烟草tga1a基因的表达对外源基因在植物中表达的影响

烟草DNA结合蛋白TGA1a可特异地作用于CaMV35S增强子的激活序列as-1(-83~-63), 并表现为转录激活功能. 为了研究tga1a基因的表达对外源基因在植物中表达的影响, 将它置于维管束特异性启动子rolC下游, 并与CaMV35S启动子控制的报道基因串联成一种反式调控系统, 构建了植物表达载体, 同时, 以CaMV35S和rolC分别控制的报道基因构建植物表达载体为阳性对照. 通过农杆菌介导方法转化烟草和分子鉴定, 证明报道基因存在于转化烟草基因组中, 分别测定了不同转基因单株的GUS活性, 结果表明: rolC控制下的tga1a的表达显著增强了CaMV35S控制下的报道基因表达, 其GUS活性明显高于CaMV35S或rolC单独调控报道基因的转化植株, 单株的最高GUS活性达到两个阳性对照的10倍以上. 组织化学定位证实该串联系统使GUS蛋白主要集中在维管束组织. 这一研究结果为提高外源基因在转基因植株中的表达水平和外源基因的组织特异性表达创立了一个新模式.     

转双抗虫基因烟草的研究

用改造的雪花莲凝集素基因GNAmm与合成的苏云金芽孢杆菌(Bt)毒蛋白cry1Ac基因构建了带有双价基因的植物表达载体,在该表达载体中这两个基因的转录分别受笋瓜PP2启动子(SPP2P)和CaMV 35S启动子的调控。通过根癌土壤杆菌介导转化法,获得了一批抗卡那霉素的转化再生烟草植株。PCR检测及基因组DNA Southern blot\,Slot blot杂交分析的结果表明Gna基因和Bt基因已整合到烟草总DNA中。用Bt毒蛋白抗血清进行Western blot分析,转基因植株均有Bt杀虫蛋白的不同程度的表达。对转化再生烟草的虫试结果表明,在所受试的19株烟草中60%的植株上的棉铃虫在5天内死亡率达到100%,而且存活幼虫的生长发育受到明显抑制;蚜虫抑制生长试验表明,多数转化再生植株具有较强的抗蚜活性,平均能够抑制桃蚜50%～60%的蚜口密度,有的高达80%以上。以上结果表明利用这两个改造过的抗虫基因可以获得既抗虫又耐蚜的转双抗虫转基因植物。     

抗草甘膦抗虫植物表达载体的构建及其转基因烟草的分析

构建了含草甘膦抗性突变基因（aroAM12）和人工合成重组Bt抗虫基因（Bts1m）的植物表达载体pCM12_s1m。aroAM12基因的表达由CaMV35S启动子控制,Bts1m基因的表达由2E_CaMV35S启动子和Ω因子控制。通过农杆菌介导,将aroAM12和Bts1m基因转化到烟草中,转基因烟草通过在含草甘膦的MS培养基上筛选而获得。Southern blot分析表明所有经过草甘膦筛选出的转化植株都整合有aroAM12基因,约70%的转化植株同时整合有aroAM12和Bts1m基因。Northern blot、Immunodot blot分析进一步证明整合的两个基因在转录、翻译水平上均进行了表达,不同植株之间表达存在着差异。草甘膦抗性和虫试实验证明,获得的转基因烟草对草甘膦和烟青虫具有很强的抗性。     

表达昆虫特异性神经毒素AaIT基因的转基因烟草的抗虫性

经改造的昆虫特异性神经毒素AaIT基因插入植物高效表达载体得到重组质粒pNGY-2。重组质粒中AaIT基因5＇端与烟草花叶病毒(TMV)的Ω序列3＇端融合,受两个串联的35S启动子控制,通过土壤农杆菌LBA4404介导转化烟草NC89,经NPTⅡ选择后再经GUS染色挑选出阳性再生植株,Southern blotting进一步证实了AaIT基因已经整合到烟草基因组中,对棉铃虫(Heliothis armigera)的抗虫实验表明,转基因烟草有显著的抗虫活性。     

