家蚕抗菌肽attacin基因植物表达载体的构建

抗菌肽是昆虫抵御外界微生物侵染的主要物质。本文利用双酶切将pBI121载体上的植物启动子CaMV35S克隆到植物表达载体pCAMBIA2300上,构建重组表达载体pCAMBIA2300-CaMV35S。PCR扩增家蚕抗菌肽attacin基因编码区全长并利用T载体成功克隆（GenBank登陆号：GU244351）,然后利用双酶切将attacin 基因亚克隆到pCAMBIA2300-CaMV35S上,通过PCR鉴定,成功构建了attacin基因的植物表达载体pCAMBIA2300-CaMV35S-attacin。为研究attacin基因在植物抗病性方面的应用奠定基础。     

pCAMBIA2300-betA-BADH双价植物表达载体的构建

将载体pCAMBIA2300-35s-OCS进行改造,增加了酶切位点Sac Ⅰ,之后,PCR扩增编码胆碱脱氢酶(CDH)的基因并用Sma Ⅰ和Xba Ⅰ将其与改造后的pCAMBIA2300-35s-OCS连接,得到单价植物表达栽体p2300-gz-betA.再通过Sal Ⅰ、Hind Ⅲ、Sac Ⅰ等酶切位点将编码甜菜碱醛脱氧酶(BADH)的基因及其启动子和终止序列连接到栽体p2300-gz-betA上,最终得到pCAMBIA2300-betA-BADH双价植物表达载体,并将其导入根癌农杆菌GV3101和LBA4404.betA和BADH是编码甜菜碱合成途径的两个关键酶的基因(CDH和BADH),也是近年来研究较多的耐盐基因,其双价植物表达栽体应用于遗传转化后,有望进一步提高转基因植物的耐盐性.     

pCAMBIA2300-betA-BADH双价基因植物表达载体的构建

摘要：本实验以载体pCAMBIA2300-35s-OCS为基础通过分子克隆手段,将甜菜碱合成途径的两个关键酶基因,即编码胆碱脱氢酶（CDH）的betA基因和编码甜菜碱醛脱氧酶（BADH）的BADH基因及启动子和终止序列构建在同一植物表达载体上,得到pCAMBIA2300-betA-BADH双价植物表达载体。并将其导入根癌农杆菌GV3101和LBA4404,进一步用于双子叶植物的遗传转化。     

拟南芥WUS基因的克隆及植物表达载体的构建与鉴定

克隆得到正确的拟南芥WUS基因,成功构建WUS基因组成型植物表达载体和WUS-EGFP融合基因诱导型植物表达载体,分别转化了根癌农杆菌EHA105,并用WUS基因组成型植物表达载体转化橡胶树愈伤组织,得到组织化学检测阳性的愈伤组织,为深入研究WUS基因生理生化功能做准备。用双酶切切下植物表达载体PBI121的35S-GUS片段,将其连接到pCAMBIA2301上成为中间植物表达载体pCAMBIA2301-35S-GUS。提取拟南芥总RNA,通过RT-PCR方法获得WUS基因,经过TA克隆连接到pEASY-T1载体上,再用双酶切切下WUS基因,将其取代pCAMBIA2301-35S-GUS中的GUS片段,形成pCAMBIA2301-35S-WUS植物表达载体。同时以质粒pCAMBIA2301-EGFP为模板,PCR获得EGFP,用重叠延伸PCR技术（gene splicing by overlap extension PCR,简称SOE PCR）获得WUS- EGFP融合基因,经双酶切将融合基因连接到诱导型植物表达载体pER8上,构成pER8-WUS-EGFP诱导型植物表达载体。通过电击转化法将这两个载体成功转入根癌农杆菌EHA105中,经过双酶切检测、PCR检测与测序分析,检测结果皆完全正确,克隆的WUS基因与WUS-EGFP融合基因序列和NCBI上公布的序列也完全一致,说明这两个植物表达载体已经构建成功。另外用pCAMBIA2301-35S-WUS植物表达载体遗传转化橡胶树易碎胚性愈伤组织,经GUS染色为蓝色,说明愈伤组织转化成功。     

FaGST基因改良菊苣新种质创制

谷胱甘肽转移酶(glutathione transferases,GSTs)在植物抵御逆境胁迫中具有非常重要的作用.从高羊茅叶片中克隆获得FaGST基因,对其进行酶切位点分析,设计酶切引物,通过Bam HI和PstⅠ双酶切将目的基因片段连接在真核表达载体pCAMBIA 1300-35 S上,成功构建pCAMBIA 13...     

