大豆11S球蛋白Gy5(A3B4)的基因克隆和序列分析

大豆11Ｓ球蛋白(Ｇｌｙｃｉｎｉｎ)是大豆种子的主要贮藏蛋白,分子量为360ｋＤ,由6对相同的蛋白亚基(每对亚基的分子量约60ｋＤ)构成。每对亚基又是由一个酸性Ａ肽(35～45ｋＤ)和一个碱性Ｂ肽(22ｋＤ)通过二硫键连接而成。Ａ肽和Ｂ肽源自同一个基因,即首先由一个大的ｍＲ?..     

Characterization of the genetic diversity of the Tall coconut (Cocos nucifera L.) in the Dominican Republic using microsatellite (SSR) markers

The predominant coconut variety cultivated in the Dominican Republic is a local Tall, known as criollo. It was never characterized genetically. The Malayan Dwarf and its hybrid with the local Tall are also present. Thirteen accessions, representing nine localities, are planted in a collection at the Instituto Dominicano de Investigaciones Agropecuarias y Forestales (IDIAF). We explored genetic diversity in 114 individuals from this collection. The main aim was to detect possible relationship with resistant varieties to coconut lethal yellowing (LY) disease. Contrarily to what happened in other Caribbean countries, LY never became an epidemic in the Dominican Republic. Thirteen simple sequence repeats markers from a kit dedicated to coconut diversity were used. In addition to diversity parameters, we used Bayesian assignment tests and cluster analysis to determine its population structure and its relationship with other coconut populations. The criollo coconut proved to be a typical Indo-Atlantic variety and is probably highly susceptible to the usual LY pathogens. Local conditions and the nature of the local phytoplasma strain probably explain the particular epidemiology of LY in the Dominican Republic. As a cross-pollinating variety, the criollo presents polymorphism within a population, but there is little if any variation among populations. The marker study confirmed the hybrid status of each member of two accessions and, thus, the reliability of the sampling.     

Development and validation of a breeder-friendly KASPar marker for wheat leaf rust resistance locus Lr21

Development and utilization of genetic markers play a pivotal role in marker-assisted breeding of wheat cultivars with pyramids of disease resistance genes. The objective of this study was to develop a closed-tube, gel-free assay for high-throughput genotyping of leaf rust resistance locus Lr21. Polymorphism identified from re-sequencing of a 2.4-kb fragment covering the functional region of the Lr21 gene from the second to the fourth indels was targeted for assay development. The generated sequence data revealed the 88- or 105-bp indel in the first intron of the Lr21 gene in the selected resistant cultivars compared to susceptible US spring and winter wheat cultivars. Allele-specific primers for a KASPar assay were designed around the junction of the indel at position 1,346 bp. The marker was tested on a panel of 384 US wheat lines and found to be effective in differentiating resistant and susceptible genotypes.     

Deletion/insertion polymorphism of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Polish Red cattle,Polish White-backed cattle and European bison (<Emphasis Type="Italic">Bison bonasus</Emphasis> L., 1758)

The aim of the present study was to identify deletion/insertion polymorphism of the bovine prion protein (PRNP) gene within the promoter sequence (23 bp indel), intron 1 (12 bp indel) and the 3′ end untranslated region (14 bp indel). The experiment was performed on three groups of animals protected under a genetic resources conservation program: 139 Polish Red (PR) cows, 79 Polish White-backed cows and 50 European bison (Bison bonasus L., 1758). White-backed cattle were characterized by a higher frequency of ins/del heterozygotes and a relatively lower frequency of ins/ins homozygotes within the promoter sequence region (23 bp indel), compared to Polish Red cattle. At the polymorphic locus of intron 1 (12 bp indel) the genetic structure of both cattle populations was similar. Monomorphism, expressed by the occurrence of one genotype variant in each of the analyzed sequence regions, was observed in European bison. Five haplotypes were found in Polish White-backed cows, four haplotypes in Polish Red cows and only one in analyzed group of bison. Differences between the observed and expected number of PRNP haplotypes were recorded in Polish Red cattle. The article is published in the original.     

Fine Mapping and in silico Isolation of the EUI1 Gene Controlling Upper Internode Elongation in Rice

Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9804 bp long, and comprises two exons and one intron. The length of the cDNA is 1931 bp containing a 1734 bp ORF, a 110 bp 5′-UTR and a 87 bp 3′-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family. Hongli Ma, Shubiao Zhang: These authors contributed equally to this work     

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene

The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3′, suggesting a similar protease specificity. Also the putative heparin binding sites and `hinge' regions are highly homologous in mouse and hPCI.     

Use of an alternate splice donor site in the human GAP gene is responsible for synthesis of the p100 isoform

GAP (GTPase-activating protein), a negative regulator of the receptor tyrosine kinase signal transduction pathway, exists as two isoforms: a ubiquitous, p120 form and a primate placenta-specific p100 form lacking the N-terminal hydrophobic domain. The cDNA species encoding p120 and p100 GAP are identical except that p100 GAP cDNA contains a 65-bp insert not present in p120 cDNA. The purpose of this study was to locate the 65-bp insert in the genomic GAP sequence, thereby determining the mechanism by which alternate splicing produces the two mRNA species. It was found that the 65-bp insert is located just 3′ to the sequence encoding the hydrophobic domain, indicating that the p100 form of GAP results from utilization of an alternate splice donor site. In addition, the sequence encoding the hydrophobic domain was found to be contained within a single large exon. The intron separating this exon from the exon encoding the 5′-portion of the SH2-N domain was determined to be at least 40 kb in length. Finally, it was found that the sequence encoding the SH2-N domain contains an intron 1006 bp long, and the sequence of this intron has been deduced. It is anticipated that the data presented in this paper will provide the basis for elucidating RNA processing mechanisms responsible for preferential expression of p100 GAP in the human placenta.     

