新疆3种藜科盐生植物NHX基因的克隆与序列分析比较

从新疆野生植物盐角草(Salicornia europaea)、盐爪爪(Kalidium foliatum)和盐穗木(Halostachys caspica)中分别克隆了约1.7kb的NHX基因cDNA片段,此片段均包含了NHX完整基因,三者之间有较高的同源性,盐角草NHX基因与盐爪爪NHX基因同源性达92.81%,盐角草与盐穗木同源性达92.19%,盐爪爪与盐穗木同源性达97.66%.它们与其它几种藜科盐生植物如滨藜、碱蓬、灰绿藜的NHX基因同源性也很高,达到80%以上,与拟南芥同源性也达到86%.此基因在植物尤其是藜科盐生植物中高度保守,其编码的功能性蛋白可能在影响植物耐盐中起作用.     

盐桦BhNHX的克隆及其与CaM在胁迫下的协同表达

以盐桦组培苗为材料,通过RACE技术克隆了液泡膜Na /H 反向运输体基因BhNHX,采用DNAman软件和BLASTN对cDNA序列进行分析并与其他植物的NHX基因进行同源性比较,最后对BhNHX和钙调蛋白基因(CaM)在各种胁迫条件下的协同表达进行半定量的RT-PCR分析.结果显示:(1)获得了BhNHX的全长cDNA序列,包含1 623 bp的开放阅读框架,编码一个540个氨基酸的多肽,有11个推测跨膜区,与已报道的液泡膜Na /H 反向运输载体蛋白跨膜区数目一致;(2)BhNHX的核酸序列与其他植物NHX具有较高同源性,与葡萄NHX序列一致性达到76%;(3)BhNHX和钙调蛋白基因CaM可同时被盐、低温、干旱所诱导;但ABA只能较明显诱导BhNHX的表达,而对CaM的诱导表达不明显.研究表明,在盐桦受到盐、低温及干旱胁迫时BhNHX和CaM可能协同表达,而在ABA诱导时两者可能具有不同的表达调控模式.     

灰绿藜和碱蒿NHX基因 3'-UTR序列的差异性分析

采用3'末端快速扩增技术,分别从新疆野生盐生植物灰绿藜和碱蒿中克隆CgNHX和AaNHX cDNA 3'末端.测序结果表明克隆获得的片段为CgNHX和AaNHX基因3'末端,并且CgNHX和AaNHX3'-UTR的不同拷贝之间存在很大的差异性.序列同源性分析结果显示,AaNHX基因3'-UTR与CgNHX基因3'-UTR同源性高达52%,两者与SsNHX(碱蓬NHX)同源性分别达到60%和54%,表明NHX基因的加工方式可能相同,同时说明该基因在野生盐生植物中的功能是很重要的.     

盐生植物碱蓬Actin基因片段的克隆及序列分析

目的:克隆盐生植物碱蓬(Suaeda glauca)Actin基因片段,为研究其它基因在碱蓬的表达和调控提供内参基因.方法:根据已知植物Acfin基因的保守序列设计一对简并性引物,采用RT-PCR的方法扩增Actin基因片段,使用分子生物学软件进行序列分析.结果:获得一段大小为598bp的基因片段,编码198个氨基酸;该序列与其它Actin基因核苷酸序列的同源性均在80%以上,与氨基酸序列的同源性达93%以上.结论:克隆的基因为Actin基因片段,将其命名为SgACT,并登录在GenBank,登录号为EU429457.     

胡杨Na+/H+反向运输载体(PeNHX2)基因的克隆与序列分析

植物液泡膜上Na /H 反向运输载体(Na /H antiporterNHX)已被证明在盐胁迫中发挥关键作用,该研究旨在从胡杨中克隆获得耐盐相关的的完整cDNA序列,为进一步比较胡杨的耐盐基因的差异,探讨胡杨的耐盐分子机理奠定了基础,并为通过转基因手段提高木本植物的耐盐能力提供侯选基因。根据已发表NHX的同源基因序列,采用特异性引物扩增核心片段,并结合5’与3’RACE技术,成功地从胡杨(populus euphratica)克隆获得PeNHX2,其cDNA全长1638bp。测序和序列分析结果表明该基因与已发表毛白杨(Populus tomentosa)PtNHX基因在核酸水平及蛋白质水平上同源性均达到最高,依次为90.6%和88.25%。利用DNAMAN软件进一步分析得知,该基因与新疆盐生植物中所克隆获得的的犁苞滨藜(Atriplex dimorphostegia)AdNHX1、灰绿藜(Chenopodium glaucum)CgNHX1、盐爪爪(Kalidium foliatum)KfNHX同源性也较高,核酸水平上依次为75.1%、74.8%、74.3%,蛋白质水平依次为78.3%、79.0%、78.3%。PeNHX2与NCBI已注册的部分PeNHX1序列,在核酸或蛋白质水平同源性(72.34%和73.72%)均较低,分析它们可能同属于胡杨NHX基因家族,相关生物学功能有待于进一步研究。     

