藏鸡主要组织相容性复合体B-LBⅡ基因第二外显子序列遗传变异分析

根据鸡主要组织相容性复合体B-LBⅡ基因序列设计特异性引物,在藏鸡基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,并通过克隆和PCR直接测序获得了该片段的核苷酸序列。发现了15个B-LBⅡ新等位基因。对18个B-LBⅡ等位基因核苷酸序列和其所编码的MHCB-LBⅡ分子β1结构域的氨基酸序列分析显示,第二外显子核苷酸序列遗传多态性异常丰富,存在着62个多态变异位点(共包括80个突变),其中41个为简约性多态位点;衡量该序列遗传多样性的π值为0.0718;反映其群体内遗传变异度的平均遗传距离为0.056±0.008,低于在5个外来品种所估算的平均遗传距离。该编码区核苷酸相对异义替换率(15.61±2.69%)显著高于其同义替换率(3.25±0.94%),进一步分析表明,基因重组和平衡选择机制可能是引起B-LBⅡ基因序列变异的主要因素。在β1结构域氨基酸序列中,存在11个同义替换和27个异义替换;在24个肽结合位点中有12个变异位点;与其他6个中国地方鸡品种和一个外来品种比较发现,有11个异义氨基酸替换仅出现在藏鸡群体中,并被认为与藏鸡的免疫特异性有关,可为鸡的抗病力研究提供分子依据。     

藏鸡胚胎的低氧适应特征及其血红蛋白突变效应

藏鸡能在4200 m以上海拔正常繁衍生息, 但低地鸡在模拟4200 m海拔(12.0%O₂)条件下孵化却只有3.0%的孵化率. 在两品种全期低氧和阶段低氧模拟孵化实验中, 4200 m全期低氧揭示4~11天是隐性白与藏鸡阶段存活率相差最悬殊的阶段, 12~21天相差极显著, 1~3天相差不显著. 但4200 m阶段低氧只能造成两品种孵化率显著差异, 而并不足以造成低氧阶段间阶段死亡率显著差异, 结果显示藏鸡具有优越的克服低氧损害后期不良影响的能力. 对藏鸡和隐性白鸡血红蛋白7条链全部编码序列进行多态分析, 筛选到α^D链编码基因的一个低氧功能突变位点(Met- 2D(B13)-Leu). 该突变基因为藏鸡独有, 其频率随着海拔升高而升高, 而且该突变基因频率越高的群体, 孵化率越高. 以鸡的HbD晶体为模板, 对突变血红蛋白结构和功能进行了同源预测, 结果表明Leu替代Met提供了一个更疏水环境, 有利于血红素的稳定, 从而提高了血红蛋白的血氧亲和力. 突变(Met-32D(B13)-Leu)发生的时间和地点与藏鸡家养化时间和地点密切相关.     

藏鸡诱导型一氧化氮合酶基因低氧适应功能分析

低氧刺激诱导型一氧化氮合酶(Inducible nitric-oxide synthase, iNOS)催化产生NO, 增加血流, 改善组织供氧。文章采用测序和PCR-RFLP技术检测藏鸡及低地鸡iNOS基因编码区、5′侧翼区(2.0 kb片段)序列和3′侧翼序列SNP, 并测定低氧和常氧孵化时鸡胚尿囊绒毛膜组织iNOS基因表达量和酶活力。结果在iNOS基因 5′侧翼区发现一个与低氧适应相关的藏鸡高频率突变SNP位点(-870C→T), 藏鸡该突变的等位基因T频率高于低地鸡种。藏鸡iNOS基因表达量和酶活力在低氧孵化环境中多高于矮小鸡。结果表明藏鸡群体iNOS基因的突变及其低氧表达量的增加是其适应低氧环境的重要基础。     

