一个小麦低分子量谷蛋白基因的分离

以新疆小麦日喀则基因组DNA为模板,采用Glu-D3位点特异引物进行PCR扩增。PCR产物插入pUCm-T载体,转化感受态大肠杆菌E．coli DH5a,获得阳性克隆。经测序发现该插入片段长度1287bp,包括了部分启动子序列和完整的编码序列。编码序列推导的蛋白质含有8个半胱氨酸残基,属于6类LMW-GS中的TypeI类,为以后这类基因的功能研究提供了基因材料。     

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a K_m value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.Many pathogenic bacteria and fungi produce toxins that interfere with various functions of plant cells and may affect plant defense mechanisms (Durbin, 1981). Toxin production is commonly associated with disease severity and can be involved in colonization or systemic invasion by the pathogen (Schäfer, 1994). Toxin resistance has been shown in most cases to be based on the ability of the plant to metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance genes that encode proteins involved in detoxification mechanisms have been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and Birch, 1997). In many cases a relationship exists between toxin tolerance and resistance to the disease (Anzai et al., 1989; Meeley et al., 1992). The availability of toxin-resistance genes will permit a greater understanding of the mechanisms causing plant disease and will also set the stage for engineering resistance to plant disease (Keen, 1993).Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by the ascomycete fungus Eutypa lata (Pers.: Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991). This disease is responsible for considerable loss in yield and is the most devastating disease of grapevine (Vitis vinifera) in many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The fungus infects the stock through pruning wounds and is present in the xylem and phloem of the vine trunk and branches (Moller and Kasamitis, 1978; Duthie et al., 1991). After a long incubation period, a canker forms around the infected wound. The toxin synthesized by the fungus in the trunk is believed to be transported by the sap to the herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates grapevine cells through passive diffusion and its accumulation in the cytoplasm has been explained by an ion-trapping mechanism related to the ionization state of the molecule (Deswarte et al., 1996b). In the cell the effects of eutypine include reduction of adenylated nucleotide content, inhibition of succinate dehydrogenase, uncoupling of oxidative phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to dwarfed and withered new growth of branches, marginal necrosis of the leaves, dryness of the inflorescence, and, finally, death of one or more branches (Moller and Kasamitis, 1981). The toxin appears to be an important virulence factor involved in symptom development of the disease (Deswarte et al., 1996a). However, the absence of toxin-deficient mutants of the fungus and its long incubation period in the trunk before symptom development have prevented a critical study of the toxin in vine plants. Determining the gene responsible for eutypine resistance would therefore be an important critical tool in determining the role of eutypine toxin in symptom development in the disease; and it has the potential to confer resistance to transgenic grapevines.Recently, Colrat et al. (1998) found detoxification to occur in grapevine cells through the enzymatic reduction of eutypine into its corresponding alcohol, eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol). We have determined that this derivative of the toxin is nontoxic for grapevine tissues. Furthermore, we have established a relationship between the susceptibility of grapevine to eutypa dieback and the ability of tissues to inactivate eutypine, suggesting that the detoxification mechanism plays an important role in defense reactions. Eutypine is enzymatically detoxified in numerous plant species and, among them, we found that the tissues of mung bean (Vigna radiata), a nonhost plant for the pathogen, exhibit an efficient detoxification activity. As a prerequisite for demonstrating the involvement of eutypine toxin in eutypa dieback, we report here the purification to homogeneity and the characterization of an ERE from etiolated mung bean hypocotyls.     

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1^st cysteine residue within the repetitive domain at 40^th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.     

