Transmembrane molecular pump activity of Niemann-Pick C1 protein

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.     

Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1 Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.     

Identification of Bphs,an autoimmune disease locus,as histamine receptor H1

Bphs controls Bordetella pertussis toxin (PTX)-induced vasoactive amine sensitization elicited by histamine (VAASH) and has an established role in autoimmunity. We report that congenic mapping links Bphs to the histamine H1 receptor gene (Hrh1/H1R) and that H1R differs at three amino acid residues in VAASH-susceptible and -resistant mice. Hrh1-/- mice are protected from VAASH, which can be restored by genetic complementation with a susceptible Bphs/Hrh1 allele, and experimental allergic encephalomyelitis and autoimmune orchitis due to immune deviation. Thus, natural alleles of Hrh1 control both the autoimmune T cell and vascular responses regulated by histamine after PTX sensitization.     

黄瓜性别控制基因CsACS1G的分子标记及其快速检测

根据黄瓜性别控制基因CsACS1G、CsACS1及BCAT基因DNA差异序列,设计CsACS1G基因特异标记引物G1/G2,采用碱处理法,快速提取黄瓜品种WI1983G、95、98-17、S-2-98及95×WI1983G的F1、F2群体植株DNA,并以此为模板进行PCR扩增.为了验证特异引物对检测CsACS1G基因的准确性,在黄瓜开花期间进行田间调查,结果表明,以快速提取的黄瓜DNA为PCR模板,特异引物G1/G2能稳定、准确扩增出CsACS1G基因特异片段,电泳检测结果与田间调查结果一致.     

谷子硝酸盐转运蛋白NRT1家族的鉴定及表达分析

[目的]为了揭示谷子硝酸盐高效吸收和利用的分子机制,本文鉴定并分析了谷子硝酸盐转运蛋白NRT1基因家族。[方法]以拟南芥中已知的11个参与硝酸盐吸收和转运的NRT1蛋白为基础,利用Blast的方法在全基因组范围内鉴定谷子参与硝酸盐吸收和转运的NRT1基因,利用MEGA7.0和MEME构建系统进化树和鉴定保守的基序,并进一步利用PlantCARE预测顺式作用元件,最后通过RT-PCR的方法研究了谷子硝酸盐转运蛋白NRT1家族基因的组织表达情况。[结果]从谷子中鉴定了8个硝酸盐转运蛋白NRT1基因,系统进化分析表明所得8个NRT1基因分别属于NPF1、NPF2、NPF4、NPF6和NPF7亚家族。谷子NRT1蛋白中都含有一个保守的QX4GX8GX3FX5P基序,可能与硝酸盐的吸收和转运相关。谷子NRT1基因的启动子中富含光响应元件,尤其是SP1元件。RT-PCR分析表明谷子8个NRT1基因表达具有组织特异性,暗示了其可能在不同组织和器官的硝酸盐吸收转运中起作用。[结论]本研究鉴定了8个谷子硝酸盐吸收和转运相关的NRT1基因,为后续谷子NRT1基因功能研究及谷子耐低氮分子机制的揭示奠定了基础。     

桉树焦枯病菌CpSit1基因的鉴定与表达

利用Blast及TCDB数据库对桉树焦枯病菌(Calonectria pseudoreteaudii)的Cpsit1基因进行鉴定;再利用SMART数据库和Prot Param、TMHMM、PHD、Pro Comp 9.0在线分析工具对Cpsit1基因编码蛋白的理化性质、跨膜螺旋、蛋白质二级结构和亚细胞定位进行预测分析;最后采用qRT-PCR方法对CpSit1基因在焦枯病菌侵染桉树过程中的表达情况进行分析.结果表明:CpSit1基因长度为1 780 bp,其编码的蛋白序列共有592个氨基酸残基组成,并将其鉴定为铁载体-铁:H+同向转运蛋白.其保守结构域为MFS_1;共含有14个跨膜螺旋;在二级结构中α螺旋占45.10%,延伸链占26.01%,无规则卷曲占28.89%.通过qRT-PCR相对定量的方法分析焦枯病菌侵染桉树24、48和72 h后CpSit1基因的表达情况,结果显示,CpSit1基因在这3个时段均发生上调表达,但24 h的表达量明显大于48和72 h.说明桉树焦枯病菌CpSit1基因在病原菌侵染寄主的过程中通过调控铁载体-铁化合物的转运来完成铁元素的摄入,协助其在寄主中的定植.     