藏鸡高原适应性的胚胎心脏组织差异表达研究

氧是机体及细胞进行有效能量代谢不可缺少的物质,低氧使机体及细胞的生理发生改变。藏鸡是我国青藏高原特有的原始地方鸡种,是世界上在高原生活历史最久的鸡种,对低氧具有稳定遗传的适应能力。在本研究中,藏鸡的全同胞种蛋分别在常氧和低氧的条件下进行孵化,寿光鸡和矮小隐性白鸡两个低地鸡种为对照,在胚胎发育的第43期取其心脏组织,与鸡的全基因组芯片杂交研究藏鸡高海拔适应的分子机理。结果表明,50个转录本在低氧的条件下表达上调,其中一些基因与细胞生长、细胞分化、肌肉收缩等密切相关;而21个转录本在常氧的情况下表达上调,这些基因主要参与细胞交流、离子运输、蛋白质的氨基酸磷酸化、信号转导等过程.有趣的是,这些差异表达基因的富集功能主要与应激过程中的免疫系统应答和离子通道行为有关。另外,层级聚类和CPP-SOM分析结果表明,藏鸡的表达模式与寿光鸡比较接近,而与矮小隐性白鸡的表达图谱相差较远。值得注意的是,本研究还筛选到12个藏鸡特异性表达的转录本。它们主要参与了能量代谢,转录时的基因启动及抑制胚胎发育的异常等过程。这些发现将有助于解释藏鸡适应高原生活的分子机制,同时,心脏组织的全基因组表达图谱也为高原医学的研究提供了线索。     

真菌中一氧化氮生物合成、降解及功能的研究进展

一氧化氮是一个有较高活性的自由基气体分子,无论在动植物还是微生物中,作为一个细胞内和细胞间的信号传导分子,它在许多的生理和病理过程中都发挥着双向的调节作用.研究发现真菌细胞可以合成一氧化氮,适当浓度的一氧化氮在真菌细胞内发挥多种重要的生物学功能,一旦一氧化氮过量累积,这个自由基分子会对细胞造成伤害,导致细胞凋亡.一氧化氮介导生成的环鸟苷酸(cGMP)作为一种重要的第二信使分子涉及到真菌细胞内多种信号途径的调控,调节了整个真菌类群的生长发育、形态发生、孢子形成和萌发、繁殖和细胞凋亡的过程,影响了真菌整个生命周期的生理活动.到目前为止,尽管一氧化氮在动植物中作用的机制得到了广泛的研究,但一氧化氮在真菌中的研究报道很有限.关于一氧化氮在真菌中的合成和降解途径,一氧化氮介导的信号传导机制的研究还不透彻,它在真菌细胞内的功能和毒理还有待于更深入的研究.     

藏鸡微卫星文库的构建与微卫星标记筛选

通过磁珠富集法构建了藏鸡基因组微卫星富集文库,分离微卫星序列并对其进行分析.将藏鸡基因组DNA经Sau3AI酶切后纯化回收,连接特定接头.用生物素标记的(CA)12探针与藏鸡基因组酶切回收片段杂交,捕获200～900 bp片段,随后将获得的片段连接到pMD 18-T载体上,转化至JM109中,成功构建藏鸡微卫星富集文库.从1200个转化子中获得了353个阳性克隆,随机挑选53个测序,根据测序结果成功设计了18对藏鸡微卫星引物,最终筛选出6个具有多态性的微卫星标记,其PIC值均大于0.5,同时可以用于研究藏鸡遗传多样性.实验结果表明磁珠富集法能够有效提高分离微卫星标记的效率.     

基于F-2群体的藏鸡羽色、胫色性状的遗传分析

采用白来航、寿光鸡分别与藏鸡进行正反交交配,F1代进行自群交配产生F2群体,观察F1和F2代中羽色、胫色的表现和分离比例。结果表明,白来航鸡的白羽和寿光鸡的黑羽对藏鸡麻羽的遗传方式是完全显性遗传;麻羽是由两个或两个以上的等位基因决定的,只有同时存在这两个或两个以上的等位基因,才可能表现出麻羽来;决定胫色性状的Id/id基因为伴性遗传,隐性基因id在纯合子时有个逐步表达的过程;本研究证实了所用白来航公鸡胫色性状的基因型为显性纯合子。     

藏鸡线粒体全基因组序列的测定和分析

通过PCR扩增,测序,拼接,获得藏鸡（Tibetan Chicken）线粒体全基因组序列并进行数据分析处理。藏鸡线粒体全基因组序列全长16783bp,共有13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个D-loop区。模拟电子酶切结果显示,藏鸡DraI酶的酶切结果和先前报道的原鸡,茶花鸡,尼西鸡和大理漾濞黄鸡的酶切结果都不相同,为藏鸡特有。基于D-loop区全序列和13个蛋白质编码基因序列,采用N-J算法与原鸡属4个种,3个亚种和3个家鸡品系构建系统进化树：初步确定藏鸡起源于红原鸡,与家鸡中的来航鸡、白洛克鸡亲缘关系最近,但是藏鸡的进化与来航鸡、白洛克鸡这两个家鸡品系又显得相对独立。推测可能原因是藏鸡的祖先在进入高原以后处于相对封闭的环境,从而形成了独特群体遗传特性。     

