拟南芥抗旱转录因子CBF4基因区域的核苷酸多样性及其分子进化分析

以生长于不同气候条件下的17个拟南芥核心生态型为材料,分析了它们的抗旱转录因子CBF4基因区域的序列多态性。结果表明：拟南芥CBF4基因区域具有高密度的单核苷酸多态性(SNP)和插入缺失(Indel),多态性频率为每35．8bp一个SNP,每143bp一个Indel,基因非编码区的多态性是编码区的4倍;在编码区,SNP的频率为每96．4bp一个SNP,其中发现25av、203av和244av 3个生态型CBF4基因区域1034位(以Gen—Bank登录号AB015478序列第19696位的核苷酸为1)碱基变化：G←→T,引起第205位氨基酸变化：gly←→val。核苷酸多样性统计分析显示,该基因内部大范围内存在连锁不平衡(linkage disequilibrium,LD),5’端非编码区有一个重组。与拟南芥等的研究结果类似,选择压力对不同的区域作用不同。3’端非编码区核苷酸多样性程度最高,是平衡性选择的结果,编码区核苷酸变化符合中性突变假说,而5’端非编码区是自然选择作用的靶位点。     

梯棱羊肚菌全基因组SNP/Indel分析及基于Indel标记的遗传连锁图谱构建

基于梯棱羊肚菌Morchella importuna两个子囊孢子培养物的全基因组测序数据,对全基因组范围内的变异位点进行分析。共鉴定到18 438个变异位点,平均每Mbp的变异位点数量为361个;变异位点以单核苷酸多态性SNP为主,共计17 104个,基因组中SNP的频率为335SNPs/Mbp;Indel多态性位点1 334个,以2-10bp的插入缺失为主;73.4% SNP/Indel位于基因间隔区域,外显子区域共检测到3 042个变异位点,占总数的16.50%;对基因功能产生确定影响的移码突变有1 088个,占5.90%,错义突变916个,占比4.97%;不同Scaffold上的SNP/Indel出现频率不同,SNP频率最大的为Scaffold80,平均每Mbp包含2 856个SNP,频率最低为Scaffold60和Scaffold75,分别为16SNPs/Mbp和30SNPs/Mbp;对≥11bp的Indel变异位点进行标记开发和多态性群体分析,成功开发出75对Indel标记。采用原生质体单细胞分离技术,获得了梯棱羊肚菌M04的两个可亲和的同核体菌株M04P01和M04P40,同时采用来自M04子囊果的58个单孢菌株作为作图群体,初步构建了包含75个Indel标记和1个交配型基因的梯棱羊肚菌遗传连锁图谱,共获得12个连锁群,连锁群总长度273.7cM。     

烟草航天诱变突变体变异检测及分析北大核心CSCD

为预测控制烟草重要性状的关键基因并研究航天诱变机理,对经航天诱变选育的烟草突变体材料NC89-M与野生型NC89进行全基因组重测序,测序深度30×,并对各种类型变异进行检测注释。NC89中检测到单核苷酸多态性位点(SNP,Single nucleotide polymorphism)1848013个,小片段插入缺失(Indel,insertion-delection)398922个,结构变异(SV,Structure variation)41969个;NC89-M中检测到SNP 1876219个,Indel 402011个,SV 42699个。NC89-M和NC89对比共得到271655个SNP,分布在8378个基因上,23450个Indel造成2156个基因突变。分析结果表明,NC89-M中SNP变异数目最多,其转换类型和颠换类型的比值为2.053,说明航天诱变对烟草基因组的变异以单碱基突变为主,突变类型以转换为主;在Indel中,插入突变数目明显多于缺失突变,证明航天诱变造成的Indel中以插入突变为主;在SV中,航天诱变主要造成了插入、缺失、倒位、染色体内部迁移和染色体间的迁移5种结构变异类型;变异基因KEGG注释表明,与代谢通路和次生代谢产物合成两方面基因突变数目最多;变异基因功能注释表明,突变体中调控开花时间的MADS-box基因,调控侧生器官发育与叶缘形状的KNOX1基因和萜类化合物合成相关基因等发生了变异。     

