Analysis of LMW-GS, α- and γ-Gliadin Gene Coding Sequences from Triticum macha

Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions of two α-gliadin, two γ-gliadin and two LMW-GS gene sequences, which designed as Gli-Mal, Gli-Ma2, Gli-Mrl, Gli-Mr2, Glu-LM1 and Glu-LM2, encoded the mature proteins with 307, 241, 348, 302, 474 and 377 amino acid residues, respectively. Gli-Mal and Gli-Ma2 were recognized as pseudogenes due to the in-frame stop codons. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α- or γ-gliadin or LMW- m type proteins with the exception of Gli-Ma2. Phylogenetic analysis showed Gli-Mal was closely related to those from T. aestivum, whereas Gli-Ma2 seemed to be more homologous with the gene sequences from Dasypyrum breviaristatum. Gli-Mrl was closely related to those from T. turgidum ssp. dicoccoides, while Gli-Mr2 was the nearest to those from T. aestivum. Glu-LM1 was closely related to those from Aegilops tauschii, whereas GIu-LM2 seemed to be more homologous with those from T. durum.     

Molecular Characterization and SNP Markers of the β-purothionin Gene in Einkorn Wheats

Forty-three gene sequences encoding purothionin were characterized from the three species or subspecies of einkorn wheats. These sequences contained 887 bp, among which 92 SNPs including 29 indel loci were detected, giving an average SNP frequency of one SNP per 9.64 bases. According to these sequences, 5 SNP markers were successfully designed, which were used to mine the variations ofpurothionin genes of 102 einkorn wheat accessions. Based on the 5 detected SNP loci, 102 einkorn wheat accessions could be divided into 21 haplotypes, among which 11 haplotypes contained a single sample. Phylogenetic analysis indicated that the purothionin genes from einkorn wheats were more closely related to those from D genome than B genome. Seven out of the 43 gene sequences were assumed to be pseudogenes by the definition of containing in-frame stop codons and small insertions/deletions leading to frameshift. In the remaining 36 amino acid sequences, the 8 Cys and Tyr-13 loci in the mature thionin domain which played important roles in the biological activities were all conserved, whereas there were some varieties occurred in some other important amino acid residues such as Lys and Arg.     

Cloning of Thymidine Kinase Gene of Duck Plague Virus Using Degenerate PCR

The DNA of duck plague virus （DPV） thymidine kinase （TK） gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H（gH） gene were used in the polymerase chain reaction （PCR） to amplify DNA product with 3 741-base-pairs （bp） in size. DNA sequence analysis revealed a 1 077-base-pairs （bp） open reading frame （ORF） encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek＇s disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.     

Characterization of a Novel 1Ay Gene and Its Expression Protein in Triticum urartu

High molecular weight glutenin subunit （HMW-GS） plays an important role in determining dough property and breadmaking quality, and the exploration of novel genes for HMW-GS will be crucial for quality improvement program. A gene coding the y type HMW-GS at Glu-A1 locus in Triticum urartu （AA, 2n=2×= 14） with an electrophoretic mobility similar to that of 1Dyl2, was cloned, sequenced, and heterologously expressed. This novel active lay gene FJ404595 was confirmed by structure analyses of nucleotide and deduced amino acid sequences combining with phylogenetic analysis. The open reading frame （ORF） of this gene was 1 830 bp, encoded a protein of 608 amino acid residues containing 46 hexapeptides and 14 nonapeptides, which was mostly similar to the lAy gene AM183223 at a high identity of 99.62% with the two substitutions of both leucine/proline and valine/glutamate, obviously different from the lAy gene EU984504 with 587 residues containing 44 hexapeptides and 13 nonapeptides in T. urartu. The amino acid （leucine） at 446 differed from that （proline） of all the eight compared active lAy subunits. The predicted secondary protein structure implied that this lAy subunit might also have positive impact on flour processing quality.     

Sweet potato and potato residual flours as potential nutritional and healthy food material

In this study,the proximate composition,mineral content and amino acid composition of starch processing residues from 10 cultivars of sweet potato and 10 cultivars of potato were determined,and the nutritional and health-related values of these residues were investigated.The residual flours contained 20.63–31.48 g and 17.14–28.57 g rich dietary fiber per 100 g dry weight for sweet potato and potato,respectively,as well as mineral elements,including potassium,ferrum,zinc and copper.The highest limiting amino acid score(AAS) of the almost balanced amino acid composition were observed to be 71.07 and 57.96 for sweet potato and potato residues,respectively.A grey relational analysis showed that the nutritional values of Jishu 4 at 0.7519 and LT-5 at 0.7281 were the highest among the sweet potato and potato residues,respectively.The evaluation of the sweet potato/potato residues,the by-products of the starch industry,based on recommended daily intake(RDI) standards,indicated that the residues have potential nutritional and health-related food values.     

