绵羊Dlx3基因启动子活性及其多态性与羊毛品质性状的关联

【目的】通过开展绵羊Dlx3基因启动子结构、活性、多态性及其与羊毛品质性状的关联等分析,揭示Dlx3基因在绵羊毛囊发育中的作用及其作用机制。【方法】采用PCR扩增Dlx3基因起始密码子上游1.5 kb区域,利用荧光素酶报告基因技术分析Dlx3基因启动子活性,采用测序方法寻找Dlx3基因启动子区SNP,并利用PCR-RFLP技术进行SNP分型。【结果】①Dlx3基因启动子近端序列的保守性较高,该区域内人、鼠及绵羊都具有23个保守的转录因子结合位点和一个CpG岛,而启动子的远端序列的保守性较低;②Dlx3基因的启动子在绵羊胚胎成纤维细胞中具有启动子活性;③Dlx3基因启动子的-1 551—-1 108 bp与-1 108—-707 bp区域对启动子活性影响较大;④Dlx3基因启动子区SNP位点（G-1166A）多态性与羊毛卷曲度显著相关。【结论】①Dlx3基因启动子在绵羊胚胎成纤维细胞中有活性;②Dlx3基因启动子的近端序列组成在人、鼠和绵羊中较为保守,而其远端序列的保守性较低,但是Dlx3基因启动子的远端序列对启动子活性影响较大;③Dlx3基因启动子区G-1166A位点是羊毛卷曲度的一个分子标记。     

猪Dlx5基因多态性及其与猪体尺和胸椎数的关系

为探讨猪远端缺失基因5(distal-less homeobox 5,Dlx5)的多态性、群体分布特性及其与体尺和胸椎数的关系,采用PCR-RFLP方法检测了猪Dlx5基因g.394C>T位点在6个中外猪品种中的多态性分布,并分析了该位点与猪体尺和胸椎数的关系。检测结果表明,经HhaⅠ酶切后在6个猪品种群体均发现了CC、CT和TT 3种基因型,其中C等位基因在莱芜黑猪中为优势等位基因,而T等位基因在里岔黑猪、鲁莱黑猪、杜洛克猪、大约克猪和长白猪中占优势。在该多态性位点,莱芜黑猪、里岔黑猪、鲁莱黑猪、杜洛克和长白猪群体均处于哈代—温伯格平衡(P>0.05),而大约克猪群体偏离平衡状态(P<0.05);基因型在群体间的分布差异极显著(P<0.01)。群体遗传特性分析表明,有效等位基因数在1.254～1.991之间;杜洛克猪的多态信息含量为0.182,属于低度多态,其余品种均在0.254～0.374之间,属于中度多态。关联分析结果表明,该多态性位点对莱芜黑猪体长、体高、屠前活重、腹围、胸围、腿臀围和胸椎数以及里岔黑猪胸椎数的影响均不显著(P>0.05)。Dlx5基因g.394C>T位点在猪群中存在多态,基因型分布在莱芜黑猪与其他猪种间不同,与猪体尺和胸椎数无显著关联。     

m6A甲基化酶相关基因在牛骨骼肌生成中的表达

【目的】近年来,RNA m⁶A甲基化修饰在肌肉发育中的作用不断被发现,通过探究m⁶A甲基化酶相关基因,包括METTL3,METTL14,WTAP,FTO和ALKBH5,在牛肌肉组织以及骨骼肌卫星细胞(skeletal muscle satellite cells, SMSCs)增殖和分化过程中的表达,同时分析体外成肌分化过程中m⁶A甲基化水平的变化,为阐明m⁶A修饰在骨骼肌发育中的作用及机制提供参考。【方法】使用实时荧光定量PCR(RT-qPCR)技术检测m⁶A甲基化酶相关基因在新生和成年牛不同部位骨骼肌中的表达。随后在秦川牛肉用新品系背最长肌中分离SMSCs,通过生长曲线、免疫荧光和RT-qPCR技术验证SMSCs的增殖和成肌分化功能,使用RT-qPCR检测m⁶A甲基化酶相关基因在SMSCs增殖期24、36、48、60、72 h和分化第0、2、4、6、8天中的时序表达谱。最后利用LC-MS/MS和Dot blot技术检测SMSCs分化过程中m^6...     

