Sequence analysis of WIS-2-1A,a retrotransposon-like element from wheat

WIS-2-1A, a 8624 bp insertion in the Glu-1A-2 locus of chromosome 1A of wheat, consists of two 1755 bp long terminal repeats enclosing a 5114 bp internal region. No long open reading frames could be found, but inspection of the predicted amino acid sequence showed regions with homology to retrotransposon structures, including a methionine tRNA initiator binding site, a nucleotide binding domain, a protease, an integrase and a polymerase. DNA replication errors have resulted in frame-shifts in the protein coding region, suggesting that retrotransposition of WIS-2-1A, if it occurs, must be mediated by trans-acting factors.     

重组泡球蚴Em18蛋白不同纯化方法的比较

目的：比较不同方法纯化重组泡球蚴Em18的效果。方法：进一步对BL21-pET41a-Em18重组菌的诱导表达条件进行摸索优化,采用改良的超声程序破碎大肠杆菌细胞,分别经不同的纯化方法进行蛋白纯化,通过SDS-PAGE对纯化结果进行比较分析,Western blot进行活性鉴定。结果：①在菌液OD600为0.8-1.0,加IPTG终浓度为1 mmol/L,37℃诱导3h,rEm18-GST重组蛋白得到成功高表达,在相对分子量为50kDa处有表达条带。②超声程序为：工作3 s,间歇4s,功率200~300W,超声体系中加入溶菌酶和蛋白酶抑制剂作用,可促进大肠杆菌细胞的破碎,降低目的蛋白的降解;加入终浓度为1%的Triton-X100作用可增加融合蛋白的可溶性。③通过比较不同方法纯化重组泡球蚴Em18的效果表明采用单纯His柱纯化可获得浓度高、纯度高的rEm18-GST重组蛋白。Western blot分析表明该蛋白能与泡型包虫病（AE）患者血清特异性反应。结论：建立了一种纯化原核表达Em18融合蛋白的较为经济和有效的方法,得到大量有生物学活性的Em18融合蛋白,为包虫病诊断试剂盒的研制奠定基础。     

Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei

In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. We have cloned and characterized one of the alleles. Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. The gene sequences differ at 15 positions, resulting in five amino acid substitutions. The calculated molecular masses of the polypeptides are 54,378 Da and 54,363 Da. These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57-59 kDa. However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat-germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE. The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41-51%. The conserved residues are not equally distributed over the polypeptide. The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved. In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable. The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome. It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids. The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only +3.     

Sequence analysis of a functional member of the Em gene family from wheat.

We report the complete sequence of one functional member of the Em gene family whose expression in wheat embryos is regulated by a complex set of environmental and developmental controls, including the phytohormone abscisic acid (ABA). The Em coding region contains one short intron, and there is an inverted repeat in the transcribed 3'-flanking region. A 646 bp fragment from the 5' promoter, which was previously shown to direct ABA-regulated expression in transformed tobacco tissue and rice cells, is characterized by: (1) three stretches of between 33 and 73 nucleotides of A/T rich (greater than 86%) boxes, (2) one copy of an eight bp palindrome (CATGCATG) which is identical to the RY repeat found in the 5' promoters of many legume genes expressed during embryo development, (3) 15 copies of a six bp repeat (PuCACGPy), found primarily in the 5' region, and (4) two sequences in the ABA-response region, CGAGCAG and a CACGT motif, both of which are conserved in 5' non-coding regions of other plant genes that are expressed in response to ABA and/or in embryos. These sequence comparisons are discussed in relation to the regulation of Em gene expression and other ABA-regulated genes.     

Microsatellite markers linked to six Russian wheat aphid resistance genes in wheat

