毛葡萄芪合成酶基因的克隆及序列分析

芪合成酶是白藜芦醇生物合成过程中起关键作用的酶.通过RT-PCR,从高抗葡萄黑痘病的中国野葡萄毛葡萄商-24克隆了芪合成酶基因家族的2个成员VqSTS1和VqSTS2,其cDNA全长均为1 179 bp,编码392个氨基酸.氨基酸序列分析表明,VqSTS1和VqSTS2编码氨基酸247-257位点的IPN（S/F）AGAIAGN ＂是芪合成酶家族固有的氨基酸保守结构域,368-378位点的GVLFGFGPGLT＂是芪合成酶与查尔酮合成酶家族固有的保守序列特征.在编码的392个氨基酸中,VqSTS1和VqSTS2仅有12个氨基酸不同,氨基酸一致性达97%.多重比较分析显示,VqSTS1与VqSTS2与五针松、河岸葡萄、白粉藤、华东葡萄及欧洲葡萄等STS在氨基酸水平的一致性分别为65%、96%、97%和98%.将VqSTS1和VqSTS2登录GenBank,登录号分别为EF158856和EF158857.     

水杨酸诱导湖北海棠全长cDNA文库的构建及应用

以'湖北海棠'为材料,经水杨酸处理后,用改良CTAB法提取总RNA,纯化后构建全长cDNA文库,并进行PGIP基因的克隆.结果表明:(1)提取的总RNA无降解,无污染;mRNA弥散带主要集中在500～2 000 bp左右,没有rRNA 残留.(2)ds cDNA弥散带主要分布于300～2 000 bp之间,PCR验证后片段大小分布于200～2 000 bp之间,说明合成ds cDNA质量较好,成功地构建了全长cDNA文库.(3)通过PCR从该cDNA文库中克隆了PGIP基因,命名为MhPGIP,GenBank登录号为FJ449708;其核苷酸序列及推导氨基酸序列与苹果的一致性分别为98%和97%,该序列含有两个串联的亮氨酸重复序列.综上所述,构建的全长cDNA文库质量很好,该文库的建成可以用于今后抗病新基因的挖掘、克隆和利用,为苹果抗病机理的研究奠定基础.     

中国北方马铃薯黑痣病立枯丝核菌的融合群鉴定

从山东、甘肃、青海、内蒙古、河北和黑龙江6省采集马铃薯黑痣病标本300余份,分离获得251个立枯丝核菌Rhizoctonia solani菌株。融合群测定结果表明,这些菌株分别属于多核的丝核菌AG‐3、AG1‐IB、AG4‐HG‐Ⅰ、AG4‐HG‐Ⅱ、AG4‐HG‐Ⅲ、AG‐5和AG‐11融合群。其中AG‐3是优势致病群,占分离菌株总数的71.31%;其次是AG4‐HG‐Ⅰ,占15.14%;AG‐11融合群菌株是国内首次从罹病马铃薯植株上分离得到。从各融合群中选取代表性的菌株进行5.8S rDNA‐ITS区序列分析,结果表明,隶属不同融合群或亚群菌株的5.8S rDNA‐ITS区序列存在较大的差异,而相同融合群(亚群)不同菌株的序列具有较高一致性。     

风信子花器官中HAP2基因的分离与表达研究(英文)

在离体条件下,以风信子花被片为外植体,通过控制激素的浓度可诱导花被片、雄蕊或胚珠的再生。近年来,在拟南芥和金鱼草等模式植物中已经分离出了许多控制花器官发育的同源异形基因,如AG,AP1,AP2,AP3等,其中AP2在控制花萼和花瓣形成过程中起重要作用,因此本文从风信子中分离AP2的同源基因,并对它在风信子再生系统中的表达进行了分析。根据AP2同源基因功能域的保守序列设计一对简并引物：5'-TGGGA（A/G）TC（G/T/C）CA（C/T）AT（C/T）TGGA-3'和5'-TCCCA(AGC)(CT)(GT)(AG)CC(AG) CA(CT)TT(AG)TG-3', 以再生的花被片为材料进行RT-PCR,扩增出大小约300bp的片段,序列分析表明该片段的氨基酸序列与AP2同源性高达89%。进而,利用5’和3’Race PCR,得到全长的cDNA。该基因命名为HAP2,GenBank登记号为AF134116,该基因全长1597bp,编码368个氨基酸(Fig.1）。与AP2相比,HAP2也含有10个氨基酸长的碱性功能域,其中KKSR为核定位信号。此外,HAP2也含有两个序列重复的68个氨基酸长的功能域（HAP2-R1,HAP2-R2）,HAP2-R1也含有能形成(-螺旋结构的核心区域,且与AP2-R1中的核心序列100%同源,而HAP2-R2中的核心区域与AP2-R2相比, 缺少9个氨基酸(Fig.2）。RT-PCR结合Southern 杂交结果表明(Fig.3）,HAP     

