水稻菠菜紫黄质脱环氧化酶基因的克隆遗传转化及其特性的研究

自发现叶黄素循环具有热耗散的作用后它被引起广泛的关注目前普遍认为叶黄素循环的色素定位于天线色素蛋白复合体上在跨膜质子梯度pH形成后玉米黄质Z和环氧玉米黄质A能够从叶绿素中吸收过多的激发能并以热能的形式耗散到体外从而保护光合器官免受强光的破坏紫黄质脱环氧化酶VDE是叶黄素循环的关键酶在较低的pH条件下它能在数分钟内将紫黄质V转变为Z和A本论文从水稻和菠菜中克隆了编码VDE酶的基因并通过转基因植物进一步研究了叶黄素循环在热耗散方面的作用主要获得了以下结果 首次从两个水稻亚种籼稻和粳稻中克隆了Rvde基因分别命名为iRvde和jRvde的全长cDNA序列分别长1647bp和1887bp两者开放阅读框的同源性为98%与其它已知vde基因的同源性在60以上推导两者均编码446个氨基酸其中转运肽序列长98个氨基酸两者成熟蛋白的氨基酸序列完全相同与已知VDE成熟蛋白的同源性在75%以上其中与小麦的同源性最高达87.4 通过PCR扩增获得了Rvde基因的核基因组DNA序列在它们的编码区中含有4个内含子其长度在jRvde中分别为105bp327bp81bp和69bp而iRvde基因的第2个内含子长425bp与jRvde的第2个内含子差别较大内含子的AT含量为6063%其两端为典型的GT/AG结构 构建了Rvde基因的原核表达载体pET-Rvde在0.4mmol/L IPTG的诱导下该基因能在大肠杆菌BL21(DE3)中大量表达SDS-PAGE和Western杂交表明表达蛋白的分子量约为 43 kDa随着IPTG诱导时间的延长蛋白量逐渐增加诱导4h后它占大肠杆菌总蛋白的25左右吸收光谱差值A502-540随反应的进行逐渐增大反应体系总色素的HPLC分析表明V逐渐降低而Z刚好相反说明表达的蛋白具有与活体VDE酶相同的功能能在体外将V转变为A和Z 从菠菜中克隆了Svde基因并构建了该基因的反义抑制植物表达载体pCB-antiSvde用根癌农杆菌介导法转化烟草获得了大量的转基因植株再生的愈伤组织经GUS染色后呈蓝色PCR扩增潮霉素抗性基因hpt和Svde基因结果显示在转基因植株T0和T1代中都分别扩增出1.0 kb和1.4 kb的目的片段而在未转化的对照植株中没有扩增转基因植株的T0代种子在潮霉素培养基上的萌发数与未萌发数的比值为3:1符合单基因的孟德尔分离规律从T1代转基因植株中筛选出抑制程度较强的一个株系A29Southern杂交结果表明外源Svde基因已整合到烟草的基因组中并且只有一个插入位点通过冻融法从该植株的类囊体中提取VDE酶其酶活性为3.2是对照植株的45.7表明VDE酶受到了抑制荧光动力学及HPLC测定结果显示强光处理后在转基因植株中Z和A的形成较少非光化学淬灭NPQ值较对照低Fv/Fm的下降较对照快表明转基因植株的热耗散能力下降进而说明叶黄素循环具有热耗散的功能 同时还建立了根癌农杆菌介导的水稻遗传转化体系并初步作了转化Svde基因的试验另外还建立了一种适合于筛选转基因植株的DNA微量提取法此方法操作快捷方便一个人在一天内能制备50多个样品100mg的植物鲜样平均可获得40µg的DNA提取的DNA可直接用于PCR反应酶切分析及Southern分析

辐照交联电线电缆生产线束流反射和偏转装置

一种用于电子束辐照交联电线电缆生产线的束流反射和偏转装置，由安装在支撑架上的两只电磁铁反向排列而成，在磁铁两端固定的磁轭上端有长方体形磁极。本实用新型置于生产线辐照窗下，形成的磁场将线缆间隙漏下的电子束流定向反射以提高束流利用率，改变线缆的缠绕排列方式。本实用新型还可用于辐照加工扇形线。