小麦新品种“川麦42”低分子量谷蛋白亚基新基因的分子克隆

采用PCR方法从小麦（Triticum aestivum L.）新品种“川麦42”中克隆得到一个低分子量谷蛋白亚基（LMW-GS）新基因,暂命名为LMWCM42-1。该基因编码区全长846 bp,编码281个氨基酸,具有LMW-GS基因的典型结构特征。推导氨基酸序列比较显示,尽管LMWCM42-1与已知LMW-GS高度相似,但在N-末端重复区部分重复单元和C-末端区中仍存在明显差异。聚类分析表明,LMWCM42-1可能是由Glu-D3位点编码的。     

Diagnostic Probes Targeting the Major Sperm Protein Gene That May Be Useful in the Molecular Identification of Nematodes

Discrimination of closely related nematode species is typically problematic when traditional identification characteristics are prone to intraspecific variation. In this study, a molecular approach that can distinguish Pratylenchus penetrans and P. scribneri is described. The approach uses universal primers in conjunction with polymerase chain reaction (PCR) to amplify equivalent fragments of the major sperm protein (msp) gene from any nematode. This gene fragment typically includes an intron of variable sequence. The presence of this highly variable segment in an otherwise conserved gene sequence allows P. penetrans and P. scribneri to be distinguished by either a species-specific amplification or by dot-blot hybridization. The approach is potentially of general utility in species-specific identification of nematodes.     

Characterization of LeMir, a Root-Knot Nematode-Induced Gene in Tomato with an Encoded Product Secreted from the Root

A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet. This gene was therefore named LeMir (L. esculentum miraculin). Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz). LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root. Rapid induction of expression upon nematode infection is localized to root tips. In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis. The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies. Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding. LeMir is also expressed in tomato callus tissue. Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction. Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms.Root-knot nematodes (Meloidogyne spp.) are endoparasites of the roots of most cultivated crops and cause significant economic losses worldwide (Sasser, 1980). This group of nematodes has a complex life cycle (Williamson and Hussey, 1996). The infective stage, the second-stage juvenile, is attracted to root tips, where it penetrates the zone of elongation and then migrates intercellularly, first toward the root tip and then up to the developing vascular tissue (Wyss et al., 1992). There the nematode initiates a feeding site, causing the formation of large, multinucleate, metabolically active giant cells (Jones, 1978; Huang, 1985). Nearby cells of the cortex, pericycle, and vascular parenchyma enlarge and divide, forming a root-knot or gall. Initiation of the gall can be seen under the microscope between 12 and 24 h after inoculation. Although tomato (Lycopersicon esculentum Mill.) is an excellent host for these nematodes, some varieties are resistant because of the presence of the dominant gene Mi. The presence of Mi is correlated with the development of a localized necrosis of host cells at the feeding site within 24 h of infection (Dropkin et al., 1969; Paulson and Webster, 1972).Because of the economic importance of the root-knot nematode, the molecular biology of the formation and maintenance of the feeding site has been studied by a number of authors (for review, see Williamson and Hussey, 1996). Several genes have been characterized that are up-regulated in the giant cell and the gall (Goddijn et al., 1993; Niebel et al., 1993, 1995, 1996; Bird and Wilson, 1994; Opperman et al., 1994). However, very little is understood regarding molecular changes that occur in the root early after infection, before giant-cell initiation or induction of the hypersensitive response. Genes induced early after nematode infection could potentially have a role in the defense against nematodes or other root pathogens. Overall, root defense systems are poorly understood compared with shoot systems. Of the cases examined, proteins induced in the root during pathogenesis are similar to antimicrobial proteins found in the shoot, such as chitinase, β-1,3-glucanase, osmotin, and ribosome-inactivating protein (Maraganore et al., 1987; Benhamou et al., 1990, 1993; Neale et al., 1990; Savary and Flores, 1994; Savary et al., 1997).To identify rapidly up-regulated, nematode-induced plant genes with a possible role in defense against nematodes or other root parasites, we developed a technique to obtain synchronously infected root tips and then produced a cDNA library from them (Ho et al., 1992; Lambert and Williamson, 1993). Several genes that are increased in expression by 12 h after nematode infection were identified by differential screening (Williamson et al., 1994; Lambert, 1995). Most of these genes appeared to be equally induced in plants independent of the presence of Mi in the genome. Some have homology to known plant defense genes, including those coding for peroxidase, chitinase, lipoxygenase, and proteinase inhibitors (Lambert, 1995; B. Ferrie and V.M. Williamson, unpublished data). One nematode-induced cDNA, clone 23a, encoded the partial sequence of a protein with high similarity to that of miraculin, a protein isolated from the berries of Richadella dulcifica, a west-African shrub.Miraculin alters human taste perception, converting sour into sweet taste (Theerasilp et al., 1989). Because of its sequence similarity to miraculin, the tomato gene was named LeMir (L. esculentum miraculin). The sequence also shows similarity to the soybean trypsin-inhibitor family. Several members of this family have anti-insect/anti-pathogen activity (Ryan, 1990), suggesting that LeMir may have a role in defense against nematodes or other pathogens/pests. Furthermore, LeMir shows very high similarity to TID91, a gene of unknown function that is highly expressed in tobacco genetic tumors (Fujita et al., 1994). In the present study, we characterized the LeMir cDNA sequence and we present information regarding its expression pattern at the mRNA and protein levels.     