杜氏盐藻RuBisCO小亚基基因的克隆和分析

根据莱菌衣藻(Chlamydomonas reinhardtii)、团藻(Volvox carteri)、伞藻(Acetabularia cliftonii)等生物的1,5-二磷酸核酮糖羧化酶/加氧酶(RuBisCO)的小亚基rbcS基因氨基酸的高度保守序列,设计一对简并引物,进行RT-PCR.209 bp的PCR产物经测序分析及进行氨基酸序列同源性比对,表明克隆的序列为盐藻rbcS基因的cDNA片段.根据该序列信息,采用RACE(rapid amplification of cDNA ends) 方法扩增其5'上游未知区和3'下游未知区.5'RACE得到的cDNA长度为约300 bp,3'RACE得到的长度约380 bp.三段序列拼接后cDNA全长为878bp,其中开放读码框包括190个氨基酸.此cDNA序列,推导成氨基酸序列与已知物种的rbcS基因相比对,同源性分别为V.carteri 78%,C.reinhardtii 75%,A.cliftonii 67%,据此可推断所克隆的序列为盐藻RuBisCO的小亚基cDNA序列,GenBank收录号为AY739272.     

猪繁殖与呼吸综合征病毒S1株基因组序列测定和分析

应用RT-PCR方法分段扩增出PRRSV上海分离株S1毒株的4条基因大片段,扩增后的产物分别克隆于pCR-XL-TOPO载体鉴定后测序,同时应用RACE方法对S1毒株的3'和5'基因末端进行了成功的扩增并克隆于pMD-18T载体进行测序,按顺序将这些序列进行拼接得到PRRSV S1株全基因组cDNA序列.测序结果表明PRRSV S1株基因组全长15441 bp,包含9个开放式阅读框,5'UTR含有189nt,3'端UTR含有181nt,其中包含30nt Poly (A).基因组序列分析结果显示该病毒与ATCC VR-2332和BJ-4分离株的核苷酸同源性分别99.5%和99.6%.与另一国内分离株CH-1a的核苷酸同源性为90.8%.     

中国东北地区玉米纹枯病菌的融合群鉴定

自东北三省采集玉米纹枯病标本300余份,分离获得286个丝核菌菌株。融合群测定及5.8S rDNA-ITS区序列分析结果表明,这些菌株分别属于多核丝核菌的AG1-IA、AG1-IB、AG1-IC、AG4-HG-I、AG4-HG-III、AG-5、WAG-Z群及双核丝核菌的AG-Ba群。其中AG1-IA是优势致病群,占分离菌株总数的38.46%,其次是WAG-Z和AG-5群,分别占26.92%及24.83%。AG4-HG-III群菌株是国内首次从罹病玉米植株上分离得到。自各融合群中选取代表性的菌株进行5.8     

玉米粗缩病毒基因组第七组份的cDNA克隆及序列分析

从采自中国河北滦城感病的玉米材料中提取玉米粗缩病毒(MRDV)的双链RNA。根据已知MRDV的部分序列设计引物,反转录、PCR扩增,克隆并测序分析了MRDV的第七片段(S7)cDNA序列。结果表明,S7 cDNA序列全长为1936bp,与国外所测的MRDV S7的序列长度相等,而且S7包含的两个阅读框(ORF1和ORF2)位置无变化。它们的核苷酸和最大开放阅读框(ORF1)同源性分别为877%和91.6%,然而,MRDV S7的片段与水稻黑条矮缩病毒(RBSDV)S8片段的核苷酸和最大开放阅读框(ORF1)有更高的同源性,分别为95.5%和93.5%。     

鳜鱼Mx蛋白全长cDNA的克隆和序列分析

Mx蛋白是一类由I型干扰素诱导表达的抗病毒蛋白.本研究以感染了鳜传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus,ISKNV)的鳜鱼为材料,提取肝脏总RNA,通过逆转录-聚合酶链式反应(RT-PCR)扩增出Mx蛋白基因的核心片段序列,再应用3'和5'快速扩增cDNA末端(RACE)方法PCR扩增Mx蛋白cDNA末端,最终获得鳜鱼Mx蛋白cDNA序列(GenBank登陆号AY392097).序列分析表明鳜鱼Mx蛋白cDNA含有2391bp,其中编码区长1881bp,编码627个氨基酸残基,推测蛋白质分子量大小为7.15kDa.鳜鱼Mx蛋白具有脊椎动物Mx蛋白共有的结构特征一个三联体GTP结合区域(GXXXSGKS/T、DXXG、T/NKXD);一个发动蛋白家族的典型结构特征序列(LPRGS/KGIVTR);以及C端高度保守的Leu拉链结构域.鳜鱼Mx蛋白全基因的获得为下一步研究鱼类Mx蛋白的抗病毒活性、作用机制,以及干扰素的检测奠定了基础.     

