玉米大斑病菌NAD(H)激酶蛋白的鉴定及分析     

唐聪  杨晓荣  赵洁  冯胜泽  巩校东  谷守芹  韩建民  董金皋 《河北农业大学学报》2016,(2):137-142

NAD(H)激酶是NADP(H)从头合成途径的关键物质,对生物体的生长、发育具有重要作用。本研究以稻瘟病菌中3个NAD(H)激酶蛋白为查询序列,搜索玉米大斑病菌蛋白质组序列库,结果发现在玉米大班病菌中均有同源序列,分别为127652、103052和163223。利用生物信息学的方法对3个基因的结构进行分析,结果表明只有163223为断裂基因,包含5个外显子和4个内含子,127652和103052为单外显子;163223和103052呈现酸性,而127652则呈现碱性;103052为稳定蛋白,而另两者为不稳定蛋白;通过高级结构分析发现这3种蛋白在N端一侧α螺旋较多,C端一侧延伸链和β转角较多,并且这3种蛋白质有2个在结构和功能上的相似的结构域。  相似文献   

利用转录组测序技术分析ADC和NOS基因mRNA在鹅等级前卵泡中的表达     

武惠岩  刘璐璐  杨辉  王子遒  刘畅  蒋明  孙永峰 《吉林农业大学学报》2017,39(4)

为研究精氨酸代谢的关键酶精氨酸脱羧酶(Arginine Decarboxylase,ADC)和一氧化氮合酶(Nitric-oxide Synthase,NOS)mRNA在鹅等级前卵泡中的表达情况。采用高通量测序技术对鹅等级前卵泡进行转录组测序分析,并通过荧光定量PCR对结果进行验证。通过FPKM值计算分析,这2个基因在鹅等级前卵泡中均有表达,且随着卵泡的发育,NOS mRNA的表达呈现先降低后增加再降低的趋势,在初级卵泡中的表达量达到最高值,在中白卵泡中的表达量最低。ADC mRNA的表达则呈现先增加后降低再增加的趋势,在初级卵泡中的表达量最低,在小白卵泡中的表达量最高。荧光定量PCR结果与转录组测序结果基本相同。  相似文献   

CD147 monoclonal antibody-mediated nanoparticles for gene therapy to target lung cancer cells     

WU Fei-long  KONG Qing-lei  CAI Song-wang  YE Zhi-qiang 《园艺学报》2016,32(9):1562-1567

AIM: In this study, CD147 antibody was used to carry out targeted modification of nanoparticles for protein kinase Cε (PKCε)-siRNA gene therapy to target lung cancer cells. The inhibitory effects of the nanoparticles on the proliferation and invasion of the lung cancer cells were observed. METHODS: The magnetic nanoparticles targeting CD147 protein were assembled as gene vector. The expression of CD147 in the lung cancer cells was observed under laser scanning confocal microscope. The cells were divided into CP group, CN group and LP group as the experimental groups. Targeted nanoparticles were used as CA group. Non-transfected cells were used as control group. The cell transfection was carried out with 250 ng plasmids/well in 6-well plate. The effect of nanocontrast agent on the cell endocytosis was observed under laser scanning confocal microscope. The mRNA expression of PKCε was detected by RT-qPCR. The protein expression of Ki67, MMP3, PKCε, Wnt1 and GAPDH was determined by Western blot. The cell proliferation ability was detected with colony formation assay. The cell invasion ability was detected by Transwell method. RESULTS: The expression of CD147 protein in the human lung cancer A549 cells was confirmed by immunofluorescence staining. The endocytosis of siRNA into the A549 cells in CP group was observed with the highest efficiency as compared with CN group and LP group. The relative mRNA expression of PKCε in the A549 cells of CP group, CN group, LP group and CA group were (9.76±0.18)%, (98.51±0.32)%, (99.17±0.16)% and (99.68±0.11)%, respectively. The difference between CP group and control group was statistically significant (P<0.05). No significant difference among CN group, LP group and control group was observed. The protein expression of PKCε, Ki-67, MMP3 and Wnt1 in CP group was significantly reduced, and the protein expression levels among CN group, LP group and control group had no significant difference. The colony number in CP group was significantly smaller than that in control group (P<0.05). The effective colony numbers in CN group, LP group and CA group had no significant difference as compared with control group. The number of the invading cells in CP group was significantly less than that in control group (P<0.05). The numbers of the invading cells in CN group, LP group and CA group had no significant difference as compared with control group. CONCLUSION: Nanogene vector targeting CD147 can carry PKCε-siRNA to conduct gene therapy efficiently on the lung cancer cells to achieve effective inhibitory effects on the proliferation and invasion of the lung cancer cells.  相似文献   

颈静脉灌注精氨酸对泌乳中期奶牛血清生化和免疫指标的影响     

丁洛阳  梁祥焕  王梦芝  张军  王洪荣  周刚  张鑫  徐巧云 《动物营养学报》2016,(6):1899-1906

本文旨在研究颈静脉灌注精氨酸对泌乳中期奶牛血清生化和免疫指标的影响。采用3×3复式拉丁方试验设计,选择6头体重、胎次、泌乳期、泌乳量和体况基本一致的荷斯坦奶牛为试验动物,随机分为3组(每组2头),分别为酪蛋白组(对照组)、精氨酸组(灌注37.66 g/d)、丙氨酸组(与精氨酸组等氮,灌注77.24 g/d);每个试验期为22 d(7 d灌注+15 d间隔),在每个试验灌注期的最后1 d晨饲前采血,测定血清生化和免疫指标。结果表明:精氨酸组的奶牛血清中总蛋白、免疫球蛋白G(Ig G)、肿瘤坏死因子α(TNF-α)浓度要显著高于其他2组(P0.05),白球比、白细胞介素1(IL-1)浓度显著高于丙氨酸组(P0.05),但是牛奶中体细胞数显著低于其他2组(P0.05),而血清中谷丙转氨酶、谷草转氨酶活性,免疫球蛋白A(Ig A)、免疫球蛋白E(Ig E)、白细胞介素6(IL-6)浓度在组间无显著差异(P0.05)。综上,灌注精氨酸能够通过提高血清总蛋白、Ig G及IL-1、TNF-α的浓度,从而一定程度上增强泌乳奶牛机体的免疫力和胎儿的被动免疫力。  相似文献   

Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat   总被引：1，自引：0，他引：1  

ZHANG Long-yu  ZHANG Gai-sheng  ZHAO Xin-liang  YANG Shu-ling 《中国农业科学(英文版)》2013,(6):941-950

To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Ade/-His/-Leu/-Trp) (QDO), and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor (TanLTP), polyubiquitin (TaPUbi), glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen (TaPCNA), CBS domain containing protein (TaCBS), actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.  相似文献   

用CODEHOP设计简并引物克隆反刍月形单胞菌K6乙酸激酶基因片段   总被引：3，自引：0，他引：3  

逄晓阳  刑鑫  刘国文  王哲 《中国兽医学报》2010,30(1)

利用CODEHOP设计细菌乙酸激酶的简并引物,选用1对引物ACKSe以高效丙酸生成菌反刍月形单胞菌K6基因组DNA进行PCR,得到749 bp PCR产物,产物经pMD18-T载体克隆转化至DH5α大肠杆菌中,测序后经Blastx比对,此DNA产物与其他菌属来源的乙酸激酶蛋白序列具有相似性,所克隆的序列即为K6的乙酸激酶基因片段。用CODEHOP程序化设计的简并引物可信性强,阳性率高。该基因的成功克隆为丙酸生成菌K6乙酸代谢工程研究提供了依据。  相似文献   

Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa     

Ochiai N  Fujimura M  Motoyama T  Ichiishi A  Usami R  Horikoshi K  Yamaguchi I 《Pest management science》2001,57(5):437-442

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.  相似文献   

荔枝霜疫霉中双组分信号转导系统的鉴定与表达分析     

王荣波  陈姝樽  刘裴清  李本金  翁启勇  陈庆河 《植物病理学报》1955,50(1):49-59

 荔枝霜疫霉(Peronophythora litchii)隶属于茸鞭生物界卵菌门疫霉属,由其引起的荔枝霜疫病是目前荔枝生产上一种最重要的病害,严重影响荔枝产量和鲜果品质。双组分信号途径在微生物中参与多种生命活动,但是在卵菌中尚未有相关研究。本研究通过生物信息学分析在荔枝霜疫霉中鉴定到2个杂合型组氨酸激酶(PlHK1、PlHK2)和1个响应调控蛋白(PlRR1)。其在卵菌中是保守存在的,并且与真菌在进化上相对独立。功能域分析表明,PlHK1和PlHK2的C端额外的融合了1个磷酸转移功能域(Hpt),这与真菌和植物的存在显著差异。转录分析表明3个基因在荔枝霜疫霉侵染阶段上调表达,并且响应渗透胁迫和氧化胁迫。以上结果揭示了双组分信号系统可能在疫霉致病过程中发挥重要作用。  相似文献   

仔猪精氨酸的内源合成及其影响因素     

郭秀兰  唐仁勇 《中国畜牧杂志》2007,43(23):45-48

由于精氨酸及其代谢产物具有广泛生物学作用,使得近年来研究者们在生理、生化和营养等方面对其作了大量的研究。母乳严重缺乏精氨酸,仔猪精氨酸内源合成对精氨酸的平衡起了重要作用,使得哺乳仔猪精氨酸的内源合成成为精氨酸营养的研究核心。通过对精氨酸内源合成路径和场所的了解,研究者们进一步研究了精氨酸内源合成的影响因素,主要有仔猪日龄、日粮中精氨酸的水平、乳酸盐和皮质醇的浓度等。本文就仔猪精氨酸内源合成的途径和场所、内源合成的量、影响因素以及精氨酸的需要量等进行了综述,重点阐述了精氨酸内源合成的影响因素。  相似文献   

Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRa and c-Met in canine primary brain tumours   总被引：1，自引：0，他引：1  

P. J. Dickinson  B. N. Roberts  R. J. Higgins  C. M. Leutenegger  A. W. Bollen  P. H. Kass  R. A. LeCouteur 《Veterinary and comparative oncology》2006,4(3):132-140

Inhibition of tumour growth and angiogenesis by targeting key growth factor receptors is a promising therapeutic strategy for central nervous system tumours. Characterization of these growth factor receptors in canine primary brain tumours has not been done. Using quantitative real‐time TaqMan polymerase chain reaction (PCR), we evaluated the expression of messenger RNA (mRNA) for five tyrosine kinase growth factor receptors (vascular endothelial growth factor receptor [VEGFR]‐1, VEGFR‐2, endothelial growth factor receptor [EGFR]‐1, platelet‐derived growth factor receptor a [PDGFRa], and c‐Met) relative to normal cerebral cortex in 66 spontaneous canine primary brain tumours. Increased expression of VEGFR‐1 and VEGFR‐2 mRNA was greatest in grade IV astrocytomas (glioblastoma multiforme) and grade III (anaplastic) oligodendrogliomas. EGFR‐1 mRNA expression was more consistently increased than the other receptors in all tumour types, while increased PDGFRa mRNA expression was mostly restricted to oligodendrogliomas. The similarities in increased expression of these tyrosine kinase growth factor receptors in these canine tumours, as compared to data from their human counterparts, suggest that common molecular mechanisms may be present.  相似文献