杨树派间不同种的遗传密码子使用频率分析

遗传密码子的简并性特征造成了不同物种使用的密码子存在偏爱性。了解不同物种的密码子使用特点,可以为外源基因导入过程中的基因改造提供依据,从而实现外源基因的高效表达。杨树是世界上广泛栽培的重要造林树种之一,已经成为林木基因工程研究的模式植物。本研究采用高频密码子分析法,对美洲山杨P.tremuloides,毛白杨P.tomentosa,美洲黑杨P.deltoids和毛果杨P.trichocarpa 4种杨树的蛋白质编码基因序列(CDS)进行了分析,计算出了杨树同义密码子相对使用频率(RFSC),确定了4种杨树的高频率密码子,发现虽然不同种类的杨树密码子使用上有一些差别,但是偏爱密码子的差别却很小,共性的密码子占绝大多数。仅有Pro,Thr和Cys等少数几个氨基酸的偏爱密码子有差别。这种“共性”提示我们,用不同种的杨树中任何一种杨树的偏爱密码子所设计的外源基因在其他杨树中也可以使用。     

杨树同义密码子用法的初步分析

杨树是世界上广泛栽培的重要造林树种之一,已经成为林木基因工程研究的模式植物。用杨树的314个蛋白编码基因,通过对应分析和ENC-plot分析探讨了若干重要因子对杨树密码子用法的效应。从分析结果中可以看出,在影响最大的第一条向量轴上,基因的坐标位置与该基因的表达水平(CAI)极显著负相关(r=-0.94**),其次是与GC3S和基因长度极显著相关(r=0.86**和r=-0.57**),说明基因表达水平高低是影响密码子发挥作用的主要因素,基因编码区碱基组成和基因长度次之。ENC-plot分析结果也证明了这一点。相对密码子使用值(RSCU)的计算结果表明,高表达基因强烈偏好以A或T结尾的密码子,并确定了TTA和ATA等10个密码子为杨树的主要偏爱密码子。将杨树的密码子使用频率与拟南芥、水稻、大肠杆菌和人等不同模式生物种比较后发现,杨树密码子的偏爱性与同为双子叶植物的拟南芥最为相似,与人和大肠杆菌之间的差异较大。     

鸟王茶基因密码子偏好性分析

生物体中普遍存在密码子使用偏好性,这对基因的异源表达效率具有较大影响.本研究使用CodonW软件对鸟王茶(Camellia sinensis var.niaowangensis)转录本中1 856个编码氨基酸的基因密码子进行分析,计算其密码子相对使用度,确定了27个最优密码子,并发现最优密码子大多以A或U结尾.通过ENC-GC3关联分析及PR2-plot偏倚分析,发现鸟王茶密码子使用模式受到突变和选择等多种因素的影响.与其他物种对比发现,鸟王茶密码子偏好性与其他物种存在不同程度的差异,其中与杨树及烟草差异最小.由于密码子具有通用性,分析结果对通过重建密码子提高茶树基因在其他植物中的表达效率从而验证鸟王茶基因功能具有一定意义.     

SARS冠状病毒的密码子偏爱性分析

为了分析SARS(Severe Acute Respiratory Syndrome)冠状病毒的密码子偏爱性(codon preference),为SARS冠状病毒基因表达中宿主系统的选择提供参考。运用EMBOSS(The European Molecular Biology Open Software Suite)的CHIPS(Codon Hetemzygosity in a Protein-codingSequence)和CUSP(Create a eodon usege table)程序对SARS冠状病毒的6个编码蛋白的基因进行分析,并将这6个编码序列拼接在一起进行全基因组的密码子偏爱性分析。分析结果与大肠杆菌、酵母及人的密码子偏爱性进行比较。结果显示SARS冠状病毒的CHIPS分析Nc(effective number of codons)值为53．338,S、E、M、N蛋白、3CL水解酶、RNA聚合酶的Nc值分别为45．733,61．000,59．040,46．618,46．924,51．902。编码SARS冠状病毒A,P,R,S,T,L等氨基酸的不同密码子使用频率有较大差异。大肠杆菌有25个、酵母有12个、人有20个密码子与SARS冠状病毒密码子使用偏爱性差异较大．因此可以得出结论：编码SARS冠状病毒氨基酸的密码子出现的频率较均一。SARS冠状病毒的密码子偏爱性与真核生物较接近,与原核生物相差较远,其基因表达选择在酵母等真核系统可能更为合适。     

