超甜玉米单倍体悬浮细胞系的建立及其生长特性

从超甜玉米的花药培养物中,筛选出28个具有高度胚胎发生能力的类型Ⅱ愈伤组织系,这种愈伤组织质地松散,易于分散,可以长期进行继代培养。将3个月龄的类Ⅱ愈伤组织接种在液体培养基中进行振荡培养,建立起分散程度很好的8个悬浮细胞系。实验结果说明,培养基中加入酪蛋白水解物(CH)和2-(N-吗啉代)乙基磺酸(MES)对悬浮细胞的生长是有利的。培养基的 pH 迅速下降吋,细胞的增殖十分缓慢,当培养基的 pH 值开始升高时,细胞的增殖也显著加快。利用滋养培养法,可以使悬浮细胞的愈伤组织形成率大大提高(比对照高11.8—19.7倍),并在低密度下达到较高的(41.3%)植板率。染色体计数表明,继代培养6个月之后,88.0%的悬浮细胞和95.2%的再生植株的染色体数保持着2n=x=10的单倍性水平。     

藏羚羊成纤维细胞系的建立及其核型分析

藏羚羊(Pantholops hodgsonii)是我国特有的国家Ⅰ级重点保护野生动物,生存在高海拔低氧环境下,具有高原生物的细胞遗传特性。实验采用组织块贴壁培养法进行原代培养,后经差异贴壁法联合消化排除法纯化,成功构建了藏羚羊成纤维细胞系,并对培养不同代次细胞的形态、生长动力学、染色体核型等进行了研究。结果表明,培养得到的藏羚羊细胞为典型的成纤维细胞,第7、10、15代细胞群体倍增时间分别为26、48、50 h。细胞的生长均为"S"型。染色体中二倍体染色体(2n=60)占主体,所占比例为65%～91%,包括性染色体X、Y在内均为端着丝粒类型,染色体组的总相对长度为193.45,平均相对长度为3.22,未发现随体、次缢痕等特殊标志性特征。     

马头山羊成纤维细胞系的建立与生物学特性分析

采用组织块贴壁培养法对马头山羊耳缘组织进行培养, 成功构建了马头山羊耳缘组织成纤维细胞系, 并对其形态学、生长动力学、细胞活力测定、中期染色体、微生物污染等特性进行了研究。结果表明: 培养细胞形态为典型的成纤维细胞, 细胞群体倍增时间(PDT)约为36 h。细胞冻存复苏后的活率为96.7%, 传代后生长状况与冻存前一致。细胞中期染色体二倍体(2n=60)占主体约为96%。微生物检测细菌、真菌、病毒支原体检测结果为阴性。细胞系各项指标均达到美国典型培养中心(ATCC)标准。此细胞库的建立在细胞水平上对马头山羊的遗传资源进行了保存, 也为今后的生物学研究以及体细胞克隆保种等研究提供了理想的实验材料。     

朱鹮游荡期的生态观察

本文对朱游荡期的夜宿地、觅食地、日活动行为进行了观察     

朱鹮集群繁殖地的生态观察

1993～2005年,朱鹮在洋县刘家庄村集群营巢,成功繁殖了112窝,产卵372枚,成活雏鸟198只.繁殖地的优势树种为油松,其平均高度为14.9 m.9个样方的香农多样性指数(H)为2.6,乔木的多样性指数为1.8;辛普生指数(D)为0.77,乔木的多样性指数为0.58.繁殖地所有树种的平均密度为0.55株/m2,乔木的平均密度为0.12株/m2.刘家庄村分布有冬水田7.10 hm2.巢位树全部是油松.     