单引物方法克隆盐角草orf25基因

采用单引物RT-PCR扩增的方法,从新疆野生植物盐角草（Salicornia europaea）中克隆获得了1.7kb的cDNA片段,经过测序和序列分析,发现基因片段包含了orf25基因完整的读码框架。采用序列同源性分析方法,结果显示新疆盐角草orf25基因与甜菜线粒体同源性高达98%,与烟草线粒体同源性达到95%,与小麦同源性为92%,与玉米同源性为88%,表明orf25基因在植物中是高度保守的一种基因,同时说明野生植物盐角草中也存在与农作物相似的雄性不育相关基因,orf25基因编码的功能性蛋白可能在影响植物雄性不育改良作物品种方面具有重要的意义。     

盐生植物盐爪爪甜菜碱醛脱氢酶基因的克隆及在盐胁迫下的BADH基因的表达

根据已发表的几种藜科植物甜菜碱醛脱氢酶(BADH)基因的同源保守区设计了一对引物,采用RT-PCR方法从盐生植物盐爪爪(Kalidium foliatum)中扩增出BADH基因的1个开放阅读框架,其核苷酸序列长1503bp,推测的氨基酸序列全长为500个氨基酸残基。核苷酸序列与藜科几种盐生植物如滨藜、碱蓬、菠菜、山菠菜和甜菜等的同源性为81%,与甜土植物水稻的同源性为69%。氨基酸序列与以上两类植物(盐生植物和甜土植物)的同源性比对为80%和71%,说明BADH基因在藜科盐生植物中是一种较高保守的基因。BADH基因编码的多肽在高等植物中行使重要的功能。用不同浓度的NaCl胁迫处理盐爪爪植株,BADHmRNA的表达水平比对照植株高,说明盐爪爪BADH基因的表达受盐诱导,间接说明甜菜碱醛脱氢酶催化合成的甜菜碱作为渗透调节的小分子物质,它的积累与盐胁迫存在紧密关联,本研究为进一步从生理和分子水平阐明盐爪爪的耐盐机制提供一定的参考。     

盐生植物盐爪爪甜菜碱醛脱氢酶基因的克隆及在盐胁迫下的BADH 基因的表达

根据已发表的几种藜科植物甜菜碱醛脱氢酶(BADH) 基因的同源保守区设计了一对引物, 采用RT-PCR 方法从盐生植物盐爪爪( Kalidium foliatum) 中扩增出BADH 基因的1 个开放阅读框架, 其核苷酸序列长1 503 bp , 推测的氨基酸序列全长为500 个氨基酸残基。核苷酸序列与藜科几种盐生植物如滨藜、碱蓬、菠菜、山菠菜和甜菜等的同源性为81% , 与甜土植物水稻的同源性为69%。氨基酸序列与以上两类植物(盐生植物和甜土植物) 的同源性比对为80% 和71% , 说明BADH 基因在藜科盐生植物中是一种较高保守的基因。BADH 基因编码的多肽在高等植物中行使重要的功能。用不同浓度的NaCl 胁迫处理盐爪爪植株, BADH mRNA 的表达水平比对照植株高, 说明盐爪爪BADH 基因的表达受盐诱导, 间接说明甜菜碱醛脱氢酶催化合成的甜菜碱作为渗透调节的小分子物质, 它的积累与盐胁迫存在紧密关联, 本研究为进一步从生理和分子水平阐明盐爪爪的耐盐机制提供一定的参考。     

RLM-RACE法扩增獐茅液泡膜Na+/H+逆向转运蛋白基因5'-cDNA末端序列

根据已获得的盐生植物獐茅(Aeluropus littoralis var.sinensis Debeaux)液泡膜Na+/H+逆向转运蛋白(Na+/H+antiporter)基因部分cDNA片段,设计2条特异引物(GSP1、GSP2),采用RLM-RACE(RNA ligase-mediated rapid amplification of 5'and 3'cDNA ends)法获得了其5'-cDNA末端序列,并找出了转录起始位点.该片段长816 bp,与芦苇液泡膜Na+/H+逆向转运蛋白基因序列同源性最高达91%,与拟南芥、盐角草、水稻、大麦、小麦的同源性分别为81%、82%、86%、87%和81%,为该基因全长的克隆及启动子的研究奠定了基础.与其它测定基因转录起始位点的方法相比,RLM-RACE方法更快捷、简便、准确.     