同义密码子用语与蛋白质结构的关系

对大肠杆菌的５４种三维结构已知的蛋白质及其对应的ｍＲＮＡ序列的集合的分析结果表明,绝大部分密码子与其编码的氨基酸在蛋白质α螺旋、β折叠和卷曲等三种二级结构中的偏好性没有显著的统计性差异。同时,又对Ａｄｚｈｕｂｅｉ提供的相应哺乳类数据集作了同样的偏好性检验,表明９种氨基酸的同义密码子携带蛋白质二级结构信息,与他们的结论一致。推测,这种差异可能由翻译机制的进化趋异造成的。这些结果对于真核基因在原核生物中实现高表达可能有重要的实际意义。     

红松查尔酮合成酶基因CHS密码子偏好性分析

查尔酮合成酶(Chalcone synthase,CHS)广泛存在于植物体内,是花色素形成过程中一种重要的酶,可以进一步催化生成黄酮类化合物。本研究采用Codon W和EMBOSS在线软件对红松查尔酮合成酶基因CHS的密码子使用偏好性进行分析,并与北美乔松等其他24种植物的CHS基因以及模式植物基因组进行比较,对认识红松CHS基因的密码子使用偏好性,为选择适宜的表达系统奠定了一定的基础。研究结果表明:红松CHS基因编码区的有效密码子数(ENC)和GC含量分别为48.92和0.548,C+G含量高于A+T含量,密码子偏好以A/T结尾;多数植物CHS基因的G+C含量高于A+T含量,且密码子更偏好C/G结尾;聚类分析表明,红松与马尾松和赤松的密码子使用偏好性的相似性较高;密码子使用频率研究发现,红松CHS遗传转化与异源表达较优的受体可能是大肠杆菌和拟南芥。     

茶树CBF1基因密码子使用特性分析

转录因子CBF(C-repeat-binding factor)广泛存在于各种植物中, 是植物抗逆过程中一个重要的调节因子。文章运用CHIPS、CUSP和CodonW在线程序对茶树(Camellia sinensis)CBF1基因(CsCBF1)序列进行分析, 并与茶树基因、模式植物基因组和其他植物CBF基因进行比较, 对了解CsCBF1基因密码子使用特性, 并为其选择合适的表达系统具有重要意义。结果表明：CsCBF1基因与70个茶树基因对密码子的使用有明显的差异, CsCBF1基因偏好使用以G/C结尾的密码子, 而筛选的70个茶树基因偏好使用以A/T结尾的密码子。在密码子使用频率上, CsCBF1基因与拟南芥(Arabidopsis thaliana)、烟草(Nicotiana tobacum)的差异小于与小麦(Triticum aestivum)、玉米(Zea mays)的差异; 因此, 拟南芥、烟草更适合作为CsCBF1基因的外源表达宿主。通过分析40种植物CBF基因编码特点可知大部分CBF基因偏好使用以G/C结尾的密码子, 这可能与基因的特殊功能有关。     

