来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

Equivalent Model Of Expansion Of Cement Mortar Under Sulphate Erosion

The expansion property of cement mortar under the attack of sulfate ions is studied by experimental and theoretical methods. First, cement mortars are fabricated with the ratio of water to cement of 0.4, 0.6, and 0.8. Secondly, the expansion of specimen immerged in sulphate solution is measured at different times. Thirdly, a theoretical model of expansion of cement mortar under sulphate erosion is suggested by virtue of represent volume element method. In this model, the damage evolution due to the interaction between delayed ettringite and cement mortar is taken into account. Finally, the numerical calculation is performed. The numerical and experimental results indicate that the model perfectly describes the expansion of the cement mortar.

Surface Stress Induced By Adatoms Adsorption On Solid Surfaces

Based on the statistical thermodynamics theory, a theoretical model of adsorbate induced surface stress of adatoms adsorption on solid surface is presented. For the low coverage, the interaction between the adsorbed molecules is entirely negligible and the adsorption induced surface stress is found to be the function of the coverage and the adsorption energy change with strain. For the high coverage, the adsorbate-adsorbate interaction contributes to the adsorption-induced surface stress effectively. In the case of carbon adsorption on the Ni(100) surface, the value of 0.5 is obtained as a characteristic coverage to decide whether to take the interaction between the adsorabtes into consideration and the results also show that the adsorption induces a compressive surface stress.

Measurement Of Fracture Toughness And Interfacial Shear Strength Of Hard And Brittle Cr Coating On Ductile Steel Substrate

The fracture toughness and interfacial adhesion properties of a coating on its substrate are considered to be crucial intrinsic parameters determining performance and reliability of coating-substrate system. In this work, the fracture toughness and interfacial shear strength of a hard and brittle Cr coating on a normal medium carbon steel substrate were investigated by means of a tensile test. The normal medium carbon steel substrate electroplated with a hard and brittle Cr coating was quasi-statically stretched to induce an array of parallel cracks in the coating. An optical microscope was used to observe the cracking of the coating and the interfacial decohesion between the coating and the substrate during the loading. It was found that the cracking of the coating initiated at critical strain, and then the number of the cracks of the coating per unit axial distance increased with the increase in the tensile strain. At another critical strain, the number of the cracks of the coating became saturated, i.e. the number of cracks per unit axial distance became a constant after this critical strain. Based on the experiment result, the fracture toughness of the brittle coating can be determined using a mechanical model. Interestingly, even when the whole specimen fractured completely under an extreme strain of the substrate, the interfacial decohesion or buckling of the coating on its substrate was completely absent. The test result is different from that appeared in the literature though the identical test method and the brittle coating/ductile metal substrate system are taken. It was found that this difference can be attributed to an important mechanism that the Cr coating on the steel substrate has a good adhesion, and the ultimate interfacial shear strength between the Cr coating and the steel substrate has exceeded the maximum shear flow strength level of the steel substrate. This result also indicates that the maximum shear flow strength level of the ductile steel substrate can be only taken as a lower bound estimate on the ultimate shear strength of the interface. This estimation of the ultimate interfacial shear strength is consistent with the theoretical analysis and prediction presented in the literature.

First-passage failure of quasi-integrable Hamiltonian systems

The first-passage failure of quasi-integrable Hamiltonian si-stems (multidegree-of-freedom integrable Hamiltonian systems subject to light dampings and weakly random excitations) is investigated. The motion equations of such a system are first reduced to a set of averaged Ito stochastic differential equations by using the stochastic averaging method for quasi-integrable Hamiltonian systems. Then, a backward Kolmogorov equation governing the conditional reliability function and a set of generalized Pontryagin equations governing the conditional moments of first-passage time are established. Finally, the conditional reliability function, and the conditional probability density and moments of first-passage time are obtained by solving these equations with suitable initial and boundary conditions. Two examples are given to illustrate the proposed procedure and the results from digital simulation are obtained to verify the effectiveness of the procedure.

Recurrence plot analysis of DNA sequences

Recurrence plot technique of DNA sequences is established on metric representation and employed to analyze correlation structure of nucleotide strings. It is found that, in the transference of nucleotide strings, a human DNA fragment has a major correlation distance, but a yeast chromosome's correlation distance has a constant increasing. (C) 2004 Elsevier B.V All rights reserved.

