基因改造Rhodobacter sphaeroides提高

[目的]通过敲除类球红细菌2.4.1基因组中八氢番茄素合成酶基因crtB,让异戊二烯前体更多流向辅酶Q10的合成.引入大肠杆菌编码的分支酸裂解酶基因ubiC和4-羟苯甲酸转移酶基因ubiA,提高4-羟苯甲酸的合成和与聚异戊二烯的连接,从而提高类球红细菌的辅酶Q10产量.[方法]以自杀型质粒pSUP202为载体,构建包含crtB基因上游2.5 kb片段,壮观霉素抗性基因,ubiC、ubiA基因和crtB基因下游2.5 kb片段的基因置换质粒,利用结合转移方法转入类球红细菌2.4.1中,利用抗性机制筛选双交换突变株,RT-PCR方法检测引入的ubiC和ubiA基因转录.用HPLC方法测定出发菌株和基因改造菌株的辅酶Q10产量.[结果]成功构建出基因置换质粒,筛选出发生基因置换的突变株,RT-PCR证实了外源基因的转录,并且突变株辅酶Q10的产量比出发菌株提高40％.[结论]大肠杆菌的ubiC和ubiA基因能够利用自身启动子在类球红细菌中表达,利用基因改造的方法能成功提高类球红细菌的辅酶Q10产量.     

Uncv无毛小鼠无毛基因的分子克隆及序列分析

目的 探讨Unev无毛小鼠的无毛性状与无毛基因(hairless gene,hr)的相关性.方法 参照Gen-Bank上公布的小鼠的hr序列,设计5对引物,用RT-PCR方法对本单位培育的Unev无毛小鼠hr的编码区序列进行了克隆与分析.结果 获得了Unev无毛小鼠及野生型hr的全部编码区序列(3546 bp).Unev无毛小鼠hr基因与野生型小鼠hr基因的长度及序列完全一致,同源性为100%.与GenBank上发表的国外小鼠hr基因序列(Z32675)相比,同源性为99.7%,共10个碱基发生了突变,其中2个碱基突变导致了相应的氨基酸突变;和昆明小鼠的hr(AY547391)相比,同源性为99.6%,共12个碱基发生了突变,其中3个碱基突变导致了相应的氨基酸突变;但这些突变是由种属差异造成的.结论 Uncv无毛小鼠的无毛性状产生与hr基因无关.     

棉铃虫多核型多角体病毒v-cath同源基因的克隆及序列分析

为获得棉铃虫多核衣壳型多角体病毒(Helicoverpa armigera multiple nucleocapsid nucleopolyhedrovirus)基因组序列,采用随机克隆方法,建立HearMNPV的质粒基因文库,并通过对插入片段进行克隆鉴定和序列分析,获得编码组织蛋白酶基因v-cath。该基因阅读框为1026bp,共编码341个氨基酸。核苷酸和氨基酸同源性比较结果表明:HearMNPV的v-cath基因与蓓带夜蛾核型多角体病毒B(Mamestra configurata NPV-B)的同源性最高,而与苹果皮小卷蛾颗粒体病毒(Cydiapomonella GV CpGV)同源性最低,由此认为,杆状病毒科的v-cath基因在进化上存在2种进化方式:一类以点突变为主,基因长度变化不明显;另一类突变以小片段的碱基增减为特征。     

离子束诱变粟酒裂殖酵母产辅酶Q_(10)的初步研究

辅酶Q10(coenzyme Q10,CoQ)对心脏充血性病人有较好的疗效,是临床常用药物之一。实验研究了离子束诱变粟酒裂殖酵母对提高CoQ10的产量的影响与作用。实验筛选出六株突变菌株,研究了突变株生理生化特性。结果表明:突变菌株的CoQ10产量都有不同程度的提高,其中编号为N1菌株产量达6.9344mg/L,是对照菌株的10倍多,最低的N2菌株的产量也是对照菌株的1.3倍。     

离子束诱变粟酒裂殖酵母产辅酶Q10的初步研究

辅酶Q10(coenzyme Q10,CoQ)对心脏充血性病人有较好的疗效,是临床常用药物之一.实验研究了离子束诱变粟酒裂殖酵母对提高CoQ10的产量的影响与作用.实验筛选出六株突变菌株,研究了突变株生理生化特性.结果表明:突变菌株的CoQ10产量都有不同程度的提高,其中编号为N1菌株产量达6.9344 mg/L,是对照菌株的10倍多,最低的N2菌株的产量也是对照菌株的1.3倍.     