杜仲基因密码子使用模式分析

为了解杜仲基因密码子使用模式,该文以杜仲基因组密码子为研究对象,运用CodonW软件对杜仲的320个蛋白编码基因进行同义密码子相对使用频率(RSCU)分析、ENC-GC3s关联分析编码基因的密码子ENC值、PR2-plot偏倚分析编码基因的密码子碱基使用频率,并运用CUSP软件与Codon Usage Database软件对杜仲基因密码子的GC含量、使用频率与代表性物种烟草、拟南芥、大肠杆菌和酿酒酵母的密码子GC含量和使用频率进行比较。结果表明:杜仲基因密码子的RSCU>1的密码子有30个,其中18个以G/C结尾、12个以A/U结尾,说明杜仲基因密码子偏好以G/C结尾,且偏好性较强;有效密码子数(ENC)范围为30~60,该范围内的密码子距离标准曲线较远,其ENC值小,偏好性较强;PR2-plot偏倚分析碱基使用频率显示,G>C、U>A;杜仲与代表性物种的GC含量分析显示,杜仲的GC12、GC3以及平均GC含量均高于代表性物种;杜仲与代表性物种的密码子使用频率分析显示,杜仲与烟草、酿酒酵母的密码子偏好较为接近,杜仲与拟南芥、大肠杆菌的密码子偏好差距较大。杜仲是我国特有的珍贵中药材,对其进行密码子使用模式分析,并研究其密码子偏好规律,为杜仲植物基因工程中外源基因的改良及表达提供了理论基础。     

用单链和PCR相结合的方法合成绿豆胰蛋白酶抑制剂基因

本文报道用单链与PCR技术相结台的合成方法合成了胰蛋白酶抑制剂基因,包括氨基酸编码顺序、起始和终止密码子,上、下游两端的EcoR Ⅰ和Pst Ⅰ限性酶识别顺序等全长共248个碱基对。合成基因的编码是参考其它植物的蛋白酶抑制剂基因的同源编码和植物基因的偏爱密码子来进行设计的。合成的基因双链经限制酶EcoR Ⅰ和Pst Ⅰ在两端切出粘性末端后克隆到pUCl9中,克隆基因结构的正确性经过限制酶图谱和DNA序列分析得到完全的证明。     

基于同义密码子使用的原核生物基因组大、小染色体及质粒进化特征分析北大核心CSCD

基于同义密码子偏好分析,对54个原核基因组大、小染色体及质粒中蛋白质编码基因的序列特征进行了对比分析。结果表明,大、小染色体中蛋白质编码基因的GC含量分布相近,质粒中蛋白质编码基因的GC含量分布与所在物种全基因组的GC含量差别较大。进一步的分析表明,大、小染色体共同偏好的密码子最多,且具有相近的起始密码子和终止密码子使用特征。基于对应分析的同义密码子使用模式分析表明,大、小染色体具有相近的序列特征,且大、小染色体及质粒之间具有不尽相同的影响因素。这些结果可为今后原核生物基因组进化研究提供可靠的方法和理论依据。     

Codon bias of the gene for chloroplast glycerol-3-phosphate acyltransferase in Camellia sinensis (L.) O. Kuntze

The analysis on codon usage bias of GPAT gene of Camellia sinensis (L.) O. Kuntze may provide a basis for understanding the evolution relationship of C. sinensis and for selecting appropriate host expression systems to improve the expression of target genes. In the present study, the coding sequence of CsGPAT was analyzed with CodonW, CHIPS and CUSP programs, and compared with the genome of C. sinensis and GPAT genes of other 9 plant species. Our results showed that the cluster tree based on CDs could reveal the evolutional relations among the 10 plant species, whereas the cluster tree based on relative synonymous codon usage (RSCU) could not. There were 31 codons showing distinct usage differences between CsGPAT and genome of Escherichia coli, 21 between CsGPAT and yeast, but 13 between CsGPAT and Arabidopsis thaliana. But there were slightly fewer differences in codon usage between CsGPAT and A. thaliana. Therefore, the A. thaliana expression system may be more suitable for the expression of CsGPAT. These results may improve our understanding of the codon usage bias and functional studies of CsGPAT.     