苹果α-法尼烯合酶基因的启动子序列及同源基因

本文通过设计引物进行PCR扩增α-法尼烯合酶(AFS)基因的5'端区段并测序,获得510bp的‘国光’苹果AFS基因启动子和5'端非翻译区(5'UTR)序列,已在GenBank注册(登录号FJ263961)。序列分析结果表明,该序列具有典型的启动子特征,在转录起始点上游-46bp处有一个TATA盒,-93bp处有一个CAAT盒,-84bp处有一个W盒和-436bp处有一个热胁迫反应顺式作用元件GAAATTTTTT。与‘皇家嘎拉’苹果的AFS基因启动子序列(GenBank登录号AY786553.1)比对,本研究发现‘国光’苹果AFS基因启动子序列中有6个碱基(-186T,-207T,-283C,-301A,-413A和-433A)发生变异,‘皇家嘎拉’苹果AFS基因启动子序列相应位置的碱基分别为碱基缺失、-206C、-282G、-300G、-412G和-432G。重要的是,其中‘国光’苹果-413A碱基变异为G发生在一个热胁迫反应顺式作用元件GAAATTTTTT中,苹果虎皮病发生和AFS基因的转录表达是否受到这一碱基变异的影响值得进一步探讨。本研究结果还表明,在苹果AFS基因启动子和5'UTR序列中存在一个正向重复序列1(R9+IR18+R9),重复单元R9长度9bp,转录起始点位于其长度18bp的IR18区段。有趣的是,本研究新发现了一个与AFS基因启动子和5'UTR序列高度同源的450bp基因片段(GenBank注册登录号FJ469631),该同源片段缺失AFS基因中的27bp序列(R9+IR18,包含转录起始点)。据我们所知,这是果树中存在AFS基因启动子同源序列的首次报道。     

NADPH- and hydroperoxide-supported 17beta-estradiol hydroxylation catalyzed by a variant form (432L, 453S) of human cytochrome P450 1B1

Human cytochrome P450 1B1 (CYP1B1) catalyzes the hydroxylation of 17beta-estradiol (E(2)) at C-4, with a lesser activity at C-2. The E(2) 4-hydroxylase activity of human CYP1B1 was first observed in studies of MCF-7 breast cancer cells. Sequencing of polymerase chain reaction products revealed that CYP1B1 expressed in MCF-7 cells was not the previously characterized enzyme but a polymorphic form with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E(2) hydroxylase activities of the 432L, 453S form of human CYP1B1, the MCF-7 CYP1B1 cDNA was cloned and the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E(2) hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol (min)(-1)(nmol cytochrome P450)(-1) for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E(2) hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities and with a lesser preference for E(2) 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3',4, 4',5,5'-hexachlorobiphenyl. These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E(2) hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.     

Joint effect of G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism on the risk of premature coronary artery disease

The study sought an association between the G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism and premature coronary artery disease (CAD), and the interactive effect on CAD risk between the G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism as well as between tested polymorphisms and traditional risk factors. 167 patients with CAD younger than 55 years were compared with 132 healthy subjects. The frequency of factor V point mutation was 7.8 % among Slovene patients with premature CAD, and 4.5 % among controls. No association was found between either the factor V point mutation (AG genotype) or M1M1 genotype of factor VII Arg/Gln(353) gene polymorphism and the risk of CAD in Slovenia using univariate analysis (factor V point mutation: OR = 1.8, 95% CI = 0.7-4.9; p = 0.25; factor VII Arg/Gln(353) gene polymorphism: OR = 1, 95 % CI = 0.6-1.7; p = 0.9). However, a joint effect on the risk of CAD was found between factor V point mutation (AG genotype) and M1M1 genotype (OR = 3.6, 95 % CI = 1-12.9; p = 0.03). Additionally, an interactive effect on CAD risk was found between AG genotype and metabolic risk factors (OR = 3.8, 95% CI = 1.1-13.6; p = 0.03). In conclusion, we provide evidence for a joint effect on CAD risk between G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism as well as between factor V point mutation and metabolic risk factors.     

Specific binding of human MSH2.MSH6 mismatch-repair protein heterodimers to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases.