猪肺炎支原体P46基因的突变及序列分析

目的:将猪肺炎支原体P46基因中编码Trp的密码子TGA突变为TGG,为P46蛋白的研究及猪肺炎支原体抗体检测方法的建立奠定基础.方法:参考GenBank登录的猪肺炎支原体(Mycoplasma hyopneumoniae Mhp)P46基因序列,利用Primer 5.0软件设计合成一对引物,对猪肺炎支原体强毒株F60 P46基因的编码区进行扩增,后又设计了三对突变引物,通过重叠延伸PCR(SOE-PCR)对猪肺炎支原体P46基因的三个位点进行定点突变.结果:得到的序列与GenBank中登录的P46基因的核苷酸及氨基酸序列进行序列比较,结果表明它们有较高的同源性.突变后测序结果表明已成功将猪肺炎支原体P46基因中编码Trp的密码子TGA突变为TGG.结论:已成功突变Mhp P46基因并且序列比较显示其与其它序列同源性较高     

叶绿体基因infA-rpl36区域在小麦族物种中的序列变异分析

利用小麦叶绿体基因组中infA-rpl36区域的序列设计引物, 对小麦族(Triticeae)的12个二倍体和多倍体的物种进行了PCR扩增和序列测定, 获得了长度为584～603 bp的12条DNA序列。序列分析表明, 供试物种在infA-rpl36基因间隔区的核苷酸变异明显高于基因编码区。基因编码区核苷酸序列同源性高达97%, 表明了目标片段具有高度的保守性。但在5个物种的infA编码区出现了较大的插入、缺失突变, 导致推导的氨基酸序列也发生了很大的变化, 证实了infA基因是叶绿体基因组中最活跃的基因之一, 而rpl36基因的变异较小, 说明不同叶绿体基因的进化速度是不同的。基于测定序列建立的种系树分析发现, 多倍体物种中间偃麦草(Thinopyrum intermedium)具有多种不同的细胞质起源, 与核基因组一样在进化上较为复杂。     

中国人Ⅱ型MPS家系IDS基因的一种新突变的鉴定

为了研究粘多糖贮积症Ⅱ型(MPSⅡ)患者发病的分子遗传学机制, 以便为今后的产前基因诊断等创造必要的前提条件, 文章先采用尿糖胺聚糖(GAGs)定性检测法对疑似MPSⅡ的先证者进行初诊, 然后采用PCR、PCR 产物直接测序法对先证者及其家系成员进行突变检测。在检出IDS基因c.876del2新突变后, 对随机采集的120例正常对照和其他非II型MPS患者包括MPSⅠ, Ⅳ, Ⅵ三型的病人共15例的IDS基因exon 6进行序列分析, 同时采用不同物种突变点序列的保守性分析法, 以及直接测定患儿及其家庭相关成员IDS酶活性的方法对该新突变进行致病性分析。结果显示: 先证者尿检呈强阳性(GAGs +++); 其IDS基因exon 6编码区内存在c.876-877 del TC新缺失突变, 为半合子突变, 而其母、其姐为杂合突变; 正常对照和其他非II型MPS患者的IDS基因exon 6的检测结果均未发现该突变; 不同物种氨基酸序列的同源性比对显示: c.876-877 del TC突变所在的位置即p.292-293的苯丙氨酸(F)谷氨酰胺(Q)高度保守; 酶活性测定的结果显示: 先证者的IDS酶活性仅为2.3 nmol/4 h/mL, 大大低于正常值, 而其父的为641.9 nmol/4 h/mL, 其母的血浆酶活性为95.8 nmol/ 4h/mL, 其姐的为103.2 nmol/4 h/mL。说明所发现的c.876-877 del TC缺失移码突变是一种新的病理性突变, 是该MPSⅡ患儿发病的根本内因。     