藏鸡群体遗传多样性研究

藏鸡的体型外貌和生活习性与红色原鸡非常相似,是具有自己独特群体遗传特性的高原地方鸡种。为了有效保护并合理利用这一遗传资源,我们采用多重PCR与半自动荧光标记微卫星聚丙烯酰胺凝胶电泳相结合的方法检测了20个微卫星基因座的多态性,并随机抽取藏鸡群体中部分个体进行个体体形特征与生产性能的统计。结果表明藏鸡群体的20个微卫星基因座的多态等位基因数为4~10个,平均值为7.25个/基因座,多态信息含量（CPI）和杂合度（H）平均值分别为0.67、0.74。大染色体较小染色体的微卫星标记多态性程度要高。藏鸡群体的微卫星基因座多态性丰富,也解释了生产性能不均,外貌表现迥异的群体遗传特性。 Abstract: Morphological traits and living habit of Tibetan chicken, which is an aboriginal chicken breed on plateau with its own characteristic populational genetic features, are in great common with the Red Jungle Fowl, the assumed ancestry of domestic chicken. To fully exploit this chicken resource, Multiplex PCR with semi-automated polyacrilamide gel electrophoresis (PAGE) using fluorescently labeled microsatellite primers was used to detect the polymorphism at 20 microsatellite loci. At the same time, we randomly test the individual morphology and performance. It showed that numbers of polymorphic alleles were 4-10, with mean value 7.25 per locus. Polymorphism Information Content (CPI) and Heterozygosity (H) had mean values 0.67 and 0.74, respectively. Macrochromosomes had relatively higher polymorphism than microchromosomes(P>0.05). In all, high polymorphisms at microsatellite loci related to the uneven production performance and morphological discrepancy of population genetic characteristics in Tibetan chicken.     

藏鸡心脏高海拔低氧适应相关酶的研究

目的：研究藏鸡心脏对高海拔低氧适应性的生理特征。方法：本研究将藏鸡、矮小隐性白和寿光鸡分别饲养在低海拔和高海拔环境,测定10周龄时心脏重量、心肌乳酸（LA）和乳酸脱氢酶（LDH）、琥珀酸脱氢酶（SDH）活性。结果：结果显示藏鸡在高海拔环境中,心脏相对重量未明显增加,心肌LA低于对照鸡,LDH与对照鸡差异不显著,而SDH活性明显高于对照鸡。结论：结果说明了藏鸡对高海拔低氧环境的适应,不是通过增加心脏器官的重量,也不是通过提高无氧代谢的水平,较高的SDH活力对藏鸡心肌低氧适应有一定的意义。SDH是藏鸡适应低氧的一种标志酶。     

Abundance and Diversity of Glacial Bacteria on the Tibetan Plateau with Environment

The relation of glacial microorganism and their living environment is concerned but less understood for both glaciologists and microbiologists. Here we present the results about glacial snow bacteria from 3 glaciers on the Tibetan Plateau. The concentrations and diversity of bacteria collected from snow pit samples of East Rongbuk, Laohugou and Hailuogou glaciers on the Tibetan Plateau were investigated by epifluorescence microscope, denaturing gradient gel electrophoresis and Shannon-Weaver index. Concentrations and community diversity of bacteria in the East Rongbuk glacier with lower concentration and smaller size of microparticle were lower than in the Laohugou with higher concentration and larger size of microparticle. Bacterial concentration in East Rongbuk Glacier was close to that in polar regions, suggesting that Mt. Everest is a bacterial background for remote regions away from direct influence of anthropogenic sources. In addition, altitude difference was another factor for higher concentrations and community diversity of bacteria in Hailuogou and Laohugou glaciers than in East Rongbuk Glacier. The highest concentrations and community diversity of bacteria in Hailuogou Glacier were attributed to its most diverse atmospheric circulations and highest temperature among the 3 glaciers. We suggest a complicated correlation between glacial bacteria and their regional living environments.     