利用GFP标记对OsWAK1蛋白胞外区的鉴定

水稻OsWAK1编码一细胞壁相联的受体类似蛋白激酶,对其推断的氨基酸进行预测,其结构组成包括胞外区、跨膜区和胞内激酶区。推断OsWAK1蛋白胞外区与配体识别并结合,是胞外信号传递到胞内的信号转导途径中的重要环节,因此OsWAK1蛋白的胞外区是OsWAK1发挥其生物学功能所必需的。将预测的OsWAK1蛋白胞外区与GFP构建为融合蛋白,通过融合蛋白在烟草细胞中的定位确定OsWAK1蛋白结构上含有预测的胞外区。     

大豆LAZ1基因家族鉴定与GmLAZ1-9基因的功能研究

通过在大豆基因组数据库中比对,获得大豆LAZ1基因家族成员,开展生物信息学分析与表达模式检测,并利用转基因拟南芥进行基因功能验证.结果显示:共鉴定到17个大豆LAZ1基因,即GmLAZ1-1～Gm-LAZ1-17.这17个GmLAZ1基因不均匀地分布在大豆的12条染色体上,在进化树上可分为3个亚家族,同一亚家族成员具有...     

重组RNA干扰表达载体的构建与鉴定

干扰(RNA interference,RNAi)是指在进化过程中高度保守的、由双链RNA诱发的、同源mRNA高效特异性降解的现象。本研究以稻纵卷叶螟可溶型海藻糖酶基因(Cm Tre1)为靶标,设计RNAi靶位点并将其命名为Cm Tre1*,在其两端分别添加Bam H I与Spe I酶切位点并进行人工合成,然后经双酶切后与p1301植物表达载体连接,构建重组表达载体p1301-Cm Tre1*。PCR、双酶切和测序鉴定结果证明重组表达载体p1301-Cm Tre1*构建成功,并将其成功转入农杆菌LBA4404,构建基因工程菌。用含有p1301-Cm Tre1*质粒的农杆菌LBA4404浸染中花11号水稻的愈伤组织,经过愈伤组织的抗性筛选、分化和植株再生,获得26株阳性转基因植株。用取食非转基因水稻的稻纵卷叶螟作为对照,用实时荧光定量PCR方法分株测定取食转基因水稻稻纵卷叶螟体内Cm Tre1基因的mRNA表达水平,并检测其体内海藻糖酶活性变化。结果显示,相较于对照组,取食转基因水稻稻纵卷叶螟体内Cm Tre1基因表达量下降了44.79%,海藻糖酶活力平均下降了14.94%。研究结果表明转基因水稻的RNA干扰效应相当明显,能对取食其叶片的稻纵卷叶螟Cm Tre1基因起到明显的沉默作用。     

Identification of a second dynamic state during stick-slip motion

Stick-slip, or interrupted, motion rather than smooth uninterrupted motion occurs in many different phenomena such as friction, fluid flow, material fracture and wear, sound generation, and sensory "texture." During stick-slip, a system is believed to undergo transitions between a static (solid-like) state and a kinetic (liquid-like) state. The stick-slip motion between various types of pretreated surfaces was measured, and a second, much more kinetic state that exhibits ultra-low friction was found. Transitions to and from this super-kinetic state also give rise to stick-slip motion but are fundamentally different from conventional static-kinetic transitions. The results here suggest practical conditions for the control of unwanted stick-slip and the attainment of ultra-low friction.     