家蝇胚胎细胞系的建立及其生物学特性

昆虫细胞系在许多领域得到广泛的应用。以家蝇Musca domestica L.卵(胚胎)为材料,用含20%胎牛血清的M3培养基培养,原代培养35d左右形成单层,每7d传代1次,连续传代60代次。细胞群体倍增时间为44h,染色体2n=12,可在液氮及4℃冰箱中保存复苏,命名为MDEC-07114。     

曼地亚红豆杉细胞系的建立与评价

红豆杉(Taxus)细胞内紫杉醇的含量低且不稳定,限制了利用细胞培养大规模生产紫杉醇的产业化进程。以紫杉醇含量较高的曼地亚红豆杉(Taxus m edia)为试材,诱导得到了曼地亚红豆杉愈伤组织,在添加抗坏血酸(VC)、聚乙烯吡咯烷酮(PVP)、活性炭(activated carbon)的改良B5培养基上,15次反复继代培养后获得了高产紫杉醇的细胞系。对新建立的曼地亚红豆杉细胞系(MC)与同期诱导的东北红豆杉细胞系(NC)及实验室继代多年的中国红豆杉细胞系(SC)在细胞生长周期、紫杉醇含量和细胞死亡率等方面进行了分析比较。结果表明,SC的生长量达5.9倍,高于MC和NC的3.6和4.2倍。在初始接种量相近的情况下,MC的悬浮培养细胞在一个周期内紫杉醇产量可达9.5 mg/L,经过茉莉酸甲酯(M J)诱导后可达到41 mg/L。     

陕西汉中野生朱鹮雏鸟的生长发育特征

2014年和2015年监测朱鹮保护区野生朱鹮(Nipponia nippon)15只雏鸟的生长发育,拟合其生长发育数据的生长曲线和相对生长公式,与1989年研究结果进行对比。结果显示,野生朱鹮雏鸟的生长发育不受孵化顺序的影响,雄性体长的生长速度快于雌性。目前朱鹮雏鸟体重、体长、翅长、嘴峰长、跗跖长和中趾长的生长均符合逻辑斯谛生长曲线,雏鸟体重生长率在13日龄左右达到最大。与1989年的研究结果比较发现,目前野生朱鹮幼鸟总体生长发育有所放缓。这可能与近年野生朱鹮种群的增长和扩散导致的环境压力加大以及繁殖期食物资源不足有关。     

朱鹮的潜在繁殖地

利用ErdasImagine8.4和ArcViewGIS对陕西省部分地区的卫片进行解译,分析其中的栖息地组成。结果表明,现有野生朱种群的最大繁殖地—花园繁殖区的栖息地组成为:马尾松林占73.1%,次生疏林占16.9%,农田占4.8%,稀疏灌木林占4.2%,油松栎类混交林占1.0%。使用彩色数字环标记重见法研究了朱繁殖期活动区的栖息地组成。根据花园繁殖地和朱鹮活动区内的栖息地组成,给各种栖息地赋予相应的适宜度值,以此作为朱鹮潜在繁殖地的评估标准。对卫片区域的评估结果表明,研究区内潜在的很适宜的繁殖地主要分布于洋县华阳镇周边、洋县花园乡周围地区、酉水河两岸山区、南郑县东南部和汉中西北部等地区,占总面积的5.2%;适宜的繁殖地占19.4%,不适宜繁殖的区域占75.4%。根据这个评估结果,野生朱鹮1995-2004年的97个巢中(花园繁殖地以外),87个(占89.7%)落入适宜的繁殖地中;野外核查的结果也表明,该评估方法的准确度达到88.4%。评估的误差主要源于遥感很难准确判别林木的生长状况(树高、胸径、密度等)和混交林中某些适于营巢的树种的存在,并忽略了小面积地块。因此,适当的野外核查可以提高该评估方法的准确度     

朱鹮迁地保护研究

朱鹮（Nipponia mppon）是当今世界最濒危的鸟类之一,现存总数不过 40余只。由于生存环境的改变和种群生命力的脆弱,朱鹮数量急剧下降。中国自1986年开始人工繁殖朱鹮,1992年人工孵化并育活3只幼鸟,这是人工繁殖朱鹮成功的首次记录。这篇文章总结了朱鹮的野外生态,实验室研究及人工饲养、繁殖。     

