米黑根毛霉脂肪酶基因密码子优化及重叠延伸PCR合成

[目的]对米黑根毛霉脂肪酶基因的密码子进行优化,并采用重叠延伸PCR合成设计的基因序列。[方法]根据毕赤酵母(Pichiapastoris)密码子使用偏好性,对米黑根毛霉脂肪酶成熟肽基因进行全面密码子优化,根据优化后的氨基酸编码序列设计合成30条长度约为48 bp的寡核苷酸片段,并采用重叠延伸PCR法扩增合成全长基因序列。[结果]经凝胶电泳和酶切鉴定、测序分析表明,合成的目的基因与设计的序列相一致。[结论]该研究为脂肪酶基因在毕赤酵母中的构建及后续表达奠定了基础。     

牛传染性鼻气管炎病毒gD基因表达质粒免疫小鼠的试验研究

基因疫苗是近几年来发展起来的一种新型疫苗，具有使用安全，生产、运输、贮存方便，免疫效果稳定持久的特点。实验采用PCR方法将从洛阳种牛场西门塔尔牛精液中分离的牛传染性鼻气管炎(Infectious bovine rhino—traeheitis virus，IBRV)洛精株gD基因进行了扩增，经序列测定确证后构建了gD基因的真核表达质粒。将其单独或连同脂质体肌肉注射于BALB／c小鼠，应用ELISA定期检测血清抗体水平。结果表明，含IBRV gD基因的表达质粒能够引起小鼠的特异性血清抗体反应。     

牛卵泡抑素基因克隆及真核表达载体构建

[目的]克隆牛的卵泡抑素基因（Follistatin,FSTN）基因,构建真核表达载体。[方法]用Trizol法从牛的卵巢中提取总RNA,反转录成cDNA,用带有酶切位点牛FSTN的特异性引物扩增其完整编码区序列,连接到T载体、测序,序列无误后亚克隆入真核表达载体pIRES2-AcGFP1中,酶切及PCR鉴定载体。[结...     

Animal genetic resource trade flows: Economic assessment

Throughout human history, livestock producers have relied on a vibrant international exchange of genetic resources to achieve improvements in the quality and productivity of their animals. In recent years, however, some observers have argued that changes in the legal, technological, and economic environment now imply that international exchanges of animal genetic resources (AnGR) systematically benefit rich countries at the expense of poor countries. It is argued that international flows of AnGR are displacing the indigenous animal genetic resources of developing countries, and also that the genetic wealth of the developing world is being expropriated by rich countries.In reaction, there have been growing calls for limitations and/or barriers to the exchange of animal genetic resources. These discussions, however, seem to be based on limited information about the magnitude and direction of current trade flows in AnGR. This paper offers an analysis of AnGR trade flows from 1990 to 2005. The paper draws on national-level data from 150 countries that reported information to the United Nations Statistics Division. Three major trade categories were evaluated: live cattle and pigs for breeding, and cattle semen.Over the period studied, Europe and North America were the primary exporters of genetic resources for the species evaluated. OECD countries accounted for 98.7, 92.5, and 95% of cattle semen, live cattle, and swine exports in 2005, respectively. In evaluating the direction of trade between developed (North) and developing (South) countries, North–North trade had the largest magnitude, followed by North–South, South–South, and South–North. The data do not support the notion that Southern genetic resources are being used on a large scale in the North. We believe that importation from South to North is limited by the vast discrepancies in production efficiency and production systems between countries in the North and South.Given the low volume of South–North exchange, it seems doubtful that sufficient revenues could be acquired through a “benefit-sharing mechanism” to have any substantial impact on in situ or ex situ conservation efforts, or to generate benefits for poor livestock keepers in developing countries. We question whether global agreements or restrictions on trade will achieve the desired goal of conserving rare breeds and threatened genetic resources. We also doubt whether these agreements will succeed in improving the well-being of the poor. We suggest that resources instead be urgently employed for conservation and that more direct measures should be taken to aid poor farmers, ranchers, and herders in their efforts to conserve genetic resources.     

家蚕G蛋白γ1亚基(BmGγ1)的克隆及其谷胱甘肽硫转移酶融合蛋白的表达与纯化

异三元G蛋白是真核细胞感知外界信号后将信号传递到胞内的重要分子,参与生物体广泛的信号转导。为了研究家蚕体内G蛋白的生理功能及其作用机制,运用生物信息学方法预测了家蚕G蛋白γ1亚基(Gγ1)的序列,设计引物验证预测序列后,克隆了家蚕Gγ1的序列,再通过酶切克隆至表达载体pET-41b(+)后,导入E.coliBL21宿主菌中,经异丙基β-D-硫代半乳糖苷(IPTG)诱导表达重组谷胱甘肽硫转移酶(glutathione s-transferase,GST)融合蛋白,并亲和层析纯化表达产物。家蚕Gγ1重组GST融合蛋白经SDS-PAGE电泳和Western blot分析,在分子质量约36 kD处出现特异性蛋白条带,重组蛋白经GST亲和层析柱纯化后,得到了高纯度的融合蛋白,说明已经成功克隆到家蚕Gγ1基因,并在E.coliBL21中高效表达。     

家蚕GH18家族几丁质酶的系统进化和BmChi的时期表达分析

昆虫GH18家族几丁质酶主要参与昆虫蜕皮、细胞增殖和免疫等生理过程。为了系统开展家蚕GH18家族基因的研究,通过多物种几丁质酶的系统进化分析鉴定了8个家蚕GH18家族成员,并根据含有糖苷水解酶18(Glyco_18)催化结构域和几丁质结合结构域的不同将其分为6类,其中,各物种的CHT5具有典型几丁质酶结构。家蚕GH18家族成员中,BmChiR-1具有5个Glyco_18催化结构域和6个几丁质结合结构域,其它7个成员均只有1个Glyco_18催化结构域,BmCHT12还有1个ChitnaseA_N端结构域。8个家蚕GH18家族成员的基因分布在7条染色体上。通过半定量RT-PCR调查家蚕CHT5基因Bm-Chi在家蚕各发育时期的转录表达模式,该基因在蜕皮、化蛹、羽化等发育时期均有高水平表达,推测BmChi在蚕体旧表皮几丁质的降解过程中发挥重要作用。     

诱导滞育的温度和光照节律对家蚕滞育激素基因Dh表达的影响

滞育激素(DH)是导致家蚕滞育生理现象出现的关键因素。研究卵期不同温度和光照节律引起家蚕滞育激素基因Dh表达的变化,探讨温度和光照对家蚕滞育调控的机制。催青期25℃持续光照(25LL)条件下,蚕卵中Dh基因mRNA高水平稳定转录;而在15℃持续黑暗(15DD)条件下,蚕卵中只有痕迹量Dh基因mRNA存在;25LL或者20℃、光照与黑暗12h交替(20LD)条件下,胚胎发育至单眼着色后(有效积温2160℃.h以后)Dh基因mRNA转录水平显著上调,与此时期胚胎对诱导滞育的温度和光照节律更加敏感一致。从催青期开始整个世代的保护温度和光照节律,呈现高温25℃显著上调整个胚后时期Dh基因mRNA转录水平,光照上调蛹期与蛾期Dh基因mRNA转录水平,且高温的作用强于光照。5龄第3天幼虫的基因芯片表达谱显示,Dh基因mRNA转录水平存在显著的性别与组织差异,雌蚕多个组织中的Dh基因mRNA转录水平都显著低于雄蚕,卵巢中的Dh基因mRNA只有痕迹量。亲代卵的催青温度与光照节律决定子代卵的滞育性,也影响到Dh基因mRNA的转录水平;但子代卵内Dh基因mRNA转录水平高低不是蚕卵滞育与否的关键。推测家蚕Dh基因表达的性别差异及在早期蚕卵中表达特征与滞育性不一致的现象,可能与Dh基因的前体多肽翻译产物剪切有关。     

蓖麻GST的基因克隆及生物信息学分析

谷胱甘肽S-转移酶(glutathione S-transferase,GST)是谷胱甘肽结合反应的关键酶,催化谷胱甘肽结合反应的起始步骤,主要存在于胞液中,有多种形式,其在病理学上有一定的重要性。本研究通过克隆GST基因以及对其进行相关的生物信息学分析。利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增该基因,经分析该基因可编码219个氨基酸,并且通过相关分析得到这219个氨基酸组成的蛋白为稳定的亲水性蛋白,有一个明显的区段存在信号肽,在该蛋白中α-螺旋和无规则卷曲是主要的二级结构方式,根据序列同源比对,蓖麻、麻疯树和榴莲聚类到一起,说明相对于其他物种,它们的同源性较高。这些分析结果为蓖麻谷胱甘肽S-转移酶生物合成及其功能的进一步研究提供一定的理论与实践依据。     

紫芽茶树类黄酮生物合成关键酶基因表达与总儿茶素、花青素含量相关性分析

儿茶素类化合物与花青素均由类黄酮代谢途径合成,紫芽茶中富含花青素。为探明紫芽茶树中类黄酮生物合成代谢流的情况,本试验以来源于湄潭苔茶后代的1株紫色芽叶茶树和1株绿色芽叶茶树为材料,测定芽下第一叶、第二叶和第三叶的叶色、儿茶素类组分和花青素总量,分析了类黄酮生物合成相关的基因表达情况及基因表达量同总儿茶素、花青素累积量之间的相关性。结果表明,紫芽茶树中各叶位中花青素含量均显著高于对照绿芽茶树,而儿茶素类总量却低于对照;类黄酮生物合成关键酶(PAL、CHS、CHI、F3H、DFR、ANS、ANR1、ANR2、F3¢H和F3’5’H)基因均呈现上调趋势。紫色芽叶中的总儿茶素与花青素,同各相关基因(LAR除外)表达水平的相关性都较高,且二者相关系数差异不大。绿色芽叶中的总儿茶素与各基因(LAR、F3’H除外)表达的相关系数,明显高于花青素同各基因表达的相关系数。     

Evaluation of seedling and adult plant resistance to stem rust in European wheat cultivars

A total of 105 European wheat cultivars were assessed for seedling and adult plant resistance (APR) to stem rust using an array of Australian isolates of Puccinia graminis f. sp. tritici. Twenty-seven cultivars were susceptible at both seedling and adult plant growth stages. Twelve catalogued seedling stem rust resistance genes (Sr7b, Sr8a, Sr8b, Sr9b, Sr9g, Sr11, Sr15, Sr17, Sr29, Sr31, Sr36 and Sr38) were detected in the remaining cultivars, and 13 cultivars carried additional seedling resistance genes that could not be postulated with the isolates used. Low levels of APR to stem rust were found in the cultivars Artaban, Forno, Mec, Mercia, Pandas and Vlada. Although the genetic identity of this APR was not determined, it was clear that the only designated stem rust APR gene Sr2 was not present in any of the cultivars tested based on the absence of the linked traits seedling chlorosis and pseudo black chaff. One of these cultivars, Forno, is believed to carry the leaf rust APR gene Lr34, previously reported to be associated with improved resistance to stem rust. A detailed genetic characterisation of the APRs in these cultivars will be needed to understand their modes of inheritance and relationships with catalogued stem rust resistance genes. Such knowledge may help in developing cultivars with effective gene combinations that confer higher levels of protection.