陆地棉栽培品种转复合启动子控制下的cry 1Ac3基因获得高效抗虫植株

以根癌土壤杆菌( Agrobacterium tumefaciens )介导法分别将植物表达载体pBinMoBc和pBinoBc导入陆地棉( Gossypium hirsutum L.)栽培品种"新陆早1号"、"晋棉7号"、"晋棉12号"和"冀合321".pBinMoBc携带有高效启动子复合OM启动子控制下的 cry 1Ac3基因,pBinoBc携带有35S启动子控制下的 cry 1Ac3基因.经过共培养、卡那霉素筛选抗性愈伤组织及体细胞胚的诱导,得到了再生植株.对T2代的PCR、Southern blotting、ELISA检测及Western blotting证明 cry 1Ac3基因已整合入受体棉花基因组并得到表达.抗虫性检测表明转基因后代对棉铃虫( Heliothis armigera ) 具有良好的抗性,转pBinMoBc T2代与转pBinoBc T2代相比,对棉铃虫具有更快的致死速度.本研究建立了一套高效的陆地棉栽培品种转化体系;进一步的检测结果表明,复合OM启动子可以提高外源基因的表达量从而增强转基因棉的抗虫性.     

一个棉花生殖器官优势表达基因的启动子功能分析

用棉花生殖器官优势表达启动子替代CaMV35S启动子是解决目前转基因抗虫棉存在“前期抗虫性强, 后期抗虫性弱”这一生产实践问题的关键. 运用反向PCR技术从陆地棉中分离到一个棉花生殖器官优势表达基因——腺苷酸核糖基化作用因子1 (Arf1)基因的启动子序列. 该启动子具有独特的结构特征, 不仅同时包含有转录起始子(initiator), TATA box, CAAT box和GC box这四种特异元件, 而且还包含有一个5′非翻译区的内含子. 通过构建四个启动子缺失突变体, 定向替换植物表达载体pBI121上的CaMV35S启动子, 并与GUS基因融合, 构建了4个植物表达载体. 用花粉管通道技术将它们导入到棉花中, 转基因棉花后代的GUS染色和荧光定量分析结果表明: Arf1启动子是一个典型的棉花生殖器官优势表达启动子, 它为棉花生殖器官性状的分子设计和抗虫棉的再创新提供了有用的工具.     

陆地棉栽培品种转复合启动子控制下的cry1Ac3基因获得高效抗虫植株(英文)

以根癌土壤杆菌 (Agrobacteriumtumefaciens)介导法分别将植物表达载体pBinMoBc和pBinoBc导入陆地棉(GossypiumhirsutumL .)栽培品种“新陆早 1号”、“晋棉 7号”、“晋棉 12号”和“冀合 32 1”。pBinMoBc携带有高效启动子复合OM启动子控制下的cry1Ac3基因 ,pBinoBc携带有 35S启动子控制下的cry1Ac3基因。经过共培养、卡那霉素筛选抗性愈伤组织及体细胞胚的诱导 ,得到了再生植株。对T2 代的PCR、Southernblotting、ELISA检测及Westernblotting证明cry1Ac3基因已整合入受体棉花基因组并得到表达。抗虫性检测表明转基因后代对棉铃虫 (Heliothisarmigera )具有良好的抗性 ,转pBinMoBcT2 代与转pBinoBcT2 代相比 ,对棉铃虫具有更快的致死速度。本研究建立了一套高效的陆地棉栽培品种转化体系 ;进一步的检测结果表明 ,复合OM启动子可以提高外源基因的表达量从而增强转基因棉的抗虫性。     