金环蛇蛇毒中的一种新型胰蛋白酶抑制剂

目的:从金环蛇蛇毒中分离纯化名为bungaruskunin 1的一种新型胰蛋白酶抑制剂,并从其毒腺的cDNA文库中克隆出该胰蛋白酶抑制剂的cDNA全序列.方法:通过Sephadex G-50, CM-Sephadex C-25, HPLC, RP-HPLC (C4 column)方法分离纯化bungaruskunin 1.样品的丝氨酸蛋白酶抑制剂活性则是在室温条件下50mmol·L-1 Tris-HCl, pH 7.8的缓冲液中通过对显色底物的水解抑制作用来检测的.金环蛇毒腺RNA用TRIZOL提取,并用SMARTM PCR cDNA synthesis kit (Clontech)建成cDNA文库.根据其信号肽的保守区域合成引物从该文库中扩增出bungaruskunin 1的cDNA全序列,进行胶回收,酶连到pMDl8-T载体中转化测序.结果:bungaruskunin 1的前体由83个氨基酸组成,其中信号肽含有24个氨基酸,成熟肽即:bungaruskunin 1合有59个氨基酸.bungaruskunin 1的cDNA序列与从红腹伊澳蛇Pseudechis porphyriacus中分离纯化得到的丝氨酸蛋白酶抑制剂blackelin的cDNA序列的相似性高达64%.bungaruskunin 1是一种含有保守Kunitz端的Kuntiz蛋白酶抑制剂家族的一员,从而能够抑制蛋白酶和弹性酶的活性.在cDNA文库中,我们同时还筛选到了2种新的β-bungarotoxin B链的序列.结论:这些发现很好地证明了蛇中Kunitz/BPTI胰蛋白酶抑制剂和毒性神经的家族可能起源于共同的祖先.

Taxonomic problems in the Drosophila melanica species group (Diptera : Drosophilidae) from southern China, with special reference to karyotypes and reproductive isolation

Karyotypes and reproductive isolation were studied in two allopatric populations of Drosophila tsigana, one from Guizhou Province in southern China and the other from Hokkaido in northern Japan, and in one population of a closely related species, D. longi

PCB-integrated optical gas sensor

A novel technological platform for multiple gas detection based on the use of PCB-integrated polymer waveguides is presented. A proof-of-principle ammonia sensor is reported integrating onto low-cost FR4 substrates all essential photonic, electronic and chemical components. The device's potential to detect multiple gases is demonstrated. © 2011 IEEE.

Habitat degradation of Rhinopithecus bieti in Yunnan, China

Black-and-white snub-nosed monkeys (Rhinopithecus bieti) are endemic to the Trans-Himalayas in Northwest Yunnan and Southeast Tibet between the upper Yangtze and Mekong Rivers. Based on field surveys and previous reports, we identified the dark-coniferous forest, the mixed coniferous and broadleaf forest, and oak patches as suitable habitats (SH) for the monkeys. Summer grazing lands (SGL), which were made by local people cutting and burning the dark-coniferous forest at the high altitude belt, replaced SH. To have a general view of the status of the SH in Yunnan, we estimated the areas of SH and SGL from satellite images in 1997, and compared with areas estimated from aerial photo-based maps (ca. 1958). The work resulted in: 1) the area of SH was 4,169 km(2) in 1997; 2) SGL was 1,923 km(2); 3) during the past 40 years, the area of SH decreased by 31% (1,887 km(2)), and SGL increased by 204% (1,291 km(2)); and 4) the mean size of forest patches decreased from 15.6 to 5.4 km(2). In addition, the area of SGL is positively correlated to local human population (R-2 greater than or equal to0.53), implying that the reduction and fragmentation of habitat for Rhinopithecus bieti is a result of population growth of humans, who mostly employ traditional modes of production. Only 11 monkey groups remained in the changing habitat. Considering that forests at lower elevation were also encroached upon by farmlands in a similar way, the forest ecosystem is highly threatened. The destruction will continue unless there is a change in the mode of production in the region.