The Russian wheat aphid (RWA), Diuraphis noxia Mordvilko, is a serious economic pest of wheat and barley in North America, South America, and South Africa. Using aphid-resistant cultivars has proven to be a viable tactic for RWA management. Several dominant resistance genes have been identified in wheat, Triticum aestivum, including Dn1 in PI 137739, Dn2 in PI 262660, and at least three resistance genes (Dn5+) in PI 294994. The identification of RWA-resistant genes and the development of resistant cultivars may be accelerated through the use of molecular markers. DNA of wheat from near-isogenic lines and segregating F₂ populations was amplified with microsatellite primers via PCR. Results revealed that the locus for wheat microsatellite GWM111 (Xgwm111), located on wheat chromosome 7DS (short arm), is tightly linked to Dn1, Dn2 and Dn5, as well as Dnx in PI 220127. Segregation data indicate RWA resistance in wheat PI 220127 is also conferred by a single dominant resistance gene (Dnx). These results confirm that Dn1, Dn2 and Dn5 are tightly linked to each other, and provide new information about their location, being 7DS, near the centromere, instead of as previously reported on 7DL. Xgwm635 (near the distal end of 7DS) clearly marked the location of the previously suggested resistance gene in PI 294994, here designated as Dn8. Xgwm642 (located on 1DL) marked and identified another new gene Dn9, which is located in a defense gene-rich region of wheat chromosome 1DL. The locations of markers and the linked genes were confirmed by di-telosomic and nulli-tetrasomic analyses. Genetic linkage maps of the above RWA resistance genes and markers have been constructed for wheat chromosomes 1D and 7D. These markers will be useful in marker-assisted breeding for RWA-resistant wheat. Received: 17 May 2000 / Accepted: 13 June 2000     

两个小麦磷转运蛋白基因的分离、功能鉴定和表达研究

磷是能量代谢、核酸以及许多生物膜合成的重要底物。在光合作用、呼吸作用等过程中发挥了重要作用。中国大多数小麦产区的土壤存在着缺磷的问题。磷饥饿给小麦生产造成了很大损失。培育耐低磷小麦是解决这一问题的一个重要途径。在磷饥饿的过程中,哪些基因的表达发生了变化．它们是如何变化的,弄清楚这些问题对于培育转基因耐低磷小麦具有重要的意义。磷转运蛋白基因在植物吸收磷的过程中发挥着重要作用。利用RT—PCR的方法,我们从普通小麦“小偃54”中分离了两个磷转运蛋白基因TaPT8和TaPHT2;1。通过与酵母突变体互补分析表明这两个基因都能够与磷吸收功能存在缺陷的酵母突变体实现功能互补,在低磷条件下有促进酵母突变体吸收磷的作用。进一步分析表明TaPT8属于Pht1家族。TaPHT2;1属于Pht2家族。运用RQRT—PCR的方法进行分析后发现TaPT8在根中表达,受磷饥饿的诱导;TaPHT2;1主要在绿色组织中表达,受磷饥饿的抑制,受光的诱导。TaPT8可能主要参与了小麦的根从土壤中吸收磷的过程。TaPHT2;1可能在磷从细胞质向叶绿体内转运的过程中发挥了重要作用。     

Differential regulation of two ABA-inducible genes from Craterostigma plantagineum in transgenic Arabidopsis plants

In Craterostigma plantagineum the CDeT-6-19 and CDeT-27-45 genes are expressed following desiccation and/or ABA treatment. Their promoters were fused to the -glucuronidase reporter gene (GUS) and tested in transgenic Arabidopsis. GUS activity was measured in mature Arabidopsis seeds, and the responsiveness to ABA in vegetative tissue was found to be limited to the early developmental stages. When transgenic plants were crossed with plants over-expressing the ABI3 gene, it was observed that ABI3 is not required for ABA induction of the CDeT-6-19 promoter, whereas it is crucial for expression of the CDeT-27-45 promoter.     

水稻高效再生体系的建立及其对大豆Em基因的转化

以粳稻丰达1号成熟胚为外植体,建立了适合水稻转化的高效再生体系,通过农杆菌介导法将大豆Em基因转化水稻.结果表明:以NMB 500 mg·L-1脯氨酸 250 mg·L-1谷氨酰氨 300 mg·L-1水解酪蛋白 2 mg·L-12,4-D为作诱导培养基诱导愈伤组织,其诱导率为97%;愈伤组织分化率为65%;农杆菌介导法转化水稻愈伤组织,获得了26株再生植株;随机选取其中4株候选转基因水稻幼苗进行PCR筛选,初步证明大豆Em基因已整合到水稻基因组DNA中;RT-PCR检测结果表明,转入的外源大豆Em基因在转基因水稻中得以表达.本实验成功地建立了适合水稻转化的高效再生体系,为通过基因工程技术培育水稻新品种奠定了基础.     

Isolation and amino acid sequence of two new PR-4 proteins from wheat

We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2. Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of these residues were determined on the three-dimensional model of wheatwin1. Residue 88 was very accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.     