青藏高原微生物多样性研究

正青藏高原被誉为世界屋脊,其内部除平原外还有许多山峰、冰川、高山湖泊和高山沼泽,是生态环境最为奇特、生物资源最为丰富的自然资源宝库之一。同时,青藏高原的微生物群落结构及其多样性与其他区域存在巨大差异,因而具有极高的科学研究价值,并逐渐被人们所关注。研究发现气候变化对青藏高原高寒草地生态系统草丛-地境界面微生物会产生重要的影响[1]。冰川雪藻的研究主要在南部的Yala冰川开展,     

蛋白质水平解析高山嵩草对青藏高原昼夜环境的响应（英文）

高山嵩草(Kobresia pygmaea)是高寒草甸的重要建群种,其生长发育同时受到年际、季节和昼夜环境变化的影响,但目前对高山嵩草响应昼夜环境变化的研究很少。本研究通过差异蛋白质组学的方法,结合抗氧化酶活性测定和蛋白质免疫印迹技术,分析了高山嵩草在一天中每4个小时的蛋白质表达变化。结果表明:在白天的高温、强光和紫外辐射,以及夜里的低温等不利条件下,高山嵩草体内的抗氧化酶、热休克蛋白和脱落酸代谢相关的蛋白质等能够被大量诱导表达,从而对细胞和机体起到保护作用。同时,受蛋白质调控的一些生命活动如光合作用会集中在较为适宜的时间段进行。通过体内蛋白质表达的可塑性和灵活性,高山嵩草能够有效地应对短时间里环境的变化。     

Diversity of Snow Bacteria from the Zangser Kangri Glacier in the Tibetan Plateau Environment

Bacterial abundance and community diversity were investigated in a snow pit on the Zangser Kangri (ZSGR) glacier on the Tibetan Plateau by epifluorescence microscopy and small subunit 16 S rRNA gene sequence analyses. Cell abundance and bacterial colony-forming units (CFUs) ranged from 3 × 10³ to 1.5 × 10⁴ cells/ml and 0 to 2.2 CFU/ml, respectively. All isolates were classified into four major groups: Actinobacteria (68%), Alphaproteobacteria (18%), Gammaproteobacteria (9%) and Firmicutes (5%). Principal component analysis of the biochemical data combined with linear correlation analysis revealed two main sources of bacteria on the ZSGR glacier. Considerable amounts of bacteria were probably emitted by vegetation of Qangtang Grassland and transported by the Indian monsoon, some of which might have originated from the Taklimakan Desert and undergone long-range transportation by westerly winds. Bacterial abundance was positively correlated with both Ca²⁺ and HCOO^? concentrations, suggesting two microbial transport carriers, including mineral and organic particles. Members of Caulobacter and Roseomonas that participate in nitrogen transformations were observed in the uppermost snow pit layer, suggesting that they are involved in nitrogen cycling on this glacial surface.     

祁连圆柏针叶解剖结构对高原寒旱环境的适应性分析北大核心CSCD

祁连圆柏(Juniperus przewalskii)是青海省森林生态系统的重要组成部分,为揭示祁连圆柏对高原寒旱环境的适应特性,该研究以青海省祁连圆柏当年生针叶为研究对象,采用石蜡切片法制片,测定其叶宽、叶厚、下角质层厚、下表皮细胞厚、叶维管束木质部及韧皮部厚,并利用回归分析及偏相关分析探讨了针叶解剖结构对经纬度、海拔、坡向的响应特征。结果表明:(1)祁连圆柏针叶解剖结构在经纬度上无显著变化,其解剖结构与经纬度均无显著相关性。(2)祁连圆柏针叶解剖结构与海拔关系存在阶段性和结构间的差异性,在环境压力较小的低海拔段(<3 200 m)无显著变化;在海拔3 200 m以上,祁连圆柏叶宽、叶厚、下角质层厚、下表皮细胞厚随海拔升高而不同程度地增厚;当海拔高于3 680 m时各针叶解剖结构增长趋势变缓,尤其是叶宽、叶厚增速下降比例更大;下角质层厚和下表皮细胞厚与海拔相关性最强,其次是叶厚、叶宽,但叶维管束木质部厚以及韧皮部厚与海拔始终均未表现出显著相关性。(3)6个地区阴坡祁连圆柏针叶下角质层厚均高于阳坡,且下角质层厚与坡向显著相关。研究认为,祁连圆柏对高原寒旱环境适应性强,经纬度变化、低海拔段和阳坡的环境压力不足以使其产生相应结构变化;在阴坡其通过增厚针叶下角质层厚来提高耐寒性,在高海拔段其通过增厚下角质层、下表皮细胞层以及叶宽、叶厚来适应极寒极旱等复杂的环境压力。     

Sequencing and alignment of mitochondrial genomes of Tibetan chicken and two lowland chicken breeds

Tibetan chicken lives in high-altitude area and has adapted well to hypoxia genetically. Shouguang chicken and Silky chicken are both lowland chicken breeds. In the present study, the complete mitochondrial genome sequences of the three chicken breeds were all sequenced. The results showed that the mitochondrial DNAs (mtDNAs) of Shouguang chicken and Silky chicken consist of 16784 bp and 16785 bp respectively, and Tibetan chicken mitochondrial genome varies from 16784 bp to 16786 bp. After sequence analysis, 120 mutations, including 4 single nucleotide polymorphisms (SNPs) in tRNA genes, 9 SNPs and 1 insertion in rRNA genes, 38 SNPs and 1 deletion in D-LOOP, 66 SNPs in protein-coding genes, were found. This work will provide clues for the future study on the association between mitochondrial genes and the adaptation to hypoxia.     