Analysis of the codon use frequency of AMPK family genes from different species

In mammals, AMP-activated protein kinase (AMPK) is involved in the regulation of cellular energy homeostasis and, on the whole animal level, in regulating energy balance and food intake. In this paper, the relative synonymous codon use frequency of 40 AMPK family genes from seven mammal species (Bos taurus, Homo sapiens, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Sus scrofa) were analyzed using correspondence analysis and hierarchical cluster method. The result suggests that gene function is the dominant factor that determines codon usage bias in AMPK family genes, while species is a minor factor that determines further difference in codon usage bias for genes with similar functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reconstruction of the spatial structure of plant phosphatases types 1 and 2A in complexes with okadaic acid

Homology modeling based on the known spatial structures of Homo sapiens protein phosphatases types 1 and 2A was implemented. The spatial structures of human protein phosphatases and their plant homologs from Arabidopsis thaliana were predicted. The quality of the models was confirmed by the root mean square deviations of the atoms and conformational analysis results. The sites of okadaic acid bindings in the molecules of plant protein phosphatases (types 1 and 2A) were verified by the data of comparative analyses and molecular dynamics.     

The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli

Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3 end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.     

Targeted overexpression of the Escherichia coli MinC protein in higher plants results in abnormal chloroplasts

Higher plant chloroplast division involves some of the same types of proteins that are required in prokaryotic cell division. These include two of the three Min proteins, MinD and MinE, encoded by the min operon in bacteria. Noticeably absent from annotated sequences from higher plants is a MinC homologue. A higher plant functional MinC homologue that would interfere with FtsZ polymerization, has yet to be identified. We sought to determine whether expression of the bacterial MinC in higher plants could affect chloroplast division. The Escherichia coli minC (EcMinC) gene was isolated and inserted behind the Arabidopsis thaliana RbcS transit peptide sequence for chloroplast targeting. This TP-EcMinC gene driven by the CaMV 35S² constitutive promoter was then transformed into tobacco (Nicotiana tabacum L.). Abnormally large chloroplasts were observed in the transgenic plants suggesting that overexpression of the E. coli MinC perturbed higher plant chloroplast division.     

Isolation,expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.     

Heterologous expression of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Triticum aestivum and Arabidopsis thaliana

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus^® cells and the pRSETB vector was successful for expression of a soluble His₆-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.     

Non-uniqueness of factors constraint on the codon usage in Bombyx mori

Background

The analysis of codon usage is a good way to understand the genetic and evolutionary characteristics of an organism. However, there are only a few reports related with the codon usage of the domesticated silkworm, Bombyx mori (B. mori). Hence, the codon usage of B. mori was analyzed here to reveal the constraint factors and it could be helpful to improve the bioreactor based on B. mori.

Results

A total of 1,097 annotated mRNA sequences from B. mori were analyzed, revealing there is only a weak codon bias. It also shows that the gene expression level is related to the GC content, and the amino acids with higher general average hydropathicity (GRAVY) and aromaticity (Aromo). And the genes on the primary axis are strongly positively correlated with the GC content, and GC3s. Meanwhile, the effective number of codons (ENc) is strongly correlated with codon adaptation index (CAI), gene length, and Aromo values. However, the ENc values are correlated with the second axis, which indicates that the codon usage in B. mori is affected by not only mutation pressure and natural selection, but also nucleotide composition and the gene expression level. It is also associated with Aromo values, and gene length. Additionally, B. mori has a greater relative discrepancy in codon preferences with Drosophila melanogaster (D. melanogaster) or Saccharomyces cerevisiae (S. cerevisiae) than with Arabidopsis thaliana (A. thaliana), Escherichia coli (E. coli), or Caenorhabditis elegans (C. elegans).

Conclusions

The codon usage bias in B. mori is relatively weak, and many influence factors are found here, such as nucleotide composition, mutation pressure, natural selection, and expression level. Additionally, it is also associated with Aromo values, and gene length. Among them, natural selection might play a major role. Moreover, the “optimal codons” of B. mori are all encoded by G and C, which provides useful information for enhancing the gene expression in B. mori through codon optimization.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1596-z) contains supplementary material, which is available to authorized users.     

Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine,serine and threonine

An intact cDNA fromArabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced. The cDNA is 729 nucleotides in length and predicts a protein ofM _r 27140. The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to theEscherichia coli protein. Construction of a molecular tree of the known APRT amino acid sequences indicates theA. thaliana andE. coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping. Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.