烟草航天诱变突变体变异检测及分析

为预测控制烟草重要性状的关键基因并研究航天诱变机理,对经航天诱变选育的烟草突变体材料NC89-M与野生型NC89进行全基因组重测序,测序深度30×,并对各种类型变异进行检测注释。NC89中检测到单核苷酸多态性位点(SNP,Single nucleotide polymorphism)1848013个,小片段插入缺失(Indel,insertion-delection)398922个,结构变异(SV,Structure variation)41969个;NC89-M中检测到SNP 1876219个,Indel 402011个,SV 42699个。NC89-M和NC89对比共得到271655个SNP,分布在8378个基因上,23450个Indel造成2156个基因突变。分析结果表明,NC89-M中SNP变异数目最多,其转换类型和颠换类型的比值为2.053,说明航天诱变对烟草基因组的变异以单碱基突变为主,突变类型以转换为主;在Indel中,插入突变数目明显多于缺失突变,证明航天诱变造成的Indel中以插入突变为主;在SV中,航天诱变主要造成了插入、缺失、倒位、染色体内部迁移和染色体间的迁移5种结构变异类型;变异基因KEGG注释表明,与代谢通路和次生代谢产物合成两方面基因突变数目最多;变异基因功能注释表明,突变体中调控开花时间的MADS-box基因,调控侧生器官发育与叶缘形状的KNOX1基因和萜类化合物合成相关基因等发生了变异。     

芸薹属作物EST-SNP的发掘与分析

芸薹属作物是十字花科中重要的蔬菜和油用作物,经济价值和食用价值比较高。本研究通过生物信息学手段,从3个芸薹属物种EST序列中获得了大量的SNP和Indel资源,对SNP与EST的数量及拼接结果之间的关系、SNP碱基置换的偏好性、Indel碱基数量与发生频率之间的关系进行了分析。分析结果对深入研究芸薹属基因组特点具有一定的参考价值,为进一步通过试验验证预测的SNP位点、开发该作物高通量SNP分子标记奠定了基础。     

香菇三个功能基因部分序列的单核苷酸多态性分析

以15个香菇栽培品种为材料,对尿嘧啶核苷酸-胞嘧啶核苷酸激酶基因(UMP-CMP kinase gene,uck1)、分裂原活化蛋白激酶基因(mitogen-activated protein kinase gene,mapk)和外切β-1,3葡聚糖酶基因(exo-β-1,3-glucanase-encoding gene,exg1)进行了部分序列的单核苷酸多态性(single nucleotide polymorphism,SNP)分析。结果表明,测序中出现的双峰,是菌丝双核体细胞中两细胞核之间的差异造成的。在采用uck1、mapk和exg1的3,126bp中,共发现48处多态性位点,发生频率为1/65bp,其中36个属于转换、12个为颠换。从群体发生频率上,38个属于超过10%的常见SNP,10个属于罕见SNP。不同基因的SNP发生频率不同,uck1、mapk和exg1的SNP发生频率分别为1.40%、0.82%和2.41%。外显子区SNP数量高于内含子,3个基因在外显子区域分布28个,内含子分布20个。外显子的28个SNP位点中,11个为错义突变,17个为同义突变。错义突变引起了编码氨基酸的改变。对SNP位点的聚类分析表明,15个栽培品种间存在的多态性位点在1–36之间,15个品种的SNP类型不同。uck1,mapk,exg1的SNP可用于香菇栽培品种的鉴别。     

中国汉族女性TLR7基因主要编码区的全长扩增及多态性分析

目的:扩增TLR7主要编码区外显子3(exon 3)的全长基因片段并进行基因多态性分析,筛选健康人群中TLR7基因的主要SNP位点.方法:采用酚-氯仿抽提方法从28例健康女性血样中提取基因组DNA,采用长片段扩增方法分别扩增TLR7 exon 3区的3个片段,测序、拼接后进行多态性分析.结果:采用LA-Taq酶体系成功扩增TLR7 exon 3区基因片段;经与Genbank数据库中TLR7参考序列比较,我国女性TLR7基因序列高度保守,仅出现了4个点突变,并发现1个SNP位点RS3853839,表现为GG、CG和CC三种基因型.结论:建立了TLR7编码区基因扩增方法,筛选到1个TLR7SNP位点RS3853839,可为分析TLR7多态性与多种病毒感染性疾病的关系提供参考.     