Genetic Diversity of Chinese Soybean mosaic virus Strains and Their Relationships with Other Plant Potyviruses Based on P3 Gene Sequences

Soybean mosaic virus （SMV）, a member of the genus Potyvirus, is a major pathogen of soybean plants in China, and 16 SMV strains have been identified nationwide based on a former detailed SMV classification system. As the P3 gene is thought to be involved in viral replication, systemic infection, pathogenicity, and overcoming resistance, knowledge of the P3 gene sequences of SMV and other potyviruses would be useful in efforts to know the genetic relationships among them and control the disease. P3 gene sequences were obtained from representative isolates of the above-mentioned 16 SMV strains and were compared with other SMV strains and 16 Potyvirus species from the National Center for Biotechnology GenBank database. The P3 genes from the 16 SMV isolates are composed of 1041 nucleotides, encoding 347 amino acids, and share 90.7-100% nucleotide （NT） sequence identities and 95.1-100% amino acid （AA） sequence identities. The P3 coding regions of the 16 SMV isolates share high identities （92.4-98.9% NT and 96.0-100% AA） with the reported Korean isolates, followed by the USA isolates （88.5-97.9% NT and 91.4-98.6% AA）, and share low identities （80.5-85.2% NT and 82.1-84.7% AA） with the reported HZ 1 and P isolates from Pinellia ternata. The sequence identities of the P3 genes between SMV and the 16 potyviruses varied from 44.4 to 81.9% in the NT sequences and from 21.4 to 85.3% in the AA sequences, respectively. Among them, SMV was closely related to Watermelon mosaic virus （WMV）, with 76.0-81.9% NT and 77.5-85.3% AA identities. In addition, the SMV isolates and potyvirus species were clustered into six distinct groups. All the SMV strains isolated from soybean were clustered in Group I, and the remaining species were clustered in other groups. A multiple sequence alignment analysis of the C-terminal regions indicated that the P3 genes within a species were highly conserved, whereas those among species were relatively variable.     

“大通”牦牛Lfcin基因克隆及生物信息学分析(英文)

[Objective] This study was to clone Lfcin gene from Datong yak, so as to provide reference for applying this gene in feed industry and breeding industry. [Method] Using PCR technology, the lactoferricin(Lfcin)-encoding gene was obtained from genome of Datong yak; then it was cloned into pGEM-T easy vector, and then sequenced; the sequencing results were subsequently aligned with the sequences of dairy cow accessed in GenBank. Moreover, amino acid sequences of Lfcin gene from various species including yak, dairy cow, human and mouse were used for sequence alignment and phylogenesis analysis. [Result] The second exon of lactoferrin(LF) from Datong yak, which is 778 bp in length, was obtained, within which the coding region of Lfcin gene is 75 bp (25 amino acid residues); sequence analysis showed that there is discrepancy of eleven bases between Datong yak and dairy cow; Lfcin proteins from various species shared high homeology, of which that from Datong yak and dairy cow were completely identical; phylogenesis analysis showed that cladogram based on Lfcin was consistent with species evolutionary law. [Conclusion] This study laid a foundation for the prokaryotic or eukaryotic expression of Lfcin gene and further understanding the activity of Lfcin protein.     

Sequence Comparison of MHC Class Ⅱβ（Exon 2） and Phylogenetic Relationship Between Poultry and Mammalian