高超声速小钝锥尾流化学非平衡辐射研究

本文主要目的是研究化学非平衡对高超声速小钝锥尾流辐射的影响。计算结果表明：激发发射辐射比化学发光辐射重要，红外辐射比可见光辐射重要。结果还表明：由化学非平衡瞬时辐射模型计算求得的ＮＯ５．３μ的辐射能比由局部平衡辐射模型求得的辐射能大３０～７０％。由此可知，小钝锥尾流的化学非平衡辐射模型对辐射的影响是不能忽略的。

高寒草甸地区牧草生长量模拟模式研究

本文讨论了土壤含水量和蒸散量对牧草生长量的影响过程，提出牧草干物质增长模式，并根据青海海北高寒草甸地区的气象条件，对牧草生长量模式进行数值模拟，同时，与实测值进行比较，模拟结果与实测值趋向一致，可为牧草产草量预报提供一定实用价值和科学依据。

集中弯矩作用下圆弧裂纹的弯曲问题

Ｓｃｈｗａｒｚ对称原理结合复应力函数的奇异性分析的方法，对于分析集中载荷问题有独特的优越性．本文利用这个方法研究了任意位置的集中弯矩作用下含共圆圆弧裂纹板的弯曲问题，获得了一般问题解答．给出一个实际重要情形的封闭形式解；最后求得了弯曲应力强度因子公式．本文公式的特殊情形，与现有经典结果吻合．

爆炸法制备Sm_2Fe_(17)N_y永磁体

本文介绍了爆炸烧结Sm_2Fe_(17)N_7永磁体的试验及其结果.试验时,在外加磁场下把Sm_2Fe__(17)N_y磁粉取向压制成形后装人金属包套内并抽真空,炸药装在包套外的纸筒内,雷管引爆炸药后,在爆轰压力作用下,磁粉被压实而烧结成磁体。试验结束后,取出磁体测量密度及磁性参数。烧结磁体密度在理论密度6的85％～96％范围内,磁能积大于88 KJ?m~(-3)。它表明,爆炸烧结工艺是制备Sm_2Fe_(17)Ny永磁体的有效工艺。

小Kc数振荡流中圆柱绕流流场结构的数值分析

本文综合Chorin的涡方法、网格涡方法和泊松方程快速解法的优点,采用固定离散涡的数目和位置,随时间变化调整离散涡强度的计算方法,对Kc数在1到5之间,β数在10到1665之间圆柱在振荡流动中的流场结构进行了数值模拟。并与U型振荡水槽中的流场涡结构显示照片比较,计算结果表明,数值解在稳定性和收敛性方面都是比较好的。与显示照片相比,其符合程度也是令人满意的。

用计算机技术标定测力传感器

本文提出用计算机技术标定压电测力传感器的方法。提高了标定精度,方便可靠地得到传感器的动态和静态性能,迅速处理大量标定数据;适于实验室中标定压电测力传感器。

定常流作用于波浪形边界附近的圆柱上的水动力

本文介绍在水洞中做的作用于波浪形边界附近的水动力特性的实验研究。在基于圆柱模型直径的 R_c=10~4～1.9×10~4 范围内,测量了圆柱在波谷、波峰和不同距离上的阻力、升力脉动变化的频率。流谱显示实验揭示了尾流结构随距离-直径比 G/D 的变化情况及圆柱与边界相互作用的机制。

自动限温加热带电—热性能的研究

研究了自动限温加热带的电—热性能,得到的结果表明:主动电阻与温度为双曲线关系,功率与温度成线性关系,二者是相互关联的.这是由于加热带通电工作时具有自动限温能力,而自动限温的能力来自加热带的被动电阻具有很高的正温度系数的(PTC)特性所致.讨论了影响加热带输出功率的因素.

地幔对流模拟实验

模拟实验是当前地幔对流研究中的一个重要手段,一个重要组成部分。本文着重介绍近年来关于地幔热对流模拟实验研究发展概况。从相似参数出发,讨论了实验装置,测试手段,并对实验结果和今后发展趋势作了评述。