小麦RING型E3泛素连接酶基因TaSDIR1-D克隆与功能分析

具有RING结构域的E3泛素连接酶SDIR1(Salt-and drought induced really interesting new gene finger1)在植物信号调节通路中发挥着重要的作用。本研究克隆得到小麦TaSDIR1-D基因,基因组序列全长4070 bp,其中编码区上游为1443 bp,编码区为2352 bp,3'端非编码区(UTR)为275 bp;该基因编码区cDNA序列长度为849 bp,预测其编码282个氨基酸,包括2个跨膜结构域和1个保守的RING型finger结构域。以野生近缘种的二倍体、四倍体和六倍体普通小麦及一套由中国春为背景的缺-四体为材料,将基因定位在染色体4D上;小麦抽穗期TaSDIR1-D在旗叶中的表达量最高;在NaCl、ABA、PEG及4℃非生物胁迫诱导下,小麦TaSDIR1-D均上调表达,可能参与植物抗逆调节通路;通过检测32份多态性较高的小麦材料TaSDIR1-D基因序列多态性,共检测到2个SNP,基因的编码区和上游序列中各存在1个核苷酸变异,其中编码区的核苷酸变异位点(G/A)位于第4外显子上,为非同义突变,使精氨酸(Agr)变为组氨酸(His)。2个SNP组成两种单倍型,根据-583 bp处的SNP(T/C)设计分子标记SNP-583,扫描自然群体262份材料的基因型,将其分为2种单倍型,分别与千粒重、倒二节长和穗长显著相关或极显著相关,单倍型Hap-4D-2为提高千粒重的优异单倍型。研究结果为小麦分子育种提供了理论依据和基因资源。     

西藏绒山羊KA P9.2基因CDS克隆、外显子区多态性及表达分析

KAP9.2基因是角蛋白关联蛋白(keratin associated protein, KAP)中的一员,在毛发的形成过程中有重要的调控作用。本研究对西藏绒山KAP9.2基因CDS进行了克隆;采用直接测序法对绒山羊200个个体KAP9.2基因外显子区的遗传变异情况进行分析;并利用Real-time PCR分析了KAP9.2基因在不同海拔山羊中的表达。结果显示,西藏绒山羊KAP9.2基因CDS序列为576 bp,编码191个氨基酸;KAP9.2基因外显子存在25处SNP位点及一处30 bp的缺失突变,其中12处SNP为错义突变,其他13处为同义突变。遗传多态性分析表明KAP9.2基因多态性丰富,遗传变异大;连锁分析发现12与388位点、54与93位点、153与159位点、273与279位点完全连锁,H1为优势单倍型;Real-time PCR显示西藏绒山羊KAP9.2基因在高海拔地区m RNA表达水平显著高于特高海拔地区,推测该基因可能促进绒毛的生长。本研究结果揭示了西藏绒山羊KAP9.2基因的遗传多态性及其在不同海拔的表达,为进一步研究KAP9.2基因潜在的功能位点提供一定的理论依据。     

新生儿无乳链球菌分离株全基因组重测序分析

无乳链球菌是新生儿细菌感染的主要病原菌之一,严重危害新生儿的健康。目前,国内鲜有从基因组的角度对无乳链球菌进行深入研究。本研究对一株新生儿无乳链球菌分离株SA1507进行了全基因组重测序和生物信息学分析。与参考基因组进行比对,SA1507存在大量的变异位点,比对发现SNP 8 850个,大多数位于外显子区域;Indel 253个,大多数位于基因上游。对SNP和Indel进行COG聚类分析和KEGG通路分析,分别得到2 272个COG功能条目和1 363个KEGG通路条目。测序与分析将为进一步研究无乳链球菌的变异,感染和耐药性等奠定基础。     

The genomic landscape of short insertion and deletion polymorphisms in the chicken (Gallus gallus) Genome: a high frequency of deletions in tandem duplicates