二倍体蔷薇FT基因序列多样性研究

以3个类群73个二倍体蔷薇属(Rosa)植物为材料,克隆获得其FLOWERING LOCUS T(FT)同源基因,并对该基因的编码区序列进行多态性分析以及多维尺度(MDS)聚类分析。结果显示,73个二倍体蔷薇植物的FT基因共检测到215个核苷酸多态性位点,其中包括214个SNP和1个缺失突变,平均185个碱基发生1次突变;氨基酸多态性分析结果显示共有35个氨基酸发生变异,平均379.6个氨基酸残基发生1次突变;突变位点统计分析结果发现39、258、426 bp位点是高频突变位点,其碱基由A或C突变为T。MDS聚类分析结果表明,3个类群FT基因编码区序列的碱基组内差异依次排序为:野生种月季组中国古老月季,氨基酸组内差异依次排序为:中国古老月季月季组野生种,推测中国古老月季在长期栽培驯化过程中,其FT基因可能经历了较强的人工选择压力,月季组的种和变种可能是古老月季的重要亲本来源。     

野猪MC4R基因的克隆及变异初步研究

黑素皮质素受体4是在人类肥胖研究中发现的重要调节因子,参与调节动物的体重、采食量和能量稳态,缺失MC4R基因的突变纯合体小鼠出现遗传性肥胖。为了进一步揭示其群体遗传变异,寻找新的遗传标记,本研究对野猪(Sus scrofa ussuricus)MC4R基因进行了克隆(GenBank accession NoDQ388767)和序列分析,并对所发现的错义突变进行了基于限制性内切酶HindⅢ的PCR-RFLP分析。序列分析表明野猪与民猪MC4R基因的编码区序列完全相同,与大白猪相比存在4个SNPs;对14头野猪的酶切多态性分析表明该突变位点是多态位点,并且3种基因型的分布符合Hardy-Weinberg定律。结果表明,野猪具有独特的遗传信息。     

Arabinan-deficient mutants of Corynebacterium glutamicum and the consequent flux in decaprenylmonophosphoryl-D-arabinose metabolism

The arabinogalactan (AG) of Corynebacterianeae is a critical macromolecule that tethers mycolic acids to peptidoglycan, thus forming a highly impermeable cell wall matrix termed the mycolyl-arabinogalactan peptidoglycan complex (mAGP). The front line anti-tuberculosis drug, ethambutol (Emb), targets the Mycobacterium tuberculosis and Corynebacterium glutamicum arabinofuranosyltransferase Mt-EmbA, Mt-EmbB and Cg-Emb enzymes, respectively, which are responsible for the biosynthesis of the arabinan domain of AG. The substrate utilized by these important glycosyltransferases, decaprenylmonophosphoryl-D-arabinose (DPA), is synthesized via a decaprenylphosphoryl-5-phosphoribose (DPPR) synthase (UbiA), which catalyzes the transfer of 5-phospho-ribofuranose-pyrophosphate (pRpp) to decaprenol phosphate to form DPPR. Glycosyl compositional analysis of cell walls extracted from a C. glutamicum::ubiA mutant revealed a galactan core consisting of alternating beta(1-->5)-Galf and beta(1-->6)-Galf residues, completely devoid of arabinan and a concomitant loss of cell-wall-bound mycolic acids. In addition, in vitro assays demonstrated a complete loss of arabinofuranosyltransferase activity and DPA biosynthesis in the C. glutamicum::ubiA mutant when supplemented with p[14C]Rpp, the precursor of DPA. Interestingly, in vitro arabinofuranosyltransferase activity was restored in the C. glutamicum::ubiA mutant when supplemented with exogenous DP[14C]A substrate, and C. glutamicum strains deficient in ubiA, emb, and aftA all exhibited different levels of DPA biosynthesis.     

Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon

The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1). Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.     

霍乱弧菌中调控aphB 的基因筛选及其功能

【目的】筛选霍乱弧菌C6706-中调控LysR家族蛋白AphB表达的基因。【方法】将霍乱弧菌埃尔托型菌株C6706-aphB启动子区克隆到2个报告质粒pBBRLux和pKP302上,并将其导入霍乱弧菌C6706-中,以此作为出发菌株。利用出发菌株与转座子pSC123接合构建LZV630-302转座子随机突变文库,通过测定化学发光强度检测aphB启动子的表达水平,筛选aphB表达受影响的突变株。利用随机PCR方法检测转座子插入位点,并测序比对分析基因。【结果】从7个转座子库中(共约4万个突变株)得到能影响aphB表达(均导致下降)的2株突变株T1和T2。测序比对发现T1中转座子插入在vc1585读码框内,T2中转座子插入在距vc1602基因末端7 bp处。【结论】获得aphB表达改变的突变株,基因vc1585和vc1602可能直接或间接影响aphB表达,为进一步研究aphB表达调控影响因素奠定了基础。     

耐高温耐酸稳定假密环菌(Armillariella tabescens) MAN47β-甘露聚糖酶体外分子定向进化

从已经建立的易错PCR初级突变文库中筛选得到的16个兼具耐酸、高温稳定、高酶活力的克隆出发,将其作为DNA shuffling的亲本基因,利用DNA shuffling技术,结合易化筛选和96微孔板通量筛选的方法获得耐酸、高温稳定的克隆。并且,对筛选出来的耐酸高温稳定的突变子进行测序分析和同源建模,比较分析β-甘露聚糖酶突变基因序列的生物学信息。经过两轮DNAshuffling,最终筛选得到一个耐酸高温稳定突变体1108,其在90℃时酶活力还能维持在70%;在pH 3.0时酶活力维持在70%;在高温80℃和pH 4.0的条件下,酶活力是野生型的10倍;常规条件下(pH 6.0,40℃),酶活力是野生型酶的5倍。序列比对发现耐酸、高温稳定突变体1108有三个碱基发生了改变(T289A、A535T、T1085C),导致相应的氨基酸发生了改变(Ser97Thr、Val362Ala、Ile179Leu)。根据同源建模结果和氨基酸性质研究发现,突变位点位于催化中心附近,推测第97位的突变与酶的耐酸性和活性有关,第179位和第362位的突变与酶的高温稳定性有关。实验结果为进一步了解β-甘露聚糖酶MAN47的结构与功能之间的关系提供有用参考。     

Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase.

In Escherichia coli, chorismate lyase catalyzes the first step in ubiquinone biosynthesis, the conversion of chorismate to 4-hydroxybenzoate. 4-Hydroxybenzoate is converted to 3-octaprenyl-4-hydroxybenzoate by 4-hydroxybenzoate octaprenyltransferase. These two enzymes are encoded by ubiC and ubiA, respectively, and have been reported to map near one another at 92 min on the E. coli chromosome. We have cloned the ubiCA gene cluster and determined the nucleotide sequence of ubiC and a portion of ubiA. The nucleotide sequence abuts with a previously determined sequence that encodes a large portion of ubiA. ubiC was localized by subcloning, and overproducing plasmids were constructed. Overexpression of ubiC allowed the purification of chorismate lyase to homogeneity, and N-terminal sequence analysis of chorismate lyase unambiguously defined the beginning of the ubiC coding region. Although chorismate lyase showed no significant amino acid sequence similarity to 4-amino-4-deoxychorismate lyase (4-amino-4-deoxychroismate----4-aminobenzoate), the product of E. coli pabC, chorismate lyase overproduction could complement the growth requirement for 4-aminobenzoate of a pabC mutant strain. Of the several enzymes that convert chorismate to intermediates of E. coli biosynthetic pathways, chorismate lyase is the last to be isolated and characterized.     

Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.     

Activation of cryptic aminoglycoside resistance in Salmonella enterica

Aminoglycoside resistance in bacteria can be acquired by several mechanisms, including drug modification, target alteration, reduced uptake and increased efflux. Here we demonstrate that increased resistance to the aminoglycosides streptomycin and spectinomycin in Salmonella enterica can be conferred by increased expression of an aminoglycoside adenyl transferase encoded by the cryptic, chromosomally located aadA gene. During growth in rich medium the wild-type strain was susceptible but mutations that impaired electron transport and conferred a small colony variant (SCV) phenotype or growth in glucose/glycerol minimal media resulted in activation of the aadA gene and aminoglycoside resistance. Expression of the aadA gene was positively regulated by the stringent response regulator guanosine penta/tetraphosphate ((p)ppGpp). SCV mutants carrying stop codon mutations in the hemA and ubiA genes showed a streptomycin pseudo-dependent phenotype, where growth was stimulated by streptomycin. Our data suggest that this phenotype is due to streptomycin-induced readthrough of the stop codons, a resulting increase in HemA/UbiA levels and improved electron transport and growth. Our results demonstrate that environmental and mutational activation of a cryptic resistance gene can confer clinically significant resistance and that a streptomycin-pseudo-dependent phenotype can be generated via a novel mechanism that does not involve the classical rpsL mutations.     

农杆菌介导转化黑曲霉分生孢子及产孢缺陷突变子T-DNA插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)T-DNA系统,建立转化黑曲霉(Aspergillus niger)分生孢子的方法,构建T-DNA插入突变子文库,为黑曲霉基因组功能注释研究打下基础。【方法】采用携带二元质粒载体pCAMBIA1301的农杆菌EHA105,诱导转化黑曲霉分生孢子,筛选具有潮霉素抗性的突变子。分析抗性稳定突变子菌株的表型,采用反向PCR方法分析T-DNA插入位点相邻位置的序列,并推测突变基因可能具有的功能。【结果】实验获得具有稳定潮霉素抗性转化子193株,转化率为5.6×102转化子/108分生孢子。部分转化子表型出现较为明显改变,其中一株不能产孢,对其T-DNA插入位点序列分析比对结果显示,突变基因属于超级转运家族(major facilitator superfamily,MFS)。【结论】本研究建立的农杆菌转化黑曲霉分生孢子平台,结合T-DNA插入突变位点分析,可以为黑曲霉基因组功能注释研究提供一种简便有效的途径。     

Characterization of calcium-binding sites in development-specific protein S of Myxococcus xanthus using site-specific mutagenesis

Protein S, the most abundant protein synthesized during development of the Gram-negative bacterium Myxococcus xanthus, assembles on the surface of the spores. It can be dissociated from the spores using divalent metal chelators and will reassemble on the spores in the presence of calcium. The amino acid sequence of protein S contains regions which have homology to the calcium-binding sites of calmodulin. Protein S was found to bind 2 mol of calcium/mol of protein with Kd values of 27 and 76 microM. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin (Ser40----Arg,Ser129----Arg), and a double mutant was also constructed. Each mutant gene was then transduced into the genome of a M. xanthus strain from which the wild-type genes had been deleted. All three mutants produced protein S normally during development. One of the mutants (Ser129----Arg) had normal amounts of protein S on its spores, whereas the other (Ser40----Arg) bound much less and the double mutant had virtually none. Analysis of the calcium binding affinities of the purified proteins showed that [Arg40]protein S and [Arg40, Arg129]protein S did not bind detectable quantities of calcium, whereas [Arg129]protein S bound less calcium than the wild-type protein and with a reduced affinity.     

Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity.