棉花抗黄萎病基因工程研究和防卫反应相关基因的克隆

系统获得性抗性(Systematic acquired resistance, SAR)是植物抵御病原菌侵染的最有效手段，利用基因工程技术导入SAR信号发生过程中的关键基因后，植物的SAR基因表达量提高并且对病原菌侵染的反应速度加快，因此植物的抗病性得以增强，与传统的抗病基因工程技术相比它对病原菌没有专一性，许多学者称之为广谱抗病基因工程，该领域已成为目前抗病基因工程研究的热点和前沿。 NDR1和NPR1基因在植物的SAR发生中起着重要的作用，前者功能定位在ROS(reactive oxygen species)的激活和随后的水杨酸(SA)诱导合成之间，突变株病原菌诱导后SA合成能力降低，SAR发生减弱，目前还没有对该基因进行过量表达分析的报道;后者功能定位在SAR信号转导级联反应之中的SA积累和随后的SAR基因表达之间。该突变株在病原菌侵染时不产生病程相关蛋白(PRs)，表现为感病，而对照抗病;过量表达该基因的转基因拟南芥对多种病原菌的侵染产生抗性，PR1等PRs蛋白的表达量也提高，异源表达该基因的水稻对白叶枯病的抗性也提高。本研究利用RT-PCR方法从拟南芥中克隆了这两种基因，序列分析表明拟南芥Wassilewskija生态型的NDR1基因与Columbia生态型相比，共有7处碱基不同，引起编码氨基酸变化4处，而NPR1基因与报道的Wassilewskija生态型来源的NPR1基因完全相同。 我们构建了35S启动子驱动的NDR1和NPR1基因的植物组成型高效表达载体，利用农杆菌介导法转化烟草，PCR和Southern鉴定外源基因已经整合到植物基因组中。抗病性分析显示过量表达NDR1和NPR1基因的烟草对晚疫病和赤星病的抗性都有明显提高，说明这两个基因的在其它植物中异源表达后，都能提高植物对多种病原菌的抗性。 本论文提出了利用这两个基因来培育抗黄萎病棉花的设想，一方面为解决这个“世纪性”难题积累新的资料，另一方面也为其它作物的抗病基因工程提供新的经验。利用35S启动子驱动的NDR1和NPR1基因的植物组成型表达载体分别对陆地棉品种石远321进行花粉管通道法转化。同时，还探讨了这两个基因在棉花中的共转化实验，希望它们的“协同增效”能进一步提高棉花的抗病性。对其中2001年夏天在南京注射所获得的5,000粒种子在三亚进行100 g/ml卡那霉素筛选，初步鉴定分别获得转NDR1和NPR1基因株系26和24棵，PCR进一步鉴定其中分别有12和7棵为转基因阳性，转基因频率分别为0.50％和0.27％，目前利用营养钵蘸根法对其二代进行抗枯、黄萎病鉴定，结果显示有转基因植株对枯、黄萎病的抗性都明显增强，进一步的鉴定正在进行中。2002年初海南注射分别获得转NDR1和NPR1基因以及共转化种子22,000、10,500和12,500粒种子，2002年夏在中国农科院植保所黄萎菌病圃筛选抗黄萎病单株，并利用100 g/ml卡那霉素初步筛选出了一批抗性植株，每种转基因株系随机挑选5株进行PCR鉴定，结果显示为阳性。进一步的抗黄萎病鉴定和筛选以及分子分析正在进行中。 同时，本文还探讨了病原菌诱导型启动子在广谱抗病基因工程应用的可能性。根据烟草的Pr1-a启动子已知序列设计引物，PCR扩增启动子序列后,构建病原菌诱导型NPR1基因植物表达载体，并对棉花进行转化，获得种子11,500粒，利用同上的筛选方法，获得了一致的结果，目前抗黄萎病鉴定、分子检测以及生物学分析正在进行中。 最后，鉴于抗生素标记在转基因植物的应用引起了许多“安全性”争论的事实，还构建了无筛选标记的表达载体对抗虫棉进行转化，这样在生产上可以直接获得抗虫棉抗黄萎病棉花新材料，也为其它作物抗病基因工程积累经验。 本研究还提出了一种较为有效的提取高质量棉花总RNA的方法，与原来一些棉花RNA纯化方法相比，该方法所用都为常规试剂，易于重复，质量高。并且利用获得的总RNA构建了黄萎菌激发子诱导的cDNA文库，滴度测定为1╳107pfμ/μg，插入片段大小在5 00~2 000 bp范围内。 鉴于NPR1基因研究的重要性，本研究还利用简并引物PCR技术从海岛棉和陆地棉的基因组中都分离到了NPR1基因的同源片段，大小都为208 bp，与拟南芥NPR1基因的相应部分的同源性分别为66％和65％，它们之间的同源性为87％，目前该基因的全长正在分离鉴定中。 多聚半乳糖醛酸酶抑制蛋白(PGIPs)在植物的防御反应中起着重要的作用，通过分析已知20余种pgip基因序列的保守区，设计简并引物，PCR扩增海岛棉（Gossypium barbadense）7124 cDNA文库，得到一条长561 bp的片段，序列测定后分析确认为pgip基因的一部分。根据此序列和棉花病原菌诱导的cDNA文库载体中已知部分设计RACE引物，扩增后，5’和3’RACE分别得到666bp和906 bp的片段。序列分析表明它具有完整的编码框，产物为330 aa的蛋白质。序列分析该蛋白具有10个串联的LRR(leucine-rich repeat)区，与柑桔(Citrus)和枳(Poncirus)的pgip基因的同源性分别为69.2%和68.7%。进一步PCR扩增得到该基因的全长阅读框，并且获得了相应的基因组片段，序列分析发现该基因没有内含子。这是从棉属植物中克隆的第一个pgip基因。

Microstructure and wear resistance of in situ TiCp composite coating by laser cladding

A new in situ method was realized by one step laser cladding to produce Ni-base alloy composite coating reinforced by in situ reacted and gradiently distributed TiCp particles. The submicron TiCp particles were formed and uniformly distributed because of the in situ reaction and trapping effect under the rapid solidification condition. And, TiCp particles were of gradient distribution on a macro scale and their volume fraction increased from 1.86% at the layer/substrate interface to a maximum 38.4% at the surface of the layer. Furthermore, the in situ generated TiCp/gamma-Ni interfaces were free from deleterious surface reactions. Additionally, the clad coating also revealed a high microhardness of gradient variation with the layer depth and the superior abrasive wear resistance.

Application of Electrical Resistance Tomography System to Monitor Gas/Liquid Two-Phase Flow in a Horizontal Pipe

The Electrical Resistance Tomography (ERT) technique possesses great potential in monitoring widely exiting industrial two/multi-phase flow. For vertical pipe flow and inclined pipe flow, some application studies with exciting results have been reported, but there is rarely a paper regarding the application of ERT to horizontal gas/liquid pipe flow. This paper addresses this issue and proposes a smart method, Liquid Level Detection method, to conventional ERT system. The enhanced ERT system using the new method can monitor horizontal pipe flow effectively and its application is no longer restricted by the flow conditions. Some experimental results from monitoring an air/water slug pipe flow are presented.

Characterization of Air-Water Two-Phase Vertical Flow by Using Electrical Resistance Imaging

The characterization of air-water two-phase vertical flow in a 12 m flow loop with 1.5 m of vertical section is studied by using electrical resistance tomography (ERT). By applying a fast data collection to a dual-plane ERT sensor and an iterative image reconstruction algorithm, relevant information is gathered for implementation of flow characteristics, particularly for flow regime recognition. A cross-correlation method is also used to interpret the velocity distribution of the gas phase on the cross section. The paper demonstrates that ERT can now be deployed routinely for velocity measurements and this capability will increase as faster measurement systems evolve.

Effect of diffusion on coating microstructure and oxidation resistance of aluminizing steel

The effect of diffuse treatment on coating microstructure and oxidation resistance at high-temperature of hot-dip aluminum were studied by means of TEM, SEM and XRD. The results show that, the diffusion temperature has significant effect on structure of coatings and its oxidation resistance. After diffusion at 750 degreesC, the coating consists of thick outer surface layer (Fe2Al5+ FeAl2), thin internal layer (FeAl + stripe FeAl2), and its oxidation resistance is poor. After diffusion at 950 degreesC, the outer surface layer is composed of single FeAl2 phase, the internal layer is composed of FeAl phase, and its oxidation resistance declines due to the occurrence of early stage internal oxidation cracks in the coating. After diffusion at 850 degreesC, the outer surface layer becomes thinner and consists of FeAl2 Fe2Al5(small amount), the internal layer becomes thicker and consists of FeAl+spherical FeAl2, and the spheroidized FeAl2 phase in the internal layer and its existing in FeAl phase steadily improve the oxidation resistance of the coating.