High abrasion resistance with sparse mineralization: copper biomineral in worm jaws

Biominerals are widely exploited to harden or stiffen tissues in living organisms, with calcium-, silicon-, and iron-based minerals being most common. In notable contrast, the jaws of the marine bloodworm Glycera dibranchiata contain the copper-based biomineral atacamite [Cu2(OH)3Cl]. Polycrystalline fibers are oriented with the outer contour of the jaw. Using nanoindentation, we show that the mineral has a structural role and enhances hardness and stiffness. Despite the low degree of mineralization, bloodworm jaws exhibit an extraordinary resistance to abrasion, significantly exceeding that of vertebrate dentin and approaching that of tooth enamel.     

马铃薯低温响应的ScmiR390-5p及其靶基因表达与结构分析

【目的】研究马铃薯富含亮氨酸重复序列(leucine-rich repeat receptor-like protein kinase LRR-RLK)类受体蛋白激酶基因SCLRRK1受miR390（ScmiR390-5p）调控响应非生物胁迫的机理。【方法】通过低温响应马铃薯miRNA测序分析与靶基因预测,发现ScmiR390-5p通过调控1个可能的LRR类受体蛋白激酶基因对低温做出响应;利用实时荧光定量PCR技术（Quantitative Real-time PCR,RT-qPCR）验证ScmiR390-5p和SCLRRK1响应低温胁迫的表达情况;利用RLM-5′RACE确定ScmiR390-5p作用于SCLRRK1的切割位点;利用PCR技术克隆得到SCLRRK1的DNA序列和CDS序列,生物信息学分析SCLRRK1的结构和功能;利用RT-qPCR分析ScmiR390-5p/SCLRRK1在马铃薯各组织中的表达情况及其响应各种非生物胁迫的表达情况。【结果】RT-qPCR结果表明,低温诱导ScmiR390-5p表达,SCLRRK1的表达则受到低温的抑制,ScmiR390-5p和SCLRRK1的表达在低温胁迫下呈负相关性;RLM-5′RACE分析表明,ScmiR390-5p作用于SCLRRK1的切割位点是ATTCCT//CCTGAGTT,马铃薯ScmiR390-5p/SCLRRK1调控模块在低温响应中起作用。克隆结果表明SCLRRK1的CDS序列长度为2 685 bp,编码894个氨基酸,DNA序列长度为3 549 bp,含有1个内含子、2个外显子、3′非编码区和5′非编码区,ScmiR390-5p的靶位点位于SCLRRK1 CDS序列内部（960—981 bp,GGAACTATTCCTCCTGAGTTT）。生物信息学显示SCLRRK1是1个富含亮氨酸重复序列(leucine-richrepeat,LRR)的类受体蛋白激酶基因,编码的蛋白属于跨膜分泌蛋白。马铃薯组织表达RT-qPCR分析显示,ScmiR390-5p在叶中的表达量最高,根次之,茎中（地上茎、块茎和匍匐茎）表达量较低;而SCLRRK1的表达情况与ScmiR390-5p相反,其在叶中的表达最低,在茎中表达最高（匍匐茎>块茎>地上茎）。多种非生物胁迫结果显示,ScmiR390-5p和SCLRRK1表达在NaCl胁迫下呈负相关,ScmiR390-5p受到NaCl胁迫诱导表达。与对照相比,ABA和6-BA处理下ScmiR390-5p表达先下降之后呈波浪小幅回调;6-BA处理下SCLRRK1表达持续升高,ABA处理下SCLRRK1表达先上升后下降。GA₃、PEG和IAA处理8 h时,ScmiR390-5p的表达达到峰值,GA₃、PEG和IAA处理都能诱导SCLRRK1表达,但其变化趋势与ScmiR390-5p相关性不强。【结论】SCLRRK1具备编码LRR类受体蛋白激酶的核酸、氨基酸序列和结构基础,是ScmiR390-5p的靶基因;ScmiR390-5p/SCLRRK1调控模块在马铃薯组织器官中具备明显作用;ScmiR390-5p在转录后水平通过抑制马铃薯SCLRRK1的表达对低温胁迫做出响应,同时,马铃薯ScmiR390-5p/SCLRRK1调控模块在NaCl和6-BA胁迫响应中起作用,但不对ABA、GA₃、IAA和PEG胁迫做出响应,ScmiR390-5p和SCLRRK1分别在转录水平受到ABA、GA₃、IAA和PEG胁迫信号调控。     