Sequencing and alignment of mitochondrial genomes of Tibetan chicken and two lowland chicken breeds

Tibetan chicken lives in high-altitude area and has adapted well to hypoxia genetically. Shouguang chicken and Silky chicken are both lowland chicken breeds. In the present study, the complete mito-chondrial genome sequences of the three chicken breeds were all sequenced. The results showed that the mitochondrial DNAs (mtDNAs) of Shouguang chicken and Silky chicken consist of 16784 bp and 16785 bp respectively, and Tibetan chicken mitochondrial genome varies from 16784 bp to 16786 bp. After sequence analysis, 120 mutations, including 4 single nucleotide polymorphisms (SNPs) in tRNA genes, 9 SNPs and 1 insertion in rRNA genes, 38 SNPs and 1 deletion in D-LOOP, 66 SNPs in pro-tein-coding genes, were found. This work will provide clues for the future study on the association between mitochondrial genes and the adaptation to hypoxia.Tibetan chicken, lowland chicken, mitochondrial genome, hypoxia.     

Contribution of Tibetan Plateau ecosystems to local and remote precipitation through moisture recycling

The ecosystems of the Tibetan Plateau (TP) provide multiple important ecosystem services that benefit both local populations and those beyond, such as through climate regulation services on precipitation for East Asia and China. However, the precipitation regulation service of the TP ecosystems for supplying moisture and maintaining precipitation is yet to be evaluated. In this study, we used the moisture recycling framework and a moisture tracking model to quantify the precipitation regulation services of TP ecosystems for their contribution to precipitation. We found TP ecosystems contributed substantially to local and downwind precipitation, with a contribution of 221 mm/year for the TP and neighboring areas through evapotranspiration (ET) (104 mm/year through transpiration), declined to <10 mm/year for eastern China and other surrounding countries. Among ecosystem types, grassland contributed most to precipitation, followed by barren and snow lands, forests, and shrublands. In terms of seasonality, precipitation contribution from TP ecosystems was greater in summer months than in non-summer months for western China, while the opposite was true for eastern China—although the magnitude was much smaller. Over the past two decades, the significant ET increases in TP translated to a widespread increase in precipitation contribution for TP and downwind beneficiary regions from 2000 to 2020. Our study provides a quantitative way to understand the precipitation regulation services of TP ecosystems through moisture recycling, substantiating their key role to maintain precipitation and the water cycle for downwind regions—effectively acting as an ecological safeguard that could be perceived by the public.     

青藏高原食草动物粪栖真菌的多样性

分别利用真菌通用引物和厌氧真菌特异引物构建了西藏地区3种反刍动物和1种单胃动物共8份新鲜粪样的ITS克隆文库,以通过系统发育分析解析其中好氧真菌与厌氧真菌的多样性。通用引物ITS文库测得324条真菌序列,分别属于子囊菌门Ascomycota 3个目、担子菌门Basidiomycota 2个目、接合菌门Zygomycota的1个目和严格厌氧的新丽鞭毛菌门Neocallimastigomycota,共24个OTUs。其中,子囊菌门相对丰度最高,占80.6%;新丽鞭毛菌门相对丰度最低,仅占0.6%。大部分OTUs与已知真菌属、种关系较远。厌氧真菌特异引物文库测得661条序列,全部属于新丽鞭毛菌门,包括所有已知的厌氧霉属Anaeromyces、盲肠菌属Caecomyces、肠霉属Cyllamyces、新丽鞭毛属Neocallimastix、奥式霉属Orpinomyces、胃梨囊霉属Piromyces 6个属和3个未培养的属级类群(NG9、NG10、NG11),共29个OTUs。其中3个已知的单中心属存在于所有反刍动物样品中,并以Piromyces相对丰度最高(37.4%)。单胃动物马粪样中全部为NG9类群。NG9是本研究新发现的属级类群,研究中同时揭示有多个未培养种和潜在的新种。研究结果证明青藏高原反刍动物粪栖真菌多样性较高,并存在丰富的未培养种和潜在的新属及新种。