A fragment spanning over exon 2 and intron 2 of major histocompatibility complex B-LB Ⅱ genes was amplified using PCR,cloned and sequenced in 13 individuals from eight Chinese indigenous chicken breeds and one introduced breed. Another 41 sequences of MHC class Ⅱβ from ten vertebrate species were cited from the NCBI GenBank. Thirteen new B-LB Ⅱ alleles were found in the chicken breeds sampled. Alignment of the exon 2 sequences revealed 91.1-97.8% similarity to each other within the chickens sampled, and the chickens shared 84.1-87.0% homology to Phasianus colchicus, 78.5-81.5% similarity to Coturnixjaponica. The sequences in poultry showed 62.6-68.1% identity to HLA-DRB1, 50-61.5% similarity to DQB (HLA-, SLA- and H2-BB), 53.7-60% to HLA-DPB and 53.3-57.8% similarity to HLA-DOB. The frequency of nonsynonymous substitutions of nucleotide was higher than that of synonymous substitutions, and the frequencies of nonsynonymous and synonymous substitutions in poultry B-LB Ⅱ genes were lower than those observed in mammalian DRB1 and DQB1 genes. The deduced amino acid sequences of MHC class Ⅱ β1 domain exhibited extreme difference in conversed region and variable region patterns among the various species, but the two conserved cysteines forming disulfide-bond were shown consistent in poultry with that in mammalian species; and the carbohydrate attachment site was found more conserved in chicken, Homo sapiens, Bos taurus, Ovis aries and Capra hircus than in Sus scrofa and rodent animals. Compared with exon 2 of DQB1 genes of Homo sapiens, ruminant species and Sus scrofa, the differentia that the deletion of six nucleotides at position195 to 200 of exon 2 of DQB1 genes, and insertion of three nucleotides at position 247 to 249 of the exon 2 existed in rodent species were found, which led to the absence of three AA residues at position 65, 66,and 67 within β1 domain of DQB1 chain, and the insertion of one AA residue at position 85. The difference of the deletion of six nucleotides at position 72 to 77 of exon 2 of DPBI genes was observed with Homo sapiens DQB1, which caused absence of three AA residues at position 24, 25, and 26 of β1 domain of DPB1 chain. The phylogenetic tree revealed that the B-LB Ⅱ sequences from poultry are not orthologous to the class Ⅱ MHC β-chain genes of mammalian species. The tree indicated that genetic evolutionary relationship of chickens with Phasianus colchicus was much closer than with Coturnix japonica, and the DQB and DPB clusters are more tightly related to each other than to the remaining clusters.     

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Du1, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Du1 and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C → T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences (PSQQQP). The peptide fraction PF(Y)PP(Q)is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Du1 seems to be more homologous with the genes on chromosome 6B.     

小麦品种“川农16”α-醇溶蛋白基因序列分析

 【目的】克隆和分析“川农16”醇溶蛋白基因,为其进一步遗传改良提供更多依据。【方法】根据已报道的α-醇溶蛋白基因序列设计引物,对小麦品种“川农16”总DNA进行PCR扩增得到约900 bp的DNA片段,分离纯化后连接到pMD18-T载体上,转化后筛选阳性克隆进行测序。【结果】获得4个不同的基因序列：Gli2-CN16-9、Gli2-CN16-12、Gli2-CN16-14和Gli2-CN16-6,GenBank登录号分别为DQ246446、DQ246447、DQ246448和DQ246449。其中,Gli2-CN16-9、Gli2-CN16-12和Gli2-CN16-14分别为861、870和900 bp,可分别编码286、289和299个氨基酸残基的成熟蛋白;而Gli2-CN16-6编码区长度为852 bp,由于存在2个提前终止密码子,不能编码有功能的成熟蛋白,为假基因。【结论】序列比较显示它们与α-醇溶蛋白基因有很高的一致性;与γ-和ω-醇溶蛋白基因差异明显。     

柱穗山羊草α-醇溶蛋白基因的克隆与序列分析

根据已知的普通小麦α-醇溶蛋白基因序列设计引物,采用PCR方法克隆基因并进行序列分析。从柱穗山羊草Y127中克隆得到1个α-醇溶蛋白基因序列Gli2-Z-2,它具有α-醇溶蛋白基因的典型结构特征,编码区全长939 bp,编码313个氨基酸。氨基酸序列比较显示,Gli2-Z-2在多聚谷氨酰胺区比已报道的α-醇溶蛋白序列有较多的谷氨酰胺残基。     

梭梭液泡膜焦磷酸酶HaVVP基因克隆及序列分析

以梭梭叶片为材料,根据其他植物液泡膜焦磷酸酶基因的保守序列设计引物,采用同源基因克隆及RACE-PCR方法克隆到1个H+-PPase基因,命名为HaVVP。HaVVP基因编码区长2 734bp,编码767个氨基酸;所得序列与GenBank中注册的高等植物H+-PPase基因核苷酸序列的同源性均在68%以上、氨基酸序列的同源性达80%以上。     

蒲公英抗氧化酶基因的克隆与盐胁迫表达分析

利用同源克隆技术从蒲公英中克隆到铜锌超氧化物歧化酶基因（Cu/ZnSOD）、抗坏血酸过氧化物酶基因（APX）和过氧化氢酶基因（CAT）3个抗氧化酶基因，采用生物信息学方法分析基因编码的氨基酸序列，利用实时荧光定量PCR方法对盐胁迫下的基因表达进行检测，以了解盐胁迫下抗氧化酶系统变化机理。结果显示：蒲公英Cu/ZnSOD、APX和CAT的编码区序列长度分别为474、852和1 479 bp，分别编码157、283和492个氨基酸残基的抗氧化酶。多序列比对和物种进化关系表明，蒲公英3个抗氧化酶基因与莴苣和向日葵中相关基因的氨基酸序列同源性最高；与0 h相比，盐胁迫处理3 h和6 h时，3个抗氧化酶基因的表达量迅速增加，12 h时又有所降低；海水复合盐胁迫对Cu/ZnSOD的影响相较于NaCl单盐胁迫有所提高。综上所述，盐胁迫快速诱导了蒲公英抗氧化酶基因的表达，以抵抗逆境胁迫，但经过一段时间的高盐胁迫后，过量的活性氧在植株内大量积累，对其细胞造成严重的损害，酶活性降低，植株正常生长受到抑制，细胞内基因的表达亦受到影响。     

