芽中的相对表达量最多。[结论]CoSOC1-like基因在油茶的花芽分化中起重要作用,也可能参与根、茎、叶和种子等器官的生长发育。本结果为进一步研究油茶成花的分子机制奠定了基础。

Chitinase 3-like 1基因-329 G/A多态性与冠心病相关性的研究

目的:探讨Chitinase 3-like 1基因-329 G/A多态性(rs10399931)与中国汉族人群冠心病的相关性。方法:收集189例冠心病患者的临床资料,应用连接酶检测反应(LDR)分析rs10399931各基因型,并与230例非冠心病者(对照组)进行比较。冠心病组按照发病年龄分为早发冠心病组(男性<55岁,女性<65岁)与非早发冠心病组,按照冠状动脉狭窄≥50%的支数分为1、2、3支血管病变亚组进行分析。结果:冠心病组与对照组rs10399931均存在CC、CT和TT 3种基因型;冠心病组与对照组CC、CT和TT基因型频率分别为39.7%、46.0%、16.3%及43.0%、43.9%、13.0%,C、T等位基因频率分别为62.7%、37.3%和65.0%、35.0%,两组间各基因型及等位基因频率差异无统计学意义(均P>0.05)。早发冠心病组、非早发冠心病组及对照组间rs10399931各基因型及等位基因频率差异无统计学意义(均P>0.05)。1、2、3支冠状动脉病变亚组间rs10399931各基因型分布频率差异无统计学意义(P>0.05)。结论:Chitinase 3-like 1基因-329 G/A多态性与汉族冠心病发病无相关性。

Variation of Cyclin B1-like Protein During the Cell Cycle of Physarum polycephalum

Physarum polycephalum L., a naturally synchronized myxomycophyta, was demonstrated to contain a cyclin B1-like protein by Western blot and immunoelectron microscopy. The content and subcellular location of the protein varied during the cell cycle. The cyclin B1-like protein was first detected in the plasmodia of S phase while it did not appear in the nuclei until late G2 phase. The content of the protein in both the plasmodia and nuclei rose gradually onwards, peaked at metaphase and disappeared abruptly at ana-telophase. The protein was found to be distributed in both the cytoplasm and nuclei in late G2 phase and metaphase. In nuclei, the protein was mainly located in the chromosomal and nucleolar areas. The results suggest that the cyclin B1-like protein of P. polycephalum begins to be synthesized at S phase, enters the nuclei at late G2 phase, accumulates in both cytoplasm and nuclei onwards and breaks down at ana-telophase. The results also suggest that the cyclin B1-like protein acts as a cytoplasmic-nuclear protein during certain phases of the cell cycle.

小麦NPR1-like基因的克隆及赤霉菌诱导下的表达分析

AtNPR1是拟南芥系统获得性抗病反应中的关键基因,对拟南芥的广谱抗性起重要调控作用。从赤霉菌诱导的小麦抗、感赤霉病近等基因系RNA差异表达谱中获得3个与AtNPR1类似的EST片段,据此检索相应序列信息并设计引物,从小麦中克隆得到3个cDNA全长序列,分别命名为TaNPR1、TaNPR2和TaNPR3,其开放阅读框分别编码580、607和601个氨基酸残基。序列分析表明,这3个小麦NPR1-like蛋白都含有保守的BTB/POZ、ANK和NPR1_like_C结构域及功能氨基酸,但仅TaNPR1具有2个对NPR1寡聚体形成十分必要的保守半胱氨酸残基。蛋白质聚类分析表明,TaNPR1与TaNPR2和TaNPR3的同源性均较低,其中TaNPR1与NPR1蛋白聚为一类,而TaNPR2和TaNPR3均与NPR1同源蛋白聚为一类。实时荧光定量PCR分析结果显示,TaNPR1、TaNPR2和TaNPR3基因都可被植物抗病相关信号分子水杨酸和茉莉酸甲酯诱导。与感病材料Apogee相比,抗病近等基因系Apogee73S2中TaNPR1和TaNPR3能够更早地响应赤霉菌的诱导并显著上调表达;而TaNPR2在感、抗材料中对赤霉菌侵染的响应都较为缓慢且变化不明显。这些结果表明,TaNPR1和TaNPR3可能在小麦对赤霉菌的防御反应中起重要作用。

谷子Hd1-like基因的克隆及其与农艺性状的关联分析

为揭示谷子光周期敏感性分子机制和拓宽谷子育种选择范围,对光周期途径关键基因进行研究。选择11个谷子品种鉴定了抽穗日数、株高、穗粗、穗粒质量、码粒数、千粒质量等9个农艺性状,利用同源克隆法克隆了这些谷子品种CO(CONSTANS)基因的同源基因Hd1-like基因,初步进行了基因突变位点与表型性状的关联分析。结果表明:多数性状在11个谷子品种间存在显著差异,表现出丰富的表型多样性;抽穗日数、株高与多个穗部性状存在显著或极显著正相关,反映了这2个性状对谷子产量潜能存在重要影响;Hd1-like基因编码区内共检测到43个突变位点,全部为SNP,主要为A/G、T/C替换,连锁不平衡分析发现,有2组最大的连锁不平衡结构,一组包括位点71、1023、1117、1406、1875、2073,另一组包括位点134、185、921、1131、1187、2098;对11个谷子品种Hd1-like基因推定的蛋白质序列分析发现,该蛋白质N端具有明显的锌指结构,C端具有明显的CCT域,Hd1蛋白在11个谷子品种间存在15处氨基酸替换突变,其中第44个氨基酸酪氨酸和半胱氨酸之间替换、第334个氨基酸精氨酸和甘氨酸之间替换,分别位于锌指区域和CCT域内,而位于锌指区的氨基酸替换可能导致了郑州12对光周期敏感度减弱;关联分析检测出12个与表型性状关联的位点,其中7个位点与千粒质量关联,1个位点与抽穗日数关联,2个位点与株高关联,2个位点与穗粗关联,说明该基因可能是一个多效基因。

玉米BEL1-like基因家族的鉴定、表达和调控分析

BEL1-like(BELL)家族蛋白是植物中普遍存在的一类具有同源异型结构域的转录因子。拟南芥中BELL家族蛋白能与KNOTTED1-like蛋白互作形成异源二聚体,并结合到特异顺式作用元件来调控基因的表达,从而影响植物生长发育进程。本文采用隐马可夫(HMM)模型,在玉米基因组中鉴定到15个BELL家族基因(Zm BELL),分布于7条玉米染色体。通过与拟南芥BELL基因的序列比较将这些基因分为两大类。在玉米8种组织中Zm BELL有不同的表达模式,具有明显的组织表达特异性。基于基因共表达分析及BELL-like蛋白特异结合的顺式元件分析,预测到86个可能受Zm BELL调控的下游靶标基因。这86个基因和12个Zm BELL表达模式相同,并且在基因启动子区存在与BEL1-like蛋白结合的顺式元件。这些结果为进一步解析玉米BELL家族基因的功能和作用机制积累了有价值的资料。