Sequence analysis of the Legionella micdadei groELS operon

A 2.7 kb DNA fragment encoding the 60 kDa common antigen (CA) and a 13 kDa protein of Legionella micdadei was sequenced. Two open reading frames of 57,677 and 10,456 Da were identified, corresponding to the heat shock proteins GroEL and GroES, respectively. Typical -35, -10, and Shine-Dalgarno heat shock expression signals were identified upstream of the L. micdadei groEL gene. Further upstream, a poly-T region, also a feature of the sigma 32-regulated Escherichia coli groELS heat shock operon, was found. Despite the high degree of homology of the expression signals in E. coli and L. micdadei, Western blot analysis with an L. micdadei specific anti-groEL antibody did not reveal a significant increase in the amount of the GroEL protein during heat shock in L. micdadei or in the recombinant E. coli expressing L. micdadei GroEL.     

Sequence of two genes in pea chloroplast DNA coding for 84 and 82 kD polypeptides of the photosystem I complex

Summary The genes encoding the two P700 chlorophyll a-apoproteins of the photosystem I complex were localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the genes and the flanking regions has been determined. The genes are separated by 25 bp and are probably cotranscribed. The 5 terminal gene (psaA1) codes for a 761-residue protein (MW 84.1 kD) and the 3 terminal gene (psaA2) for a 734-residue protein (MW 82.4 kD). Both proteins are highly hydrophobic and contain eleven putative membrane-spanning domains. The homology to the corresponding polypeptides from maize are 89% and 95% for psaA1 and psaA2, respectively. A putative promoter has been identified for the psaA1 gene, and potential ribosome binding sites are present before both genes.     

普通小麦中双脱氢抗坏血酸还原酶(TaDHAR)基因的克隆与生化特性分析

利用同源克隆技术从六倍体普通小麦中获得了两个不同的双脱氢抗坏血酸还原酶(TaDHAR)基因的cDNA克隆。器官表达模式分析表明,这两个TaDHAR基因(暂时命名为TaDHAR1和TaDHAR2)在小麦根、茎、叶、幼穗以及开花后10d、20d和30d的种子中均有表达,为组成型表达基因。原生质体表达实验表明,两个基因的产物均可能定位在细胞质中。在细菌中表达并提纯了两个基因的重组蛋白。体外生化测定表明两个重组蛋白均具有将双脱氢抗坏血酸还原成抗坏血酸的能力,其最适pH为7.5,在37oC时的活性比25oC高,但25oC条件下pH6.0和7.0时,两个DHAR蛋白的活性显著不同。本研究的结果为进一步揭示TaDHAR基因在小麦抗坏血酸代谢中的生理作用奠定了基础。     

Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution

In plant species with large genomes such as wheat or barley, genome organization at the level of DNA sequence is largely unknown. The largest sequences that are publicly accessible so far from Triticeae genomes are two 60 kb and 66 kb intervals from barley. Here, we report on the analysis of a 211 kb contiguous DNA sequence from diploid wheat (Triticum monococcum L.). Five putative genes were identified, two of which show similarity to disease resistance genes. Three of the five genes are clustered in a 31 kb gene-enriched island while the two others are separated from the cluster and from each other by large stretches of repetitive DNA. About 70% of the contig is comprised of several classes of transposable elements. Ten different types of retrotransposons were identified, most of them forming a pattern of nested insertions similar to those found in maize and barley. Evidence was found for major deletion, insertion and duplication events within the analysed region, suggesting multiple mechanisms of genome evolution in addition to retrotransposon amplification. Seven types of foldback transposons, an element class previously not described for wheat genomes, were characterized. One such element was found to be closely associated with genes in several Triticeae species and may therefore be of use for the identification of gene-rich regions in these species.     

A chromosome-specific DNA sequence which reveals a high level of RFLP in wheat

Summary An unusual genomic DNA clone, PSR454, was isolated from a partial genomic library of wheat. This sequence is moderately repeated and detects at least 30 related sequences, all located in a tight linkage block on the long arm of chromosome 3B. When used as a RFLP probe, PSR454 detects a high level of polymorphism between wheat varieties that carry the sequence. There is no detectable hybridisation to sequences in one-third of the varieties tested, providing an on-off polymorphism that can be detected on dot blots, rather than the more resource-consuming conventional Southern analysis.