笼养朱鹮繁殖期的鸣声特征

于2005年4月采集到笼养朱鹮(Nipponia nippon)4种行为状态的鸣叫声,对其繁殖期求偶鸣叫、交尾鸣叫、假交尾鸣叫和惊叫的鸣声特征进行了分析,探讨了不同行为状态的鸣声差异。求偶鸣叫振幅小,主要频率范围在156±13.2 Hz-1 568±120.3 Hz之间(n=30),声谱谐波丰富,频带呈直线型和拱型。交尾鸣叫由7-21声连续、急促的鸣叫和一声长鸣组成,声音宏亮有力,频域特征表现为主频呈现双峰,主要频率范围在346±16.7 Hz-2 396±138.2 Hz之间(n=14),音节高度重复并多有谐波。假交尾叫声有三种声模式,模式1包含2-10声短促鸣叫声和一声长鸣;模式2仅发出多声短促鸣叫声;模式3仅一声长鸣,频域特征在三种声模式下表现不同,模式1主频呈现双峰,模式2和模式3仅有一个峰值,主要频率范围在328±18.2 Hz-2 067±189.1 Hz之间(n=38),声谱多谐波,频带形状呈下滑状或直线状。遇险惊叫声信号振幅最大,每一声信号中包含多个脉冲组,惊叫声主频较高,主要频率范围在375-2 300 Hz之间,声谱多谐波,低频频带呈直线型,高频频带呈半拱形,声音末尾有明显的降调。     

一个人工朱曦种群的遗传多态性

利用蛋白质电泳、RAPD和微卫星DNA技术,对我国濒危保护动物———人工饲养朱进行遗传多态性研究。结果表明,40只朱在前白蛋白(Pre)、白蛋白(Alb)、后白蛋白(Pa)、运铁蛋白(Tf)共4个蛋白质位点上没有发现多态性。用40个随机引物对40只朱进行RAPD分析,其中35个引物扩增出完全相同的RAPD带纹,仅筛选到5个多态性引物,朱群体的平均带纹相似率为0.86。利用其它物种的22对微卫星引物对30只朱的DNA进行检测,共筛选出4对多态性微卫星引物,扩增出13个等位基因。朱在这4对微卫星位点的平均杂合度为0.3760,平均多态信息含量为0.3382,平均等位基因数为2.075。表明朱人工饲养群体的遗传多态性比发现之初得到了一定程度的恢复,但仍然比较贫乏     

年龄对朱鹮繁殖成功率的影响

本文对陕西洋县16年间朱鹮(Nipponia nippon)年龄对繁殖成功率的影响进行了研究,结果表明:朱鹮首次参加繁殖的平均年龄为3.89±1.40年(n=73),雌雄分别为3.64±1.36年(n=37)和4.17±1.47年(n=36),差异不显著。2-3龄亲鸟的平均窝卵数、孵化率和出飞率显著低于4-10龄的繁殖个体;>10龄繁殖个体的产卵数和卵的受精率显著降低,但雏鸟成活率较高。三个年龄组之间的繁殖成功率无显著差异(F2,146=1.963,P=0.144)。随着雌雄亲鸟年龄的增长,幼鸟的出飞率显著增加。刚参加繁殖的2龄雌性产卵数很低(2.63±0.74,n=8),至5龄时达到最大值(3.2±0.92,n=10)。6-9龄的雌雄个体产卵数量处于相对的低峰,10龄左右有所恢复(3.0±0,n=3),此后维持一个相对较低的水平(2.55±0.99,n=20)。刚参加繁殖的雄性朱鹮(2年)形成的配对卵的孵化率较低(0.67±0.29,n=3),但在第二年迅速达到高峰,而且能维持5-6年的时间。9-10年时略有下降(0.88±0.18,n=2;0.93±0.17,n=3),10年以后维持在一个较低的水平上(0.56±0.51,n=19)。     