Microstructure and high-temperature wear and oxidation resistance of laser clad gamma/W2C/TiC composite coatings on gamma-TiAl intermetallic alloy

There are very strong interests in improving the high-temperature wear resistance of the y-TiAl intermetallic alloy, especially when applied as tribological moving components. In this paper, microstructure, high-temperature dry sliding wear at 600 degrees C and isothermal oxidation at 1000 degrees C on ambient air of laser clad gamma/W2C/TiC composite coatings with different constitution of Ni-Cr-W-C precursor mixed powders on TiAl alloy substrates have been investigated. The results show that microstructure of the laser fabricated composite coatings possess non-equilibrium microstructure consisting of the matrix of nickel-base solid solution gamma-NiCrAl and reinforcements of TiC, W2C and M23C6 carbides. Higher wear resistance than the original TiAl alloy is achieved in the composite coatings under high-temperature wear test conditions. However, the oxidation resistance of the laser clad gamma/W2C/TiC composite coatings is deceased. The corresponding mechanisms resulting in the above behaviors of the laser clad composite coatings are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Effects of contact resistance on thermal-conductivity of composite media with a periodic structure

The thermal conductivity of periodic composite media with spherical or cylindrical inclusions embedded in a homogeneous matrix is discussed. Using Green functions, we show that the Rayleigh identity can be generalized to deal with thermal properties ot these systems. A new calculating method for effective conductivity of composite media is proposed. Useful formulae for effective thermal conductivity are derived, and meanings of contact resistance in engineering problems are explained.

Measurements of Flow Patterns and Velocities of Gas -Water Two -Phase Flows Using Electrical Resistance Imaging

This paper presents a measurement of flow patterns and flow velocities of gas-water two-phase flows based on the technique of electrical resistance tomography (ERT) in a 40m horizontal flow loop. A single-plane and dual-plane ERT sensor on conductive ring technique were used to gather sufficient information for the implementation of flow characteristics particularly flow pattern recognition and air cavity velocity measurement. A fast data collection strategy was applied to the dual-plane ERT sensor and an iterative algorithm was used for image reconstruction. Results, in respect to flow patterns and velocity maps, are reported.

MEASUREMENT OF SOLID SLURRY FLOW VIA CORRELATION OF ELECTROMAGNETIC FLOW METER, ELECTRICAL RESISTANCE TOMOGRAPHY AND MECHANISTIC MODELLING

The study presented here was carried out to obtain the actual solids flow rate by the combination of electrical resistance tomography and electromagnetic flow meter. A new in-situ measurement method based on measurements of the Electromagnetic Flow Meters (EFM) and Electrical Resistance Tomography (ERT) to study the flow rates of individual phases in a vertical flow was proposed. The study was based on laboratory experiments that were carried out with a 50 mm vertical flow rig for a number of sand concentrations and different mixture velocities. A range of sand slurries with median particle size from 212 mu m to 355 mu m was tested. The solid concentration by volume covered was 5% and 15%, and the corresponding density of 5% was 1078 kg/m(3) and of 15% was 1238 kg/m(3). The flow velocity was between 1.5 m/s and 3.0 m/s. A total of 6 experimental tests were conducted. The equivalent liquid model was adopted to validate in-situ volumetric solids fraction and calculate the slip velocity. The results show that the ERT technique can be used in conjunction with an electromagnetic flow meter as a way of measurement of slurry flow rate in a vertical pipe flow. However it should be emphasized that the EFM results must be treated with reservation when the flow pattern at the EFM mounting position is a non-homogenous flow. The flow rate obtained by the EFM should be corrected considering the slip velocity and the flow pattern.

Modified porous silica antireflective coatings with laser damage resistance for Ti:sapphire

Porous SiO2 antireflective (AR) coatings are prepared from the colloidal silica solution modified with methyltriethoxysilane (MTES) based on the sol-gel route. The viscosity of modified silica suspensions changes but their stability keeps when MTES is introduced. The refractive indices of modified coatings vary little after bake treatment from 100 to 150 Celsius. The modified silica coatings on Ti:sapphire crystal, owning good homogeneity, display prominent antireflective effect within the laser output waveband (750-850 nm) of Ti:sapphire lasers, with average transmission above 98.6%, and own laser induced damage thresholds (LIDTs) of more than 2.2 J/cm2 at 800 nm with the pulse duration of 300 ps.

Reversible Resistance Switching Effect in Amorphous Ge1Sb4Te7 Thin Films without Phase Transformation

We demonstrate a reversible resistance switching effect that does not rely on amorphous-crystalline phase transformation in a nanoscale capacitor-like cell using Ge1Sb4Te7 films as the working material. The polarity and amplitude of the applied electric voltage switches the cell resistance between low- and high-resistance states, as revealed in the current-voltage characteristics of the film by conductive atomic force microscopy (CAFM). This reversible SET/RESET switching effect is induced by voltage pulses and their polarity. The change of electrical resistance due to the switching effect is approximately two orders of magnitude.