口蹄疫病毒VP1基因在胡萝卜中的表达

为了生产一种可以直接口服的口蹄疫疫苗,以胡萝卜(Daucus carota L.var.sativa)无菌幼苗下胚轴诱导的愈伤组织为受体材料,通过农杆菌LBA4404介导的遗传转化,在CaMV双35S启动子的驱动下,将口蹄疫病毒(foot-and-mouth disease virus,FMDV)结构蛋白VP1基因的活性肽片段(包括2个141~160位肽和1个200~213位肽的基因片段)导入胡萝卜愈伤组织细胞。经PCR和PCR-Southern杂交等方法鉴定转化苗,确认口蹄疫病毒结构蛋白VP1基因已整合到胡萝卜染色体中,转化效率达到52%。SDS-PAGE试验结果初步证明,在转化苗总蛋白中有目的基因表达,胡萝卜种子中可溶性蛋白含量最高,达3.57 g/L,而根中可溶性蛋白含量最低,仅有0.17 g/L;靠近心叶的叶片可溶性蛋白含量可达到1.475 g/L,而最外部叶片可溶性蛋白含量仅有0.37 g/L,相差近5倍。Dot-ELISA和ELISA检测结果表明,转化苗中表达的口蹄疫病毒结构蛋白VP1可以与FMDV灭活疫苗免疫动物制备的抗体特异结合。以上试验结果证明,口蹄疫病毒结构蛋白VP1基因在转基因胡萝卜中表达成功。     

小麦病程相关蛋白1基因的亚细胞定位及信号肽鉴定

前期研究克隆获得了一个叶锈菌诱导的小麦病程相关蛋白1基因TaLr35PR1,明确了其基因结构和表达特征。本研究首先预测TaLr35PR1的信号肽和亚细胞定位情况,进而通过试验予以佐证。将信号肽编码的核酸序列克隆、连接到酵母信号肽诱捕载体pSUC2T7M13ORI上,并转化到酵母蔗糖酶缺陷菌株YTK12中,明确了TaLr35PR1基因的信号肽具有分泌功能。同时,利用酶切连接的方法成功构建亚细胞定位重组体pCamA-TaLr35PR1-GFP,利用基因枪轰击技术瞬时转化洋葱表皮细胞。结果表明,pCamA-TaLr35PR1-GFP融合蛋白主要在细胞外表达,与亚细胞定位预测的结果一致。这些研究结果丰富了小麦病程相关蛋白1基因的研究内容,为进一步探索该类基因的生物学功能及其作用机制提供了研究基础。     

线虫fat-1基因的原核表达及其抗体的制备

通过RT-RCR得到线虫f a t-1基因全长,将其N端亲水区克隆至原核表达载体pGEX-4T-2中,构建重组质粒pGEX-f a t1-N,将其转化大肠杆菌,并进行诱导表达。对表达产物G ST-f a t1-N融合蛋白进行纯化,制备其抗体,并对抗体效价进行了检测。结果表明,线虫f a t-1基因能在大肠杆菌中表达,制备的抗体能识别在原核表达系统内表达的G ST-f a t1-N融合蛋白,且效价很高,达到1∶107。     

The SNPs C.513A>T in the MHC B-F gene and rs15001532 in the SPOCK1 gene are associated with Salmonella pullorum disease resistance in chickens

This study was designed to identify molecular markers single nucieotide polymorphisms(SNPs) of MHC B-F gene and SPOCK1) associated with Salmonella pullorum disease susceptibility/resistance.A two-stage case-control association study was used.In the first study,a small population comprising 401 Partridge chickens(201 cases and 200 controls)was used,and a total of 118 SNPs genotyped.In the second study,a bigger population comprising 1 075 Partridge chickens(527 cases and 548 controls) was used,and SNPs with significant effect determined in the first study were further analysed.In the first study,8 SNPs were significantly associated with S.Pullorum disease susceptibility/resistance,however,after further analysis,only the SNPs rs15001532 and C.513AT were found to be significantly associated with S.Pullorum disease susceptibility/resistance.The relative risk test demonstrated that the AA genotype of rs15001532 resulted in a higher risk of S.Pullorum infection,whereas birds with the TT genotype of C.513AT were more susceptible to S.Pullorum infection.This research was based on the researches on human complex diseases.With help of these train of thoughts,some common animal diseases can be studied effectively and the process of candidate gene research for animal disease can be improved.     