LFY基因双T-DNA植物表达载体的构建

LFY基因是从从野生型拟南芥中克隆出的一个与调控花分生组织启动相关的基因,它有促进植物提早开花的作用。用PCR方法从克隆载体上扩增出LFY基因并在两端添加XhoⅠ酶切位点,将其连接在用XhoⅠ切除了hyg基因的pCAMBIA1301表达载体上,得到去除选择标记的LFY基因植物表达载pCAM-BIA1301-LFY。质粒pCAMBIA1301经HindⅡ/EcoRⅠ双酶切补平自连后得到去除多克隆位点的质粒pCAMBIA1301-,再经SacII/SphI双酶切后插入到经SacⅡ酶切的pCAMBIA1301-LFY中。建成双T-DNA植物表达载体pCAMBIA1301-LFY-hyg-,经酶切鉴定证明了所构建载体的正确性。将此双T-DNA载体用农杆菌介导法转化菊花叶片,期望得到无选择标记含LFY基因的转基因菊花。     

几丁质酶基因chiB 的克隆及表达分析

几丁质酶能够通过破坏昆虫组织中的几丁质起到抗虫作用。为获得高表达的植物几丁质酶基因载体,通过同源克隆的手段,从粘质沙雷氏菌中分离到了chiB基因,长度为992 bp,构建了pET28表达载体,并用IPTG诱导了该基因的表达。经分析发现该基因表达产物属于糖基水解酶（glycosyl hydrolases）18家族,并利用pCAMBIA2300构建了植物表达质粒pCAM-35S-chiB。     

稻瘟菌蛋白激发子基因pemG1植物表达载体的构建及其转化烟草的研究

设计含MluⅠ酶切位点的接头,将其连接入经KpnⅠ和BamHⅠ酶切后的质粒pCAMBIA2300-U-bi-Ocs,得到pCAMBIA2300-Ubi-adaptor-Ocs。设计含有KpnⅠ和MluⅠ酶切位点的引物,以稻瘟菌基因组DNA为模板扩增得到pemG1序列。将其连接入pMD18-T得到pMD18-T-pemG1。最后,用KpnⅠ和MluⅠ从pMD18-T-pemG1切下pemG1基因片段,将其连接入pCAMBIA2300-Ubi-adaptor-Ocs的KpnⅠ-MluⅠ酶切位点,构建成稻瘟菌蛋白激发子基因pemG1的植物表达载体体pCAMBIA2300-Ubi-pemG1-Ocs。用冻融法将pCAMBIA2300-Ubi-pemG1-Ocs导入根癌农杆菌菌株AGL-1,再通过根癌农杆菌介导转化获得了转基因烟草株系。用PCR,Northern和Western杂交验证了pemG1基因在受体烟草基因组中的整合,转录和表达。此研究为下一步通过稻瘟菌蛋白激发子基因pemG1的表达来提高转基因烟草的抗病性打下了基础。     

两种抗病基因双T-DNA区双元载体的构建

选择标记基因的删除是植物转基因工程中重要的步骤之一,双T-DNA区双元载体转化法以其共整合频率高、删除标记基因效率高的优点在植物遗传转化中应用非常广泛。本文通过构建天麻抗真菌蛋白基因、脂质转移蛋白基因双T-DNA区双元载体,为培育不含选择标记基因的抗病新材料奠定基础。通过PCR的方法克隆GAFP基因,产物纯化测序确定序列准确后与pSB130-35S载体进行BamHⅠ、SacⅠ双酶切;pCAMBIA2300-LTP、pSB130-35S经EcoRⅠ、HindⅢ双酶切,酶切产物纯化后连接,连接产物转化大肠杆菌。采用基因特异性引物进行菌液PCR鉴定阳性克隆,重组质粒pSB130-GAFP、pSB130-LTP双酶切产物大小分别为540bp、1200bp,与预期产物大小一致,pSB130-GAFP、pSB130-LTP双T-DNA区双元载体构建成功。载体pSB130-GAFP、pSB130-LTP转化根癌农杆菌后可以直接用于植物的遗传转化。     

棉花纤维素合酶基因GhcelA1克隆及其载体构建

 根据GeneBank 中GhcelA1（U58283）序列,利用RT-PCR技术,从棉纤维中克隆到GhcelA1 cDNA全长。运用基因重组技术,构建pCAM2300-35S-GhcelA1过表达载体;融合基因表达载体pCAM2300-35S-GhcelA1-EGFP,以及pCAM2300-35S-GhcelA1-RNAi载体,这些载体均通过限制性酶切鉴定和测序验证。农杆菌介导法将融合基因转化棉花子叶,通过激光共聚焦荧光显微镜,在蓝色激发光下观察诱导的愈伤,发现在转化的活体细胞核与质膜内侧有绿色荧光,说明融合基因载体EGFP构建正确,可以在棉花中正常表达。所构建的系列载体可用于转化棉花,促进纤维素在棉纤维中合成与积累。     

冷冻干燥速溶蛋花汤的加工技术

介绍了运用冷冻干燥技术进行速溶蛋花汤的加工,研究了生产工艺、特点以及影响产品质量的主要因素。试验表明,采用冷冻干燥技术加工的速溶蛋花汤,具有营养丰富、风味独特、方便快捷等特点。     

Measuring of Quantity of Matter-mass and the Measuring of Inertia of Matter-momentclm

A new definition of inertia,i.e. the momentum is the measuring of inertia of matter, is proposed. The article through and etc., subjects, the correctness of the new notion of inertia:The monentum is the measuring of inertia has been demonstrated, and the unsolved contradictions of the traditional definition of inetia. And the mass is the measuring of inertia has been revealed.     

香蕉遗传转化方法研究进展

香蕉是热带亚热带发展中国家重要的粮食作物和碳水化合物来源。但近年来,香蕉生产受到严重的病虫危害。大多数香蕉栽培品种是三倍体,生长周期长,而且不孕。由于没有种子,给繁殖和育种带来一定的困难。遗传转化技术的发展为香蕉品种的改良提供了一种有效的手段。香蕉的遗传转化方法有电激法、基因枪法、农杆菌介导法等。农杆菌介导法的应用是香蕉品种改良的一个重大突破。香蕉遗传转化的外植体也发展到多种,有原生质体,胚性细胞悬浮系,分生组织,以及横切薄片等。近几年,随着分子生物学的发展,出现了转化效率更高,重复性更好的香蕉遗传转化技术。如农杆菌和基因枪结合法,离心辅助农杆菌介导法、真空渗透技术等。这些新技术新方法的出现,必将推动香蕉产业高速发展。     

The Simulation of Comfort of Nine DOF of Automobile

Automobile comfort dynamic simulation is necessary when we predict and evaluate the comfort of a automobile or optimize performance of automobile. The nine DOF vibration model of automobile is established , which acts root mean square value of body in multiple work conditions as comfort evaluation indices. Based on the comfort simulation, the Hongyan 1160 is predicted in the way of comfort. The programmes can be used in many different automobiles . The computational result indicates that the model can simulate vibration of automobile truly. It is significant to research automobile comfort evaluation to optimize performance of automobile and comfort simulation .The computational program can be applied to optimize or simulate which has some reference value.     

Efficiency of water use of early plantings of sunflower

Rain fed crop production in Mediterranean environments depends to a large extent on strategies that avoid the intense summer drought. Early plantings of sunflower have given consistently higher yields in such environments, but the basis for such yield increases has not been explored. We conducted two field experiments at Cordoba (Spain) to investigate the effects of an early and a late planting date on the components of water-limited crop productivity; namely, water use (T), water use efficiency (TE) and harvest index (HI) of sunflower. The results were generalized by simulating rain fed sunflower yields, under early (1 January) and late (15 March) plantings, for a 25-year period with the aid of a simulation model of the Ceres type (OILCROP-SUN) which has been validated in Cordoba. Experimental seed yields of early plantings in 1989 and 1996 were 2.0 and 3.0 t ha⁻¹, while late plantings yields were 1.3 and 2.4 t ha⁻¹, for the 2 years. Average simulated yields were 2.7 ± 1.1 and 1.9 ± 0.7 t ha⁻¹ for early and late plantings, respectively. For the 2 years, T of early plantings was higher than that of late plantings, but the response of TE and HI to planting date was not the same in the two experiments. In the simulation exercise, T and TE of early plantings were consistently higher than those of late plantings, while there were no differences in the HI for the two planting dates. We conclude that early plantings of sunflower increase rain fed yields by increasing both T and TE, while the impact of planting date on HI very much depends on the crop water stress pattern, which is quite variable from year to year even in the predictable Mediterranean environment.     

Establishment of High-frequency Regeneration System of Explants of Alfalfa

The cotyledon and hypocotyl of eight varieties of alfalfa( Medicago sativa L. )are used to study the effect of different cultivation procedures and genotypes on embryogenic callus and embryoid induction,plant development and transplant of alfalfa.The varieties of alfalfa with high embryoid induction rate and high differentiation rate are screened. A high frequency regeneration system for alfalfa tissue culture is established:which is benifical to the gene transformation of alfalfa.     

影响核桃贮藏品质因素的分析

介绍了几种核桃贮藏方法,综述了影响核桃贮藏品质的几个主要因素,以及针对上述影响因素提出若干解决对策.     

南瓜降糖功能及其系列食品加工技术

阐述了南瓜在糖尿病辅助治疗方面的功能特性,介绍了南瓜降糖主要制品南瓜全粉、南瓜脆片、南瓜果酱和南瓜肉汁的工艺流程和操作要点。     

壳聚糖对石榴果汁澄清效果的影响

利用壳聚糖的絮凝作用对壳聚糖在甜石榴汁澄清中的应用效果进行了研究,试验结果表明,壳聚糖用量为0.3g/L、澄清时间为60min、温度为35℃时,甜石榴汁透光率可达85%以上,且通过对壳聚糖处理前后可溶性固形物、总酸、VC等营养成分含量的比较,确定以上工艺参数为甜石榴汁澄清的最佳选择。     

酸枣幼根原生质体的制备

以生长6～9 d的酸枣种子幼根为试验材料,0.7 mol·L-1甘露醇作为渗透压稳定剂、pH值5.5～6.0、28℃恒温水浴条件下,以光学显微镜观察的水解总时间、初始产生原生质体的时间、原生质体产量及细胞碎片量为指标,研究通过酶水解来制备酸枣幼根原生质体的方法.结果表明,在含1％果胶酶+4％纤维素酶R-10+3％纤维素...