Previous studies have demonstrated recognition of DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee, E., and Hays, J. B. (1991) Genetics 129, 1007-1020) and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760-769) long-patch mismatch-repair systems. Mismatch repair directed specifically against incorrect bases inserted during semi-conservative DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-T cis-syn-cyclobutane pyrimidine-dimers or T-T pyrimidine-(6-4')pyrimidinone photoproducts, with all four possible bases opposite the respective 3'-thymines in the photoproducts, were analyzed for the ability to compete with radiolabeled (T/G)-mismatched DNA for binding by highly purified human MSH2.MSH6 heterodimer protein (hMutSalpha). Both (cyclobutane-dimer)/AG and ((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of photoproduct/(A(T or C)) mismatches also showed higher hMutSalpha affinity than photoproduct/AA "matches"; the apparent affinity of hMutSalpha for the ((6-4)photoproduct)/AA-"matched" substrate was actually less than that for TT/AA homoduplexes. Surprisingly, although hMutSalpha affinities for both non-photoproduct UU/GG double mismatches and for (uracil-cyclobutane-dimer)/AG single mismatches were high, affinity for the (uracil-cyclobutane-dimer)/GG mismatch was quite low. Equilibrium binding of hMutSalpha to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced similarly by ATP (in the absence of magnesium).     

孔石莼(Ulva pertusa)质体蓝素基因的克隆及基因特征分析(英文)

采用cDNA末端快速扩增的办法,从孔石莼(Ulva pertusa)中克隆获得质体蓝素基因。该基因完整的cDNA为787bp,包括40 bp 5’端非编码区和327 bp的3’端非编码区,以及一个420 bp的开放阅读框架,编码139个氨基酸的蛋白质。该基因编码质体蓝素的前体肽,其N端41个氨基酸残基为信号肽,后面为98个氨基酸残基的成熟肽。从Genbank中选择了13个质体蓝素的前体肽基因进行序列比对分析和构建进化树。孔石莼质体蓝素基因与其它质体蓝素基因的同源性为48.2%至78.8%。该进化树将来源于6种藻类植物的7个质体蓝素基因聚类在一起,显示出它们较近的进化关系。同样,也表现出11种生物的分子进化关系。序列比对结果显示,在质体蓝素的基因序列中存在两个高度保守的基序,它编码质体蓝素蛋白的铜结合活性位点。     

Regulation of mammalian nitric oxide synthases by electrostatic interactions in the linker region of calmodulin

Calmodulin (CaM), the ubiquitous Ca(2+)-sensing protein, consists of two globular domains separated by a flexible central linker that properly orients CaM's globular domains to bind and regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. In the present study we determined that the charge and length of the central linker of CaM has an effect on the binding and activation of the NOS isozymes by using a variety of charge CaM mutants (T79D, S81D, T79D/S81D, S101D and E84R/E87K) and CaM mutants with residues removed (Delta84, Delta83-84, and Delta81-84). Our kinetic and spectropolarimetry results demonstrate that the NOS enzymes are not adversely affected by the CaM mutants with the exceptions of S101D, E84R/E87K and the deletion of residue 84. Electrostatic interactions in the central linker between residues 82-87 in combination with hydrophobic interactions in the globular domains of CaM are important for its tight association to inducible NOS.     

cDNA cloning and characterization of the novel genes related to aldehyde dehydrogenase from wild Chinese grape (Vitis pseudoreticulata W. T. Wang).

mRNA differential display was employed to study the gene differential expression of wild Chinese grape (Vitis pseudoreticulata W. T. Wang) infected by Uncinula necator in different periods, a cDNA fragment of T11AC/B0319-456 coded by aldehyde dehydrogenase (ALDH) gene has been obtained. 5' RACE and 3' RACE have been used to clone the whole cDNA sequences of ALDH which consists of three cDNA sequences, whose sizes are 1887, 1956 and 1961 bp, and they encoded a polypeptide size of 537, 524 and 477 designated as VpALDH2a, VpALDH2b and VpALDH1a, respectively. The deduced amino acid sequence shared highly identity with other plants and Human ALDH. Both VpALDH2a and VpALDH2b protein contain putative mitochondrial targeting sequence except VpALDH1a, it indicates that VpALDH2a and VpALDH2b are mitochondrial enzymes, and VpALDH1a is cytosolic enzyme. The VpALDH2a was subcloned into the expression vector pGEX-4T-1, transformed into E.coli BL 21-coden plus induced by IPTG, and about Mr. 85 kD of GST-ALDH fusion protein displayed in SDS-PAGE gel.     

Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: An example of matrix metalloproteinases

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence⁴⁰⁰His-(X)₃-His-(X)₂₈-Asp-Asp-(X)₂-⁴³⁶Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ substrate by 79%. The wild-type and D432G and D433E mutant enzymes had similarK _m values for the synthetic substrate and similarK _i values for the competitive inhibitor, GM6001. Thek _cat/K _m values for D432G and D433E mutant enzymes, however, were reduced by a factor of 4 and their K _a ^Ca values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.Abbreviations MMP Matrix metalloproteinase - TIMP tissue inhibitor of metalloproteinase - IPTG isopropyl-D-thiogalactoside - APMA 4-aminophenyl-mercuric acetate - PCR polymerase chain reaction - Dpa 3(2,4-di-nitrophenyl) diaminopropionic acid - Mca 7-methoxycoumarin acetic acid