Eight polymorphic microsatellite loci for the critically endangered crested ibis,Nipponia nippon (Ciconiiformes: Threskiornithidae)

The Chinese crested ibis, Nipponia nippon, is a critically endangered bird. The current population of this species has developed from four wild individuals rediscovered in 1981. Given its conservation status, there is considerable interest in assessing the genetic diversity and individual relatedness in this species. For this purpose, a set of eight polymorphic di‐ or trinucleotide microsatellite loci was developed for the crested ibis. The expected heterozygosity at these loci ranges from 0.01 to 0.50, with less than four alleles being observed at individual loci, a reflection of the serious population bottleneck experienced by this species.     

Major histocompatibility complex variation in the endangered crested ibis Nipponia nippon and implications for reintroduction

The major histocompatibility complex (MHC), with its extraordinary levels of genetic variation, is thought to be an essential aspect of the ability of an organism to recognize different parasites and pathogens. It has also been proposed to regulate reproductive processes in many aspects. Here we examine the genetic variation of the second exon of the MHC class II B genes of the crested ibis, an endangered species known to descend from just two breeding pairs rediscovered in 1981. Only five alleles are identified by single-strand conformation polymorphism (SSCP) analysis of 36 samples taken from both wild and captive populations, and a comparatively low level of divergence between MHC alleles is observed. We suggest that representative sampling of individuals with most of the different MHC allele genotypes to constitute a founder population, together with the monitoring of the pathogen status of candidate sites before release, is of great importance for raising the success rate of reintroduction for the crested ibis.     

Current state and the future of the crested ibis (Nipponia nippon): A case study by population viability analysis

There is only one population of the crested ibis (Nipponia nippon) surviving in the wild, which has not exceeded 40 individuals since it was rediscovered in 1981. We used detailed data of the annual population size and natality collected by the Crested Ibis Conservation and Observation Station (CICOS) over the past 17years. We were able to obtain a more accurate census of age structure and mortality as all of the fledglings have been identified by leg bands in 1987. Consequently, we analyzed the current state of the crested ibis and predicted its future using Population Viability Analysis (PVA) with the help of the computer simulation model VORTEX (Version 7.2). The results indicated that the extinction probability of the crested ibis was 19.7% in 100years. Sensitivity analysis showed that the extinction probabilities were sensitive to catastrophes and environmental variation, and the long-term existence of the population was dependent on the carrying capacity of the habitat and inbreeding depression. Conservation efforts are critical for the survival of the crested ibis. The prior conservation project involves controlling hunting and predation, as well as improving the life level of these few remnant creatures.     

Cloning of the gene for the thyrotropin beta subunit in the Japanese crested ibis,Nipponia nippon

We isolated a putative gene for the thyrotropin beta subunit (TSHbeta) from two types of genomic libraries of the Japanese crested ibis, Nipponia nippon. Exon-intron structure was deduced by comparing the determined sequence with those of TSH beta cDNA of other birds. The deduced amino acid sequence shows extensive similarities to those of the other birds, which assures our assumption that the acquired nucleotide sequence represents the TSHbeta gene. The assembled genomic fragment is 4192 bp in size and consists of 1937 bp of putative 5' flanking region followed by exon-intron structure with three exons and two introns, similar to those observed in rat, human and goldfish counterparts. Locations of introns are also similar to those in mammals and goldfish. Comparison of the 5' flanking region of the ibis TSHbeta gene with those of mammals reveals that several regulatory sequences, such as negative thyroid hormone responsive element (nTRE), Pit-1 responsive element, and AP-1 responsive element, which were characterized in mammalian TSHbeta genes, are also found in the promoter region. This is the first report on the exon-intron structure and 5' flanking region of the TSHbeta gene in an avian species.