It is increasingly recognized that insertions and deletions (indels) are an important source of genetic as well as phenotypic divergence and diversity. We analyzed length polymorphisms identified through partial (0.25x) shotgun sequencing of three breeds of domestic chicken made by the International Chicken Polymorphism Map Consortium. A data set of 140,484 short indel polymorphisms in unique DNA was identified after filtering for microsatellite structures. There was a significant excess of tandem duplicates at indel sites, with deletions of a duplicate motif outnumbering the generation of duplicates through insertion. Indel density was lower in microchromosomes than in macrochromosomes, in the Z chromosome than in autosomes, and in 100 bp of upstream sequence, 5'-UTR, and first introns than in intergenic DNA and in other introns. Indel density was highly correlated with single nucleotide polymorphism (SNP) density. The mean density of indels in pairwise sequence comparisons was 1.9 x 10(-4) indel events/bp, approximately 5% the density of SNPs segregating in the chicken genome. The great majority of indels involved a limited number of nucleotides (median 1 bp), with A-rich motifs being overrepresented at indel sites. The overrepresentation of deletions at tandem duplicates indicates that replication slippage in duplicate sequences is a common mechanism behind indel mutation. The correlation between indel and SNP density indicates common effects of mutation and/or selection on the occurrence of indels and point mutations.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

新疆东部天山蝶类多样性及其垂直分布

2006-2008年研究了新疆东部天山蝶类多样性和垂直分布.结果表明:研究区域内共记录蝴蝶7科43属63种,占新疆已记录蝶类种数的24.80％,区系组成主要是古北种,占73％;其次是广布种,占27％,没有发现东洋种.其中蛱蝶科的物种数最多,为11属19种,蚬蝶科的物种数最少,只有1属1种.按海拔将生境分为5个垂直自然带,包括低山灌木草原带、山地森林草原带、亚高山草甸带、高山草甸带、垫状植被带.蝶类物种数和个体数排序为亚高山草甸带＞山地森林草原带＞低山灌木草原带＞高山草甸带＞垫状植被带.采用Shannon-Wiener指数和G-F指数对蝶类物种和科、属的多样性进行了分析评价,结果显示亚高山草甸带的蝶类多样性最为丰富,其次是山地森林草原带和低山灌木草原带,而高山草甸带和垫状植被带的蝶类多样性相对较低,物种和科、属多样性分析结果均一致.蝶类垂直分布明显,物种数和个体数随海拔变化的趋势类似,均为先增加后下降.蝶类区系成分随着海拔升高发生改变,广布种的比例逐渐降低,高山草甸带和垫状植被带只有古北种分布.研究结果显示,生境改变对蝴蝶群落影响明显,保护生境是保护蝴蝶生存的最主要措施.     

A double-screening method to identify reliable candidate non-synonymous SNPs from chicken EST data

Discovery of non-synonymous single nucleotide polymorphisms (nsSNP), which cause amino acid substitutions, is important because they are more likely to alter protein function than synonymous SNPs (sSNP) or those SNPs that do not result in amino acid changes. By changing the coding sequences, nsSNP may play a role in heritable differences between individual organisms. In the chicken and many other vertebrates, the main obstacle for identifying nsSNP is that there is insufficient protein and mRNA sequence information for self-species referencing and thus, determination of the correct reading frame for expressed sequence tags (ESTs) is difficult. Therefore, in order to estimate the correct reading frame at nsSNP in chicken ESTs, a double-screening approach was designed using self- or cross-species protein referencing, in addition to the ESTScan coding region estimation programme. Starting with 23 427 chicken ESTs, 1210 potential SNPs were discovered using a phred/phrap/polyphred/consed pipeline process and among these, 108 candidate nsSNP were identified with the double screening method. A searchable SNP database (chicksnps) for the candidate chicken SNPs, including both nsSNPs and sSNPs is available at http://chicksnps.afs.udel.edu. The chicken SNP data described in this paper have been submitted to the data base SNP under National Center for Biotechnology Information assay ID ss4387050-ss4388259.     

PEG模拟干旱胁迫下马铃薯茎段转录组分析

该研究以‘青薯9号’马铃薯无菌苗为材料,采用转录组测序技术分析模拟干旱胁迫下马铃薯茎段的差异表达,探究茎段在干旱胁迫下的分子机制。结果表明:(1)不同程度干旱胁迫下,马铃薯叶片脯氨酸、可溶性糖以及可溶性蛋白含量明显增加;马铃薯茎段差异表达基因下调的数量均多于上调,其中3种处理条件下共有的差异表达基因有657个。(2)GO富集分析表明,马铃薯茎段差异表达基因主要集中在氧化还原过程、激素响应、氧化还原酶活性以及糖基水解酶活性;Pathway富集分析表明,马铃薯茎段差异表达基因主要集中在植物激素信号转导、苯丙酸生物合成、玉米素生物合成、苯丙氨酸代谢、淀粉和蔗糖代谢以及次生代谢产物的生物合成。(3)实时荧光定量PCR验证结果表明,6个差异表达基因在不同程度干旱胁迫中的差异表达与转录组分析的结果基本一致,证明转录组数据的可靠性。该结果对进一步研究马铃薯干旱胁迫响应机制有一定参考价值,也丰富了马铃薯抗旱育种的基因资源。     