靶向猪内质网应激通路的microRNAs预测与验证

【目的】预测并验证靶向猪内质网应激通路中关键基因的microRNAs(miRNAs),为进一步研究miRNAs对猪内质网应激信号通路调控提供理论基础。【方法】首先利用伪狂犬病毒(PRV)感染猪肾上皮(PK15)细胞,高通量测序检测差异表达的miRNAs。然后通过TargetScan预测靶向内质网应激通路关键基因ATF6、IRE1、PERK、GRP78、XBP1的miRNAs。构建含有候选miRNAs作用位点的双荧光素酶报告基因重组载体,并分别与miRNA-mimics共转染幼仓鼠肾(BHK-21)细胞,通过测定荧光素酶活性来验证内质网应激通路关键基因与候选miRNAs的靶标关系。然后在PK15细胞中分别过表达候选miRNAs,利用qRT-PCR和Western blot检测候选miRNAs对内质网应激通路关键基因mRNA和蛋白表达的影响。【结果】miRNA测序结果显示,PRV感染后共引起35条miRNAs差异表达。TargetScan预测显示,靶向ATF6、IRE1、PERK、GRP78、XBP1的交集miRNAs为miR-142-5p、miR-145-5p、miR-150和miR-1...     

小麦ZY96-3籽粒灌浆过程中籽粒发育相关基因的表达分析

【目的】 研究小麦ZY96-3籽粒在灌浆过程中与籽粒发育相关基因的表达模式,为了解基因调控籽粒灌浆的分子机制提供参考。【方法】 采用实时荧光定量PCR(qRT-PCR)技术,分析小麦ZY96-3籽粒灌浆期间6个与籽粒发育相关基因的表达动态。【结果】 从小麦ZY96-3开花后6个目标基因的表达模式均是先增后降,且都在花后7 d开始表达;与粒重相关基因TaTGW6在花后7 d表达量最高,与籽粒大小相关基因TaCYP78A5、蛋白质合成基因TaPBF-D和淀粉合成酶基因GBSSⅠ、SBE1、AGPase1都在花后15 d左右表达量达到最大;自花后19 d开始,各基因的表达量均显著下降。【结论】 与粒重相关基因TaTGW6主要在小麦ZY96-3籽粒灌浆初期起关键作用,而其余5个基因主要在籽粒灌浆中后期发挥功能。基因在小麦ZY96-3籽粒灌浆期间的表达模式。     