小偃麦异附加系TAI-13中来自中间偃麦草的低分子量麦谷蛋白亚基基因的分子克隆

通过分析小麦(Triticum aestivum L.)-中间偃麦草(Agropyron intermedium(Host)Beav)异附加系TA1-Ⅰ系列的麦谷蛋白SDS-PAGE电泳图谱和基因组DNA的PCR扩增产物,发现在异附加系TAI-13中附加的中间偃麦草染色体上具有编码高分子量和低分子量麦谷蛋白亚基基因的位点,属于第一同源群.随后,采用RT-PCR方法,从TAI-13的未成熟子粒中克隆了4个来自中间偃麦草的低分子量麦谷蛋白亚基基因.序列分析表明,13003、13006和13054是包括信号肽编码序列的全长基因,而13514没有信号肽编码序列.根据由核苷酸序列推导的蛋白质分子的N-末端氨基酸序列,这4个基因编码的麦谷蛋白亚基可分为3种类型,即Ai-M型(由基因13514编码,命名为LAi1)、Ai-Q型(由基因13006和13045编码,分别命名为LAi2和LAi3)和Ai-Ⅰ型(由基因13003编码,命名为LAi4).通过与小麦的低分子量麦谷蛋白亚基分子比较,发现Ai-M和Ai-Q是两种未见报道的新的低分子量麦谷蛋白亚基类型,而Ai-Ⅰ型与小麦的Ⅰ型亚基相似.氨基酸序列分析发现,基因13514编码的蛋白质亚基分子LAi1有较长的重复区(26个重复模块)和较多的半胱氨酸残基(9个),推测其可形成3个分子间二硫键,可能对增强面团的强度和粘弹性有正面效应.     

小偃麦异附加系TAI-13中来自中间偃麦草的低分子量麦谷蛋白亚基基因的分子克隆

通过分析小麦(TriticumaestivumL.)-中间偃麦草(Agropyronintermedium(Host)Beav)异附加系TAI-Ⅰ系列的麦谷蛋白SDS-PAGE电泳图谱和基因组DNA的PCR扩增产物,发现在异附加系TAI-13中附加的中间偃麦草染色体上具有编码高分子量和低分子量麦谷蛋白亚基基因的位点,属于第一同源群。随后,采用RT-PCR方法,从TAI-13的未成熟子粒中克隆了4个来自中间偃麦草的低分子量麦谷蛋白亚基基因。序列分析表明,13003、13006和13054是包括信号肽编码序列的全长基因,而13514没有信号肽编码序列。根据由核苷酸序列推导的蛋白质分子的N-末端氨基酸序列,这4个基因编码的麦谷蛋白亚基可分为3种类型,即Ai-M型(由基因13514编码,命名为LAi1)、Ai-Q型(由基因13006和13045编码,分别命名为LAi2和LAi3)和Ai-I型(由基因13003编码,命名为LAi4)。通过与小麦的低分子量麦谷蛋白亚基分子比较,发现Ai-M和Ai-Q是两种未见报道的新的低分子量麦谷蛋白亚基类型,而Ai-I型与小麦的I型亚基相似。氨基酸序列分析发现,基因13514编码的蛋白质亚基分子LAi1有较长的重复区(26个重复模块)和较多的半胱氨酸残基(9个),推测其可形成3个分子间二硫键,可能对增强面团的强度和粘弹性有正面效应。     