高效产脂肪酶菌株C1的分离鉴定与酶学性质研究

采用平板筛选的方法从油菜地土壤中分离到一株高效产脂肪酶菌株C1,通过生理生化实验及16S rDNA序列分析鉴定为Burkholderia cepacia。研究其酶学性质,得出该菌脂肪酶的最适温度为65℃,最适pH为8.0,具有优良的耐温性和pH稳定性;对正己烷、乙醇耐性较好,对乙腈和乙酸耐性较差;K+、Mg2+对其酶活性有明显促进作用,Ca2+、Cu2+对其酶活性有严重抑制作用。     

Obstructive lung disease and alpha-1-antitrypsin deficiency gene heterozygosity

The phenotypes of serum alpha(1)-antitrypsin were determined by antigenantibody crossed electrophoresis. There were five homozygotes and 25 heterozygotes for the deficiency gene found in a group of 103 patients with obstructive lung disease. The frequency of heterozygotes was 14 and 9 percent in two control groups with different mean ages of 36 and 80. There was only one heterozygote among 39 healthy males over 70 years of age. Heterozygosity may be a predisposing factor in chronic obstructive lung disease, especially in the male population.     

猪抗酶1基因核心启动子区鉴定及单倍型转录活性比较

通过双荧光素酶报告基因载体系统检测猪抗酶1(AZ1)基因启动子转录活性,分析蓝塘和长白猪群体AZ1基因启动子SNP及单倍型,在此基础上比较不同单倍型转录活性。结果表明:-453/+79为核心启动子区;在2个群体中检测到AZ1基因启动子8个SNPs,分别为-713 TC、-584 AC、-476 GA、-365 TC、-348 CT、-294 GC、-288 TG和-234 TC,蓝塘猪群体具有更丰富的单核苷酸多态性,而长白猪群体仅在-288座位存在GG和GT 2种基因型,其他7个SNPs均为纯合状态;蓝塘猪群体LTH1单倍型(TAGCCGTC,-713/-234)频率为0.526,为蓝塘猪优势单倍型,其次为LTH2(TAGCCGTT),频率为0.166;长白猪群体优势单倍型为Landrace-H(TAGTCGGT),频率为0.939。LTH1转录活性高于LTH2和Landrace-H,推测-234 TC、-365 TC和-288 TG座位是造成不同单倍型转录活性差异的原因。     

广东菠萝心腐病病原鉴定

为了鉴定广东湛江地区菠萝心腐病病原菌,研究了病原菌的致病性、菌丝体及孢子囊等形态特征,分析了核糖体DNA-ITS序列同源性。结果表明,该病原菌属于疫霉属真菌,同源性分析显示,该菌与GenBank中烟草疫霉ITS序列的同源性达99%以上;采用烟草疫霉特异性引物进行PCR检测,结果确认该病原菌为烟草疫霉菌(Phytophthora nicotianae)。     

浙江省水稻品种对条纹叶枯病抗性鉴定和Stvb-i基因的SSR检测

以浙江省11个粳稻品种(系)对条纹叶枯病抗性进行自然鉴定,并进而用一个与水稻条纹叶枯病抗性基因Stvb-i紧密连锁的RM209微卫星标记进行了分子检测。结果表明,春江051、丙00502SJ、丙00502HSF47、HGP5和HPG6共5个水稻品种(系)检测到Stvbi-抗性基因,其中丙00502HSF47田间表现感病,其它4个品种(系)均表现出良好的田间抗性。Z5、HGP3和HGP4虽未检测到Stvb-i抗性基因,但也表现出较强的田间抗性,推测这3个品种可能含有新的抗病基因或具有其它某种抗病机制。