用绿色荧光蛋白监测转基因植物中选择标记基因的消除

绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。     

Vip3a基因植物表达载体的构建及其对烟草的遗传转化

为了研究Vip3A基因在转基因抗虫植物中的应用,利用PCR技术克隆了苏云金芽孢杆菌的Vip3A基因和烟草的EF1α启动子,以pB1121质粒为基本载体,构建了分别由组成型CaMV35S启动子和花特异表达的EF1α启动子驱动Vip3A基因的植物表达载体pBIVip3A和pBIEFVip3A,并通过农杆菌介导的方法对烟草进行了遗传转化。经PCR检测,外源基因已整合到烟草基因组中。     

HPV-16L1植物表达载体的构建及HPV-16L1在转基因烟草中表达的鉴定

利用PCR技术克隆人乳头瘤病毒HPV-16L1蛋白编码基因 ,将其重组于pUCmT和pBI12 1中 ,构建含HPV 16L1基因的植物双元表达载体pBI L1,L1基因由CaMV 35S启动子控制表达。采用叶盘共培育法经根瘤农杆菌介导转化烟草 (NicotianatobacumL .) ,获得HPV-16L1转基因烟草植株。经PCR及Southern杂交分析 ,HPV 16L1基因整合到烟草基因组中 ;Westernblot和ELISA分析检测显示转基因烟草叶片蛋白可与HPV 16L1单克隆抗体特异性反应 ,且定位于 5 5kD处 ,其最高表达量占烟草叶片总可溶蛋白的0.076 %。小鼠红细胞凝集试验 (HA)及小鼠红细胞凝集抑制试验 (HAI)显示转基因烟草叶片蛋白可引起小鼠红细胞凝集。结果表明已成功地构建了HPV-16L1的植物双元表达载体 ,并证实了利用转基因烟草植物能够表达出HPV-16L1蛋白 ,所表达的L1蛋白具有良好的抗原性并具有介导小鼠红细胞凝集的生物活性.     

High Yield and High Selectivity of Reactions in the Frozen State--the Acceptor Reaction of Dextransucrase

Acceptor reactions catalysed by dextransucrase in general yield mixtures of oligosaccharides. Specificity improves with decreasing reaction temperature. Reactions in the frozen state surprisingly give much better yield and selectivity by factors of 2-5.     

High Yield and High Selectivity of Reactions in the Frozen State--the Acceptor Reaction of Dextransucrase

Acceptor reactions catalysed by dextransucrase in general yield mixtures of oligosaccharides. Specificity improves with decreasing reaction temperature. Reactions in the frozen state surprisingly give much better yield and selectivity by factors of 2-5.     

冬小麦高产优质高效栽培决策模拟研究

在产量和品质形成规律及投入产出效益规律基础上,应用模糊集方法,构建了一个综合的冬小麦高产优质高效栽培优化决策模拟模型.并以Window XP为平台,采用VB(Visual Basic)语言编程,建立了相应的可视化决策支持系统.实现了在不同时空、自然、社会、经济、技术条件下,进行多目标、可综合、可选择、可调控的冬小麦田间水肥管理决策的目标.通过检验证明模拟模型是可行的.     

Responses of the Antioxidant Systems and Xanthophyll Cycle in Phaseolus vulgaris to the Combined Stress of High Irradiance and High Temperature

Changes in the activities of enzymes involved in scavenging active oxygen species were followed after exposing bean seedling leaves (Phaseolus vulgaris L.) to various cross stresses of irradiance and temperature. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (AsAPOD, EC 1.11.1.11) increased to different extent with prolonged irradiation of the leaves, and were stimulated by high temperature (HT). The activity of catalase (CAT, 1.11.1.6) decreased when exposed to strong irradiance (HI), and the decrease was further exacerbated when HI was combined with HT. CAT activity was more sensitive to HT than to HI. Ascorbate (AsA) content slightly decreased and then increased during the treatment of HI, but decreased under the cross stress of HI and HT. On the contrary, glutathione (GSH) content increased all the time during various treatments of irradiance and temperature. The increase in the combined stress was even more pronounced. Irradiance is the major reason in triggering the operation of xanthophyll cycle, which was difficult to be started by HT. The antioxidant systems tended to be inactivated with prolonged exposure to the cross stress of HI and HT. The de-expoxidated state of xanthophyll cycle, however, was increasing all the time, which indicated that the zeaxanthin-dependent thermal dissipation was one major energy dissipation pathway during the cross stress of HI and HT.     

银耳子实体多糖高温高压提取工艺研究

通过对银耳子实体多糖高温高压提取工艺中,提取温度、料液比、提取时间,提取次数等影响因素的实验分析,确定银耳子实体多糖高温高压提取的最佳条件为:提取温度110℃(对应压力0.04 MPa);料液比为1∶70;提取时间为2 h;提取次数为2次,在此条件下,银耳子实体多糖提取率可达36.5%。     

High blood triiodothyronine and the euthyroid state

A high excess of circulating T3 was observed in an euthyroid woman. Agarose gel electrophoresis of serum preincubated with 125I-T3 revealed an abnormal T3-binding in gamma-globulin zone. This binding interfered with the hormone radioimmunoassay. Immunological characterization identified this protein as an IgG-K and IgG-lambda polyclonal antibody that bound T3 but not T4. Scatchard analysis of 125I-T3 binding to the gamma-globulin fraction isolated showed a single class of binding sites with a high affinity Ka = 0.4 X 10(9) L/M and maximal binding capacity of 5.2 X 10(-9) M.     

Maintenance of High Photosynthetic Rates during the Accumulation of High Leaf Starch Levels in Sunflower and Soybean

Sunflower (cv. “Mammoth Greystripe”) and soybean (Merr. cv. “Amsoy 71”) leaves were exposed to continuous light for at least 52 hours in an attempt to determine the relationship between leaf starch levels and photosynthetic rates. Immature rapidly expanding and relatively mature slowly expanding sunflower leaves were studied. After 52 hours continuous light, the rapidly expanding leaves accumulated high starch levels (3.3 milligrams per square centimeter, 43% of dry weight) with only about a 10% decline from the initial photosynthetic rate of 42 milligrams CO₂ per square decimeter per hour. Under the same conditions, the slowly expanding leaves accumulated less starch, but the photosynthetic rate declined 30%. Soybean leaves, which were slowly expanding, accumulated less starch than sunflower leaves (2.1 milligrams per square centimeter, 34% of dry weight), and their photosynthetic rates declined only about 10% after 54 hours continuous light.     

High energy solar radiation and the origin of life

Recent satellite observations of young, sun-like stars allow an estimation of the ultraviolet and X-ray radiation environment of the primitive Earth. Energy from these sources is found to be much higher than previously believed. We suggest that the influence of high energy radiation on the early development of life be reexamined.     

The Effect of High Temperatures on the Photosynthetic Activity of Intact Barley Leaves at Low and High Irradiance

Light curves of CO₂ fixation by barley seedling leaves preliminarily heated at 30–43°C for 5 min were measured. The slope of the linear part of the light curve decreased after leaf heating at temperatures above 35°C; whereas, at a high light level, the photosynthesis rate decreased only at temperatures of 40°C and higher. The linear relationships between the photosynthetic CO₂-fixation rate and a photon flux density up to 1400 mol/(m² s) were found in leaves preheated at 42°C; this indicates the strong nonphotochemical dissipation of absorbed light quanta. The lowering of the oxygen concentration from 21 to 1% led to a CO₂ fixation maximum quantum yield and a photosynthesis-rate increase at the highest light intensity in leaves preheated at temperatures above 40°C as compared to the control leaves. Nevertheless, the linear relationship between the photosynthetic CO₂ fixation and the light intensity was found in leaves heated at 42°C at O₂ concentrations of both 21 and 1%. The latter fact suggests that the proton gradient of the thylakoid membrane, which causes an increase in the nonphotochemical dissipation of the quanta absorbed, could also be formed due to the cyclic electron transport over photosystem I.