四川小麦地方品种AS1643中α/β醇溶蛋白基因

用PCR方法从四川小麦地方品种AS1643中克隆到3个α/β-醇溶蛋白基因，即Gli-AS1643-1（GenBank No.DQ166376）、Gli-AS1643-2（GenBank No.DQ166377）和Gli-AS1643-3（GenBank No.DQ166378）。其中，Gli-AS1643-1和Gli-AS1643-2的编码区长度分别为873bp和852bp，可编码270和263个氨基酸残基的成熟蛋白。Gli-AS1643-3由于在编码区内有一个提前终止密码子，为不可编码成熟蛋白的假基因。序列比较显示Gli-AS1643-1、Gli-AS1643-2和 Gli-AS1643-3分别与GenBank中的α/β-醇溶蛋白基因具有较高的一致性，且序列结构非常相似。它们的N-端氨基酸序列与各种α-、β-、γ-和α/β-醇溶蛋白的基本一致，但与ω-醇溶蛋白和低分子量谷蛋白亚基的明显不同。N-端12肽串联重复紧密相关的5个脯氨酸框和类似于微卫星序列编码的2个多聚谷氨酰胺区域。在Gli-AS1643-2的N-端存在腹泻疾病活性序列，C-端含有12型腺病毒感染序列。Gli-AS1643-1、Gli-AS1643-2和Gli-AS1643-3各由6个保守的半胱氨酸残基形成3个分子内二硫键。     

小麦地方品种半截芒Glu-B1位点HMW-GS基因的克隆及序列分析

高分子量谷蛋白亚基(HMW-GS)与小麦的品质特性紧密相关,挖掘小麦中HMW-GS新基因,对小麦品质改良具有重要意义。以小麦地方品种半截芒为材料,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)分析其HMW-GS组成,并设计特异性引物,利用PCR技术克隆其Glu-B1位点x型和y型亚基基因。序列分析表明,2个基因具有完整编码框,长度分别为2 367bp和2 106bp(GenBank登录号分别为KJ579439和KJ579440),编码789个氨基酸和702个氨基酸的蛋白,被命名为1Bx14*和1By15*。同源性搜索结果显示2条氨基酸序列与典型的HMW-GS有较高的同源性,且与普通小麦亚基1Bx14和1By15的同源性均为95%。系统进化树分析表明,1Bx14*和1By15*分别与1Bx14和1By15的遗传距离较近,聚类到同一分支上。     

龙眼TFL1基因的克隆与表达

以‘红核子’龙眼叶芽为材料,克隆了龙眼TFL1-1和TFL1-2基因的c DNA序列和基因组DNA序列,进行序列分析,并对这两个基因在花芽分化过程中的表达进行了研究.结果显示,龙眼TFL1-1基因c DNA开放阅读框共519 bp,编码173个氨基酸,TFL1-2基因c DNA开放阅读框共525 bp,编码175个氨基酸;两个基因DNA序列均含有4个外显子和3个内含子;序列分析和系统进化树分析表明,龙眼TFL1-1和TFL1-2都是TFL1同源基因,龙眼TFL1-1基因与柑橘、梨的TFL1基因亲缘关系较近;基因表达结果表明,龙眼TFL1-1和TFL1-2基因的功能都与抑制花芽分化有关.     

毛果杨Eukaryotic translation initiation factor 5A（eIF5A） 同源基因的生物信息学分析

为研究毛果杨eIF5A基因间的同源性及其进化关系,以毛果杨的eIF5A基因为研究对象,利用生物信息学软件及网站对其进行碱基分布、氨基酸组成、亲疏水性、保守区以及二级结构和三级结构的预测与分析,并与其他物种的eIF5A氨基酸序列进行多重比对与进化分析。结果表明,4个毛果杨eIF5A基因定位于不同染色体上,且都只含有5个外显子;研究还发现不同成员间氨基酸数目、氨基酸序列间的疏水性存在一定的差异;亚细胞定位分析表明,4个eIF5A蛋白均定位于细胞质上;二级结构预测结果显示,4个eIF5A氨基酸序列以无规则卷曲、扩展链和α-螺旋为主要组成部分,且4条氨基酸序列三维结构十分相似。上述结果均为毛果杨eIF5A基因家族的进一步功能分析提供了一定基础。