拒盐型盐生植物小花碱茅肌动蛋白基因片段的克隆及序列分析

本研究根据其它植物Actin基因的保守序列设计一对简并性引物,以拒盐型盐生植物小花碱茅根部总RNA为模板,采用RT-PCR的方法扩增出Actin基因片段并克隆到PUCm-T载体,阳性克隆经PCR检测后进行测序,在GenBank中注册;序列分析结果表明:该片段长约600 bp,编码198个氨基酸;所得序列与GenBank中注册的其它植物Actin基因序列同源性均在84%以上,与其它肌动蛋白的氨基酸序列同源性达94%以上.     

Identification of novel genes associated with conidiation in Beauveria bassiana with suppression subtractive hybridization

The conidiation of the entomopathogenic fungus Beauveria bassiana (Hyphomycete) is a complex process that involves the stage- and cell-type-specific expression of hundreds of genes. The suppression subtractive hybridization method was used to target genes involved in conidiation. Seventeen genes were cloned that potentially were involved in conidia formation. Six of them demonstrated differential expression between conidial and vegetative cultures. Sequence analysis showed three cDNA fragments had similarity to known genes involved in either cellular metabolism or cell regulatory processes. The other cDNA fragments showed low or no similarity to any genes previously described. The full-length cDNA and genomic sequence of a gene designated A43 was isolated. The A43 protein is composed of 180 amino acids and has 34% identity to a RNA-binding region-containing protein. The temporal expression pattern was consistent with the gene being involved in conidiation. The colony morphology of the A43 knock-out mutant had more floccus mycelium than the wild-type and also produced fewer conidia, indicating the A43 gene is involved in B. bassiana conidiation.     

Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.)

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in λgt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca²⁺-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.     

A calmodulin endoproteinase from mitochondrial membranes

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.     

Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD

To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 alpha-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated.     

烟草中一条新的S-腺苷甲硫氨酸合成酶基因的克隆及表达分析

利用差异显示技术从烟草中分离到一个受乙烯利诱导表达的cDNA片段,结合RACE扩增技术获得该基因的全长序列。该全长cDNA共含1350个碱基,编码区有1170 bp,通过GenBank同源性比较发现它与一条已知的烟草S-腺苷甲硫氨酸合成酶(SAMS1,EC2．5．1．6)的基因编码区存在90％的同源性,编码的氨基酸顺序96％同源,而在cDNA的5’及3’非编码区同源性很低,且有一些短片段的缺失。设计合适的引物分别对这两个SAMS基因进行RT-PCR分析表明,SAMS1基因受高温诱导表达增强,但不受甲基紫晶诱导的氧化胁迫及盐胁迫影响,而新分离到的这个基因(SAMS2)的表达同时受高温、甲基紫晶及高盐3种胁迫的诱导。     

Molecular profiling of bacterial species in the rabbit caecum

The diversity of bacteria present in the caecum of the rabbit was investigated. Partial bacterial 16S rRNA genes from a digested sample collected from the caecum of an adult rabbit were amplified by PCR. Sequence analysis of the amplified fragments indicated highest similarity was to bacterial sequences previously described from other gut environments. However, only one sequence showed significant identity (97% threshold) to any previously described bacterial 16S rRNA genes. Furthermore, most of the sequences clustered together in groups lacking representatives from sequences already described, suggesting that the rabbit caecal flora contains organisms not previously described.     

草莓果实MADS-box基因保守片段的克隆与序列分析

根据多种植物MADS-box基因保守区序列设计简并引物,应用PCR技术从草莓绿果中分高出33条MADS-box基因cDNA片段.序列分析表明,这些片段长度在137 - 146 bp之间,包含基因起始密码子.推导的氨基酸序列与已登录的草莓的MADS-box基因同源性超过87％.推导的氨基酸序列与已知的革莓和其他物种调控果实发育成熟的MADs-box基因以及拟南芥的MADS-box家族基因进行系统发育分析,可将这些基因片段分科归入拟南芥MADS-box基因不同亚家族中,证明草莓果实中存在各类MADS-box家族基因,克隆的部分片段可能参与调控果实发育和成熟软化的调节.     

The amino acid sequence of glutamate decarboxylase from Escherichia coli. Evolutionary relationship between mammalian and bacterial enzymes.

The amino acid sequence of glutamate decarboxylase from Escherichia coli was solved by a combination of automated Edman degradation of peptide fragments derived by proteolytic and chemical cleavage and sequencing of DNA. Correct alignment of three peptides, for which no peptide overlaps were available, was achieved by sequencing a 1.1-kbp fragment of DNA produced by a polymerase-chain reaction using primers corresponding to sequences known to be in amino-terminal and carboxy-terminal regions of the protein. Sequence similarity (24% identity) with mammalian glutamate decarboxylase was found to be limited to a 55-residue sequence around the lysine residue that binds the coenzyme. Stronger similarity (38% identity), again confined to the same region, is seen with bacterial pyridoxal-phosphate-dependent histidine decarboxylase.