非洲猪瘟病毒MGF110-5L-6L蛋白诱导宿主细胞翻译阻滞和应激颗粒形成的作用机制

【背景】非洲猪瘟（African swine fever,ASF）是由非洲猪瘟病毒（African swine fever virus,ASFV）感染引发的一种猪烈性传染病,是全球公认的养猪业“头号杀手”,至今尚无安全有效的疫苗和药物。病毒作为专性细胞内寄生物,必须通过“劫持”宿主翻译系统为病毒蛋白合成服务。其中翻译起始因子eIF2α作为翻译调控的核心节点,控制细胞应激反应和翻译重编程走向,对病毒毒力、嗜性、致病性及免疫逃逸等具有重要影响,eIF2α磷酸化调控无疑是病毒与宿主细胞竞争翻译资源的重要阵地之一。然而,关于ASFV编码蛋白与eIF2α磷酸化作用关系的认知极度匮乏。【目的】探究非洲猪瘟病毒MGF110-5L-6L蛋白对宿主细胞翻译阻滞和促进应激颗粒形成的作用机制,为深入揭示非洲猪瘟病毒的致病机制研究提供科学依据。【方法】在前期利用荧光素酶报告基因载体和绿色荧光报告载体,筛选发现外源表达MGF110-5L-6L极显著上调eIF2α磷酸化水平的基础上。选择猪肺泡巨噬细胞3D4/21和猪肾细胞PK-15作为研究用细胞系,利用质粒转染和特异性化学药物处理等方法,结合免疫印迹和激光共聚焦...     

GM-CSF与生长抑素融合表达质粒对小鼠肌肉组织GHR和IGF-I mRNA表达的影响

选择70只小白鼠,随机分为7组,每组10只,雌雄各半。其中第1组为对照组,第2-4组分别肌肉注射pcS/2SS、pGM-CSF/SS和pGM-CSF+pcS/2SS DNA疫苗;第5～7组分别口服以减毒沙门氏菌为载体的pcS/2SS、pGM-CSF/SS和pGM-CSF+pcS/2SS DNA疫苗,以β-actin作为内参,利用相对半定量RT-PCR,检测小鼠肌肉组织GHR和IGF-I mRNA的表达。结果表明:第3、5和6组的GHR mRNA表达显著高于1组和7组(P<0.05),第5和6组的IGF-I mRNA表达显著高于1组、2组、4组和7组(P<0.05),GM-CSF与SS的融合表达质粒(pGM-CSF/SS)的GHR和IGF-I mRNA表达高于pGM-CSF+pcS/2SS共同免疫,同种DNA疫苗,口服免疫组的GHR和IGF-I mRNA表达总体上比肌肉注射组要高。这些结果证明,GM-CSF可促进SS DNA疫苗的免疫效果,提高肌肉组织中GHR和IGF-I mRNA的表达;pGM-CSF/SS效果优于pGM-CSF+pcS/2SS,口服免疫组要优于肌肉注射免疫组。     

转AtDME1基因‘84K’杨的获得及目的基因诱导表达分析

目的DNA甲基化是一种重要的表观遗传标记，在植物生长发育、环境响应等过程中发挥重要作用。本研究将拟南芥去甲基化基因AtDME1导‘84K’杨基因组中，通过化学诱导表达实验，研究AtDME1基因在转基因杨树中的诱导表达特性，为建立杨树甲基化诱导变异体系，进而实现杨树品种改良等奠定良好基础。方法以白杨派优良品种84K杨无菌苗叶片为受体材料，采用农杆菌介导法将化学诱导启动子与AtDME1基因导入‘84K’杨；经过潮霉素筛选、常规PCR检测及DNA测序等方法对抗性植株进行鉴定。通过化学诱导剂17-β-雌二醇对随机挑选的1个转基因株系离体叶片进行诱导处理，处理时间为0、3、6、12、24、48、96、144 h，采用qRT-PCR检测处理叶片中外源基因AtDME1的表达量变化。结果本研究中，潮霉素分化筛选共获得了抗性芽224个，经生根筛选获得6株抗性植株，经分子检测证实这6株抗性植株均为转AtDME1基因植株，扩繁后分别标号为转基因AD-1 ~ 6。qRT-PCR检测结果显示，化学诱导剂17-β-雌二醇处理3 h时，目的基因AtDME1的表达量基本达到最大值，效果持续至12 h后逐渐减弱。结论化学诱导剂17-β-雌二醇能迅速有效地调控转基因杨树中AtDME1基因的表达，为进一步研究DME1基因在杨树基因组甲基化调控方面的作用机制奠定基础，也为杨树化学诱导表达特性的研究做好了铺垫。      