Drd2 expression in the high alcohol-preferring and low alcohol-preferring mice

The high alcohol-preferring (HAP) and low alcohol-preferring (LAP) mice were selectively bred for differences in alcohol preference and consumption. Recently, a large-effect QTL was identified on chromosome 9. The peak for this QTL is near the Drd2 (dopamine receptor 2) locus. The present study examined Drd2 mRNA expression differences between the HAP1 and LAP1 mice in brain regions important in the dopaminergic-reward pathway, including the nucleus accumbens, hippocampus, amygdala, and septum. Results show that alcohol-naïve HAP1 mice exhibited lower levels of Drd2 mRNA expression in the nucleus accumbens and the hippocampus compared to LAP1 mice. No differences were found in the amygdala or septum. To determine if a sequence difference might underlie the expression difference, the Drd2 cDNA was sequenced in each line and one single nucleotide polymorphism (SNP) was identified in the 3′ UTR. Both HAP and LAP 3′ UTR were cloned in the luc-pGL3-promoter-luc vector. The polymorphism in the Drd2 3′ UTR was assessed to determine its functional significance in modulating expression. In vitro expression analysis using neuroblastoma SK-N-SH cells resulted in a significant decrease in expression of the HAP 3′ UTR luc construct compared with the LAP 3′ UTR construct. This decreased expression is consistent with lower levels of Drd2 expression in the nucleus accumbens and the hippocampus as evidenced by qRT-PCR. These results suggest that the SNP may play a role in the differential expression of Drd2 between the HAP and LAP mice and that the polymorphism in Drd2 may contribute to alcohol preference.     

Probability of Globodera rostochiensis Spread on Equipment and Potato Tubers

The probability of spreading cysts of Globodera rostochiensis on farming equipment and potato tubers was investigated in naturally infested field plots. The number of cysts recovered from soil that adhered to equipment differed significantly between different pieces of equipment. These differences were related to initial nematode density and, in most cases, to the volume of soil that adhered to the equipment. At an initial density of 0.04 egg/cm³ of soil, significantly more cysts were recovered from a potato digger than from a potato hiller, cultivator, or plow. At an initial density of 0.90 egg/cm³ of soil, significantly more cysts were recovered from the plow than from the other equipment. Although the population density was 22 times greater, only 10 times more cysts adhered 3 to equipment used in soil with a density of 0.90 egg/cm³ of soil than when used in soil infested at 0.04 egg/cm³. The number of potato tuber samples (4.5 kg) that contained cysts with viable eggs was positively correlated with the initial densities of G. rostochiensis in soil in which they were produced. The percentage of tuber samples with cysts containing viable eggs was 10-12% for tubers harvested from soil with densities less than 1 egg/cm³ and 30-76% for tubers harvested from soil with densities greater than 4 eggs/cm³ of soil.     

Predation on Colorado Potato Beetle Eggs by Generalist Predators in Research and Commercial Potato Plantings

Field studies quantified predation on Colorado potato beetle [Leptinotarsa decemlineata(Say)] eggs and determined the relationship between predation and egg mass abundance in research and commercial potato plantings in eastern North Carolina. Predator exclusion experiments were conducted weekly in research plantings. In addition, egg mass density and predation on egg masses were monitored throughout the season in research plots and commercial potato fields. Predation was an important source of mortality for Colorado potato beetle eggs. Survivorship of eggs exposed to predators was consistently, significantly lower than survivorship of eggs protected from predation. Averaged over 2 years, the mean survivorship of eggs protected from predation was 69%, compared with 26% survivorship of eggs exposed to predation. Regression analysis failed to detect any relationship between egg mortality due to predation and egg abundance. These results imply that efforts to reduce Colorado potato beetle populations selectively will not be offset by an according decline in abundance of natural enemies and therefore should be fully compatible with naturally occurring biological control.