小麦高分子量麦谷蛋白5亚基基因的克隆及其表达载体的构建

小麦麦谷蛋白5亚基直接影响面包的烘烤品质,但我国大部分小麦品种的蛋白中缺少这种亚基。通过引物设计、PCR扩增,从Cheyenne中得到了小麦麦谷蛋白5亚基结构基因(sub5)的全长核苷酸序列(2750bp)和小麦麦谷蛋白5亚基基因启动子(Psub5)的核酸序列(630bp)。测序结果表明：得到了小麦籽粒中特异表达启动子——Psub2和用于改良小麦品质的结构基因——sub5。通过选择和改变相应的酶切位点,在构建6个中间载体的基础上,最后得到了含有目的基因的表达载体pCAM—BIAl301—Psub5—sub5—nos。酶切电泳及PCR鉴定表明：已成功地合成了sub5的表达载体。有希望通过基因工程的方法将该表达载体用于小麦的品质改良。     

小麦HMW-G12亚基基因启动子克隆及序列分析

为了研究高分子量谷蛋白基因启动子在种子中的特异性表达,以小麦品种“东农7742”的基因组DNA为模板,根据已发表序列设计并合成引物,用PCR的方法克隆了小麦贮藏蛋白中高分子量谷蛋白12亚基基因的上游调控序列。序列测定结果表明：所克隆的启动子片段大小为424bp与Thomspon报道的序列比较,同源性为97.9%,有9个核苷酸发生了改变。推测的TATA box位于-27— -30bp,Prolamin-box位于-175— -181bp,认为该元件可能与转录速率的调控有关。     

小麦高分子量谷蛋白亚基Glu-B1位点沉默基因的克隆与序列分析

二粒小麦（Triticum turgidum L．var．dicoccoides）具有极其丰富的遗传多样性,是栽培小麦品种改良的巨大基因库。在高分子量谷蛋白基因的组成上,它具有许多栽培小麦不存在的变异类型,在Glu—B1位点上的变异更大。我们利用种子贮藏蛋白的SDS—PAGE方法从原产于伊朗的二粒小麦材料PI94640中观察到缺失Glu—B1区的高分子量谷蛋白亚基。利用Glu-1Bx基因保守序列设计PCR引物,对该材料的总DNA扩增,获得了X型亚基编码基因（Glu-1Bxm）的全序列,其全长为3442bp含1070bp的启动子区。序列比较发现,Glu-1Bxm在启动子区序列与Glu—1Bx7的最为相似。而在基因编码区,我们发现Glu—1Bxm仅编码212个氨基酸,由于开放阅读框中起始密码子后第637位核苷酸发生了点突变,即编码谷酰胺的CAA突变为终止密码TAA,可能直接导致了该高分子量谷蛋白亚基的失活,这是我们在小麦Glu—B1位点基因沉默分子证据的首次报道。将Glu—1Bxm全序列与Glu—B1位点其他等位基因进行了系统树分析,发现Glu—1Bxm是较为古老的类型。本文还对该特异高分子量谷蛋白亚基变异类型对品质遗传改良研究的意义进行了讨论。     

A Relaxed Specificity in Interchain Disulfide Bond Formation Characterizes the Assembly of a Low-Molecular-Weight Glutenin Subunit in the Endoplasmic Reticulum