一种低成本的农业现场视频/ 图像快速采集系统马翠

农业现场图像信息采集是农作物长势和病虫害分析的重要手段。结合嵌入式技术与B/S 架构,设计了1 个采用高清晰图像传感器罗技Pro9000摄像头的低成本农业现场视频/图像快速采集系统,并对系统的体系结构、通信子系统、采集、压缩与传输子系统进行了详细设计。系统采用大功率WiFi和快速ARM 平台S3C6410 处理器,基于V4L2 技术采集图像,采用JPEG 图像数据压缩技术能够大大地减少传输数据量,使得视频传输更加流畅。试验结果表明,本系统传输640伊480 大小的图像到远程服务器的图像传输成功率在300 m范围内可达到92%以上;在50 m内可达到视频20 帧/秒以上,在300 m的距离也能够达到6帧/秒以上的视频帧率,可以满足视频监控的需求。由于本方案基于ARM 平台设计,成本低,易于集成应用,非常适于大范围农业上开展应用。     

Faster interprotein electron transfer in a [myoglobin, b⁵] complex with a redesigned interface

Direct measurements of electron transfer (ET) within a protein-protein complex with a redesigned interface formed by physiological partner proteins myoglobin (Mb) and cytochrome b(5) (b(5)) reveal interprotein ET rates comparable to those observed within the photosynthetic reaction center. Brownian dynamics simulations show that Mb in which three surface acid residues are mutated to lysine binds b(5) in an ensemble of configurations distributed around a reactive most-probable structure. Correspondingly, charge-separation ET from a photoexcited singlet zinc porphyrin incorporated within Mb to the heme of b(5) and the follow-up charge-recombination exhibit distributed kinetics, with median rate constants, k(f)(s) = 2.1 × 10(9) second(-1) and k(b)(s) = 4.3 × 10(10) second(-1), respectively. The latter approaches that for the initial step in photosynthetic charge separation, k = 3.3 × 10(11) second(-1).     

Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis

Neoplastic transformation has been associated with a variety of structural changes in cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant glycoproteins or how these transformation-related changes in oligosaccharide biosynthesis may affect the malignant phenotype. Here it is reported that a cell surface glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching and that the expression of these oligosaccharide structures is directly related to the metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in clones of a nonmetastatic murine mammary carcinoma correlated strongly with acquisition of metastatic potential. The results indicate that increased beta 1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important feature of tumor progression related to increased metastatic potential.     

杭州湾北部有明银鱼仔稚鱼脊柱和附肢骨骼发育研究

2014年6月至2015年5月,在杭州湾北部水域设置10个采样点。于每月大潮期间使用大型仔稚鱼网进行仔稚鱼采集,共拖网120次,采集有明银鱼(Salanx ariakensis)仔稚鱼1 972尾。本研究使用硬骨-软骨双染色技术,观察了有明银鱼仔稚鱼(体长范围为9. 7～34. 2 mm)脊柱及附肢骨骼的形态发育特征。有明银鱼背鳍、臀鳍、胸鳍、腹鳍的支鳍骨,髓弓、脉弓维持为软骨。匙骨、上匙骨、后颞骨、尾下骨、尾杆骨与尾椎髓体为硬骨。骨骼形成的顺序依次为肩带,臀鳍支鳍骨和尾下骨,背鳍支鳍骨,脉弓和髓弓,腹鳍支鳍骨和尾上骨。胸鳍支鳍骨无后匙骨。髓弓数目为73,脉弓数目为22,尾下骨(包括侧尾下骨)数目为7,臀鳍支鳍骨数目为27,背鳍支鳍骨数目为13。本研究结果表明有明银鱼具有其他银鱼类似的骨骼系统骨化程度弱的"幼态持续"现象。     

Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate

The limb blastemal cells of an adult salamander regenerate the structures distal to the level of amputation, and the surface protein Prod 1 is a critical determinant of their proximodistal identity. The anterior gradient protein family member nAG is a secreted ligand for Prod 1 and a growth factor for cultured newt blastemal cells. nAG is sequentially expressed after amputation in the regenerating nerve and the wound epidermis-the key tissues of the stem cell niche-and its expression in both locations is abrogated by denervation. The local expression of nAG after electroporation is sufficient to rescue a denervated blastema and regenerate the distal structures. Our analysis brings together the positional identity of the blastema and the classical nerve dependence of limb regeneration.     

Serotonin transporter genetic variation and the response of the human amygdala

A functional polymorphism in the promoter region of the human serotonin transporter gene (SLC6A4) has been associated with several dimensions of neuroticism and psychopathology, especially anxiety traits, but the predictive value of this genotype against these complex behaviors has been inconsistent. Serotonin [5- hydroxytryptamine, (5-HT)] function influences normal fear as well as pathological anxiety, behaviors critically dependent on the amygdala in animal models and in clinical studies. We now report that individuals with one or two copies of the short allele of the serotonin transporter (5-HTT) promoter polymorphism, which has been associated with reduced 5-HTT expression and function and increased fear and anxiety-related behaviors, exhibit greater amygdala neuronal activity, as assessed by BOLD functional magnetic resonance imaging, in response to fearful stimuli compared with individuals homozygous for the long allele. These results demonstrate genetically driven variation in the response of brain regions underlying human emotional behavior and suggest that differential excitability of the amygdala to emotional stimuli may contribute to the increased fear and anxiety typically associated with the short SLC6A4 allele.     

少根根霉Δ6-脂肪酸脱氢酶基因在转基因油菜中的表达

 【目的】验证少根根霉△6-脂肪酸脱氢酶基因在转基因油菜中的表达情况，为进一步基因工程化生产γ-亚麻酸打下基础。【方法】将少根根霉△6-脂肪酸脱氢酶基因和植物表达载体pCPN2301连接，构建重组表达质粒pCPNRAD6，采用农杆菌介导的油菜子叶节转化法，将该基因导入甘蓝型油菜H165，获得转基因植株，最后通过气相色谱检测基因的表达情况。【结果】PCR、GUS组织化学染色和Southern杂交分析结果表明，目的基因已经整合到油菜基因组中，Northern杂交分析进一步表明目的基因在RNA水平获得表达，气相色谱分析检测到转基因油菜叶片中γ-亚麻酸和十八碳四烯酸生成，证明目的基因获得功能表达。同样，用改变少根根霉Δ6-脂肪酸脱氢酶基因的转译起始密码子周边序列的基因RAD6-1所构建的转基因酵母中，也得到类似的结果，而且各种目的脂肪酸的含量均有提高。【结论】初步建立了少根根霉△6-脂肪酸脱氢酶基因在转基因油菜中的表达体系。     

Size of the great white shark (carcharodon)

The maximum length of 36.5 feet (11.1 meters) attributed to the white shark (Carcharodon carcharias) by Günther and others is a mistake. Examination of the jaws and teeth of the specimen referred to by Günther and comparison with the jaws of white sharks of known length revealed a length of about 17 feet ( approximately 5 meters). The largest white shark reliably measured was a 21-foot (6.4-meter) individual from Cuba. Bites on whale carcasses found off southern Australia suggest that white sharks as long as 25 or 26 feet (7 (1/2) or 8 meters) exist today. The size of extinct Carcharodon has also been grossly exaggerated. Based on a projection of a curve of tooth size of Recent Carcharodon carcharias, the largest fossil Carcharodon were about 43 feet ( approximately 13 meters) long.