Structure of the chicken interferon-γ gene,and comparison to mammalian homologues

The sequence of the chicken interferon-γ (ifn-γ) gene was determined, one of the first non-mammalian cytokine gene structures to be elucidated. Initial genomic clones were amplified from chicken genomic DNA and were used to isolate a cosmid clone covering the entire gene for sequencing. The exon:intron structure of chicken ifn-γ is very similar to those of its mammalian homologues, with the exception of the third intron, which is markedly shorter in the chicken. The first exon contains both 5′ UTR and signal sequence and the first 22 aa of the mature protein. The remainder of the coding region lies in exons 2–4. Exon 4 also encodes the stop codon and the 3′ UTR, including two possible polyadenylation signals. A number of potential regulatory sequences similar to those found in mammals have been identified, in the promoter, in each intron and in the 3′ UTR. In the promoter, these include the TATAATA- and CCAT-boxes, a consensus GATA motif in the reverse orientation and a potential NF-κB binding site. Other regulatory elements identified in the promoters of mammalian ifn-γ genes are absent. Internal to the gene structure, regulatory sequences identified include elements found in the DNase I hypersensitivity region of the first intron of the human ifn-γ gene and several potential NF-κB binding sites. The 3′ UTR contains an AT-rich sequence, including nine repeats of the `instability' motif ATTTA. As in mammals, chicken ifn-γ is a single copy gene. The gene is highly conserved, with no polymorphisms yet identified using either RFLP or SSCP in the coding region. However, promoter sequence polymorphisms between different inbred lines of chickens have been identified, with possible links to disease resistance.     

Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: gaining insight into evolutionary mechanism

At least 25 nondisulfide-bridged peptides (NDBPs) have been identified and characterized from scorpions. However, the genomic organization of the genes that encode these peptides have not been reported yet. BmKa1, BmKa2 and BmKb1 are three novel genes that code for NDBPs identified by our group from Mesobuthus martensii Karsch. Based on their cDNA sequences, the genomic DNA sequences encoding these peptides were obtained using the PCR method. Sequence analysis showed that three distinct genomic structural patterns are used to encode these three peptides. The BmKa1 gene is not interrupted by any introns. However, the BmKa2 gene is composed of two exons, interrupted by a 67 bp intron that is located in the DNA region encoding the mature peptide. Two genomic homologues of the BmKb1 cDNA sequence, named BmKb1′ and BmKb2, respectively, were obtained. The BmKb1′ gene contains one intron of 593 bp, inserted into the DNA region that encodes the signal peptide. Similarly, the BmKb2 gene also contains an intron that interrupts the exon that encodes the NDBP signal peptide. The amino acid sequences deduced for BmKb2 and BmKb1′ differ only at one position. The data suggest that the genomic organizational pattern of NDBPs displays more divergence than that exhibited by the genes that encode disulfide-bridged peptides from scorpions.     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

The human FE65 gene: genomic structure and an intronic biallelic polymorphism associated with sporadic dementia of the Alzheimer type

The FE65 protein binds to the intracellular domain of the beta-amyloid precursor protein (ßPP) and may modulate the internalization of ßPP. This gene is highly expressed in regions of the brain specifically affected in dementia of the Alzheimer type (DAT). As a prelude to further investigations of the role of FE65 in the metabolism of ßPP and in the pathogenesis of DAT, we have determined the entire genomic structure and sequence of human FE65 and have discovered several polymorphisms in this gene. Human FE65 contains 14 exons ranging in size from 6 to 735 bp. All splice sites conform to consensus sequences except for the donor site of intron 10. The 5’ end of FE65 mRNA was identified by rapid amplification of the cDNA 5’ end and is 31 bp longer than the previously published cDNA sequence. The 5’-flanking region of this gene is TATA-less and is very GC-rich with at least five putative Sp1 binding sites. In comparison to the genomic rat FE65 sequence, the human FE65 5’-untranslated region is 134 bp longer and has an extra exon (exon 1, 86 bp). To identify mutations/polymorphisms of the coding regions of this gene, we performed blinded analysis of 457 Caucasian case-control samples from a large epidemiological study of sporadic DAT. Screening was conducted by single-strand conformation polymorphism. Four minor variants were found within the coding region, with frequencies between 0.002 and 0.015; two of the four result in amino acid substitutions. The more informative biallelic polymorphism (a trinucleotide deletion and a single base substitution) was found within intron 13 (84 bp), which interrupts two exons encoding the βPP binding site. The frequency of the minor allele in this intron was 0.097 in DAT cases and 0.161 in controls (Χ²=7.78, P=0.0054). Having at least one copy of the minor allele was associated with a decreased risk for DAT (Χ²=9.20, P<0.005, odds ratio=0.49, 95% CI 0.31–0.77). Multivariate analysis showed that this association was independent of the APOE genotype. These results suggest that either FE65 itself or a closely linked gene influences the pathogenesis of sporadic DAT. The interaction of FE65 with βPP and the association of a FE65 polymorphism with DAT lend credence to the hypothesis that the metabolism of βPP is central to the pathogenesis of common sporadic forms of DAT.