Wheat (Triticum spp.) grains contain large protein polymers constituted by two main classes of polypeptides: the high-molecular-weight glutenin subunits and the low-molecular-weight glutenin subunits (LMW-GS). These polymers are among the largest protein molecules known in nature and are the main determinants of the superior technological properties of wheat flours. However, little is known about the mechanisms controlling the assembly of the different subunits and the way they are arranged in the final polymer. Here, we have addressed these issues by analyzing the formation of interchain disulfide bonds between identical and different LMW-GS and by studying the assembly of mutants lacking individual intrachain disulfides. Our results indicate that individual cysteine residues that remain available for disulfide bond formation in the folded monomer can form interchain disulfide bonds with a variety of different cysteine residues present in a companion subunit. These results imply that the coordinated expression of many different LMW-GS in wheat endosperm cells can potentially lead to the formation of a large set of distinct polymeric structures, in which subunits can be arranged in different configurations. In addition, we show that not all intrachain disulfide bonds are necessary for the generation of an assembly-competent structure and that the retention of a LMW-GS in the early secretory pathway is not dependent on polymer formation.The unique ability of wheat (Triticum spp.) flour to form a dough that has the rheological properties required for the production of leavened bread and other foods is largely due to the characteristics of the proteins that accumulate in wheat endosperm cells during seed development (Gianibelli et al., 2001). Among these endosperm proteins, a major role is played by prolamines, a large group of structurally different proteins sharing the characteristic of being particularly high in Pro and Gln.On the basis of their polymerization status, wheat prolamines can be subdivided into two groups, the gliadins and the glutenins. While gliadins are monomeric, glutenins are heterogeneous mixtures of polymers where individual subunits are held together by interchain disulfide bonds (Galili et al., 1996; Tatham and Shewry, 1998). The subunits participating to the formation of these large polymers have been classified into four groups according to their electrophoretic mobility (Gianibelli et al., 2001). The A group is constituted by the so-called high-molecular-weight glutenin subunits (HMW-GS), while polypeptides in groups B, C, and D are collectively termed low-molecular-weight glutenin subunits (LMW-GS). While only three to five HMW-GS are expressed in common wheat endosperm, LMW-GS include a very large number of different polypeptides.Different models of glutenin assembly have been proposed (see Gianibelli et al., 2001 for a review), but the determination of their precise structure and M_r distribution has been hampered by their large size and complex subunit composition. Crucially, because disulfide bonds appear to be the major factor affecting polymer stability, it would be very useful to know whether the pairing between specific Cys residues, rather than random assembly, controls glutenin polymer formation. Indeed, data obtained with HMW-GS indicate that the formation of certain types of intermolecular disulfide bonds is particularly favored (Tao et al., 1992; Shimoni et al., 1997). In the case of LMW-GS, at least two functionally distinct types of subunits can be distinguished. Subunits of the first type, to which the majority of B-type subunits belong, would act as chain extenders, because they contain two Cys residues that remain available for the formation of interchain disulfide bonds. Subunits of the second type, containing a single Cys residue able to form an interchain disulfide bond, would instead act as chain terminators (Kasarda, 1989). Most of the members of this second group are indeed modified gliadins that participate to polymer formation thanks to the presence of extra Cys residues (D''Ovidio and Masci, 2004). Given the complexity of the situation found in wheat endosperm, where many different subunits are synthesized at the same time and can participate in the formation of complex high-M_r polymers, the study of glutenin polymer formation can take advantage of the use of heterologous expression systems where the behavior of individual subunits can be more easily monitored. For instance, the expression of HMW-GS in transgenic tobacco (Nicotiana tabacum) has provided insights into the rules governing the assembly of some of the subunits belonging to this class (Shani et al., 1994; Shimoni et al., 1997). In this work, we have used heterologous expression of wild-type and modified LMW-GS in tobacco protoplasts to study the assembly of this class of gluten polypeptides. Our results confirm that disulfide bonds are crucial for the assembly of these proteins and indicate that a relaxed specificity in Cys pairing from different subunits can drive the formation of complex glutenin polymers.     

Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing of x-type Gene on Glu-B1

Understanding the molecular structure of high-molecular-weight glutenin subunit (HMW-GS) may provide useful evidence for the study on the improvement of quality of cultivated wheat and the evolution of Glu-1 alleles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the subunits encoded by Glu-B1 were null, named 1Bxm, in a Triticum turgidum var. dicoccoides line PI94640. Primers based on the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of line PI94640. The PCR products were sequenced, and the total nucleotide sequence of 3 442 bp including upstream sequence of 1 070 bp was obtained. Compared with the reported gene sequences of Glu-1Bx alleles, the promoter region of the Glu-1Bxm showed close resemblance to 1Bx7. The Glu-1Bxm coding region differs from the other Glu-1Bx alleles for a deduced mature protein with only 212 residues, and a stop codon (TAA) at 637 bp downstream from the start codon was present, which was probably responsible for the silencing of x-type subunit genes at the Glu-B1 locus. Phylogenetic tree based on the nucleotide sequence alignment of HMW glutenin subunit genes showed that 1Bxm was the most ancient type of Glu-B1 alleles, suggesting that the evolution rates are different among Glu-1Bx genes. Further study on the contribution of the unique silenced Glu-B1 alleles to quality improvement was also discussed.     

TcLr35小麦中病程相关蛋白1基因的克隆及分析

根据已发表的植物病程相关蛋白1基因设计引物,利用RT-PCR技术,从被小麦叶锈菌诱导的小麦抗叶锈病基因近等基因系材料TcLr35中获得一个病程相关蛋白1基因cDNA片段,长度为489bp,3′端包含21个poly(A),暂命名为PR12。利用5′RACE技术获得了818bp的PR12全长,该基因包含495bp的开放读码框,147bp的5′非翻译区(non translated region,NTR),155bp的3′非翻译区和21bp的多聚腺苷酸尾。编码164个通读的蛋白质氨基酸序列,基因产物具有植物防御体系中病程相关蛋白SCP保守结构域,与GenBank中多个植物病程相关蛋白1基因具有较高的同源性。Southern杂交显示,该基因在小麦基因组中为单拷贝。     

Development, Characterization, and Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat

A Cd²⁺-selective vibrating microelectrode was constructed using a neutral carrier-based Cd ionophore to investigate ion-transport processes along the roots of wheat (Triticum aestivum L.) and two species of Thlaspi, one a Zn/Cd hyperaccumulator and the other a related nonaccumulator. In simple Cd(NO₃)₂ solutions, the electrode exhibited a Nernstian response in solutions with Cd²⁺ activities as low as 50 nm. Addition of Ca²⁺ to the calibration solutions did not influence the slope of the calibration curve but reduced the detection limit to a solution activity of 1 μm Cd²⁺. Addition of high concentrations of K⁺ and Mg²⁺ to the calibration solution to mimic the ionic composition of the cytoplasm affected neither the slope nor the sensitivity of the electrode, demonstrating the pH-insensitive electrode's potential for intracellular investigations. The electrode was assayed for selectivity and was shown to be at least 1000 times more selective for Cd²⁺ than for any of those potentially interfering ions tested. Flux measurements along the roots of the two Thlaspi species showed no differences in the pattern or the magnitude of Cd²⁺ uptake within the time frame considered. The Cd²⁺-selective microelectrode will permit detailed investigations of heavy-metal ion transport in plant roots, especially in the area of phytoremediation.     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子.利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因.该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74%的同源性.在多肽链的N-端具有在真核生物中高度保守的UFD1结构域.我们将该基因定位在小麦的第六染色体群并将其命名为TUFD1.South-ern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的.无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源.除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域.TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达.     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子。利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因。该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74％的同源性。在多肽链的N-端具有在真核生物中高度保守的UFD1结构域。我们将该基因定位在小麦的第六染色体群并将其命名为了UFD1。Southern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的。无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源。除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域。TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达。