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Molecular characterization and phylogenetic analysis of three odorant binding protein gene transcripts in Dendrolimus species （Lepidoptera： Lasiocampidae）

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Insect Science 2014年第5期21卷 597-608页

作者： Su-Fang Zhang Zhen Zhang Xiang-Bo Kong Hong-Bin Wang Key Laboratory of Forest Protection Research Institute of Forest Ecology Environment and Protection Chinese Academy of ForestryState Forestry Administration Beijing China

Pine caterpillar moths, Dendrolimus spp. （Lepidoptera： Lasiocampidae）, are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant-binding proteins （OBPs） participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs - one pheromone-binding protein and two general odorant-binding proteins - from male antennae of four Dendrolimus species, D. superans （Butler）, D. punctatus （Walker）, D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. ka/ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. la＇kuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure.

关键词： Dendrolimus general odorant-binding protein ka/ks pheromone-binding proteins phylogenetic sex pheromone

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Genome-wide identification, characterization and evolution of cuticular protein genes in the malaria vector Anopheles sinensis （Diptera： Culicidae）

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Insect Science 2018年第5期25卷 739-750页

作者： Bai-Qi Liu Liang Qiao Qi-Yi He Yong Zhou Shuang Ren Bin Chen Chongqing Key Laboratory of Vector Insects Institute of Entomology and Molecular Biology Chongqing Normal University Chongqing China

Thirteen cuticular protein （CP） families have been recognized in arthropods. In this study, 250 Anopheles sinensis CP genes were identified and named based on genome and transcriptome sequences. They were classified into 10 families based on mo- tifs and phylogenetic analyses. In 11 other insect species, nine had CP numbers 〉 150 while Apis mellifera and Tribolium castaneum had CP numbers less than 52. The CPs of eight species occupied 〉 1.4% of the total genomic gene number, whereas in three species the CPs occupied 〈 1%. The phylogenies for each CP family in An. sinensis were constructed and discussed. The 250 CPs each had 1-8 exons with 144 CPs （57.6%） having two exons. The intron length ranged from 66--3888 bp with 174 introns （54.0%） being 66--100 bp long. Except for two CPs on two contigs, 248 CPs were mapped onto 28 scaffolds with 136 genes （54.4%） restricted to five scaffolds. A total of 107 CPs were clustered and located at 27 loci. The CPR family had the conserved motif GSYS- LVEPDGTVRTV. The RR- 1 subfamily had an additional 21 amino acid （aa） motifs with the YVADENGF sequence that is common in insects. The RR-2 subfamily had an additional 50 aa motifs with two additional regions RDGDWKG and G-x（3）-VV. A comparison with 115 orthologous counterparts of An. gambiae CPs suggested purifying selection for all of these genes. This study provides basic information useful for further studies on biological functions of An. sinensis CPs as well as for comparative genomics of insect CPs.

关键词： Anopheles sinensis characterization cuticular protein genes genome-wideidentification phylogenetics ka/ks

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Characterization and comparison of chloroplast genomes from two sympatric Hippophae species(Elaeagnaceae)

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Journal of Forestry Research 2021年第1期32卷 307-318页

作者： Luoyun Wang Jing Wang Caiyun He Jianguo Zhang Yanfei Zeng State Key Laboratory of Tree Genetics and Breeding and Key Laboratory of Tree Breeding and Cultivation of the State Forestry and Grassland Administration Research Institute of ForestryChinese Academy of ForestryBeijingPeople’s Republic of China Collaborative Innovation Center of Sustainable Forestry in Southern China Nanjing Forestry UniversityNanjingPeople’s Republic of China

The genus Hippophae includes deciduous shrubs or small trees,which provide many ecological,economic,and social benefi ts.We assembled and annotated the chloroplast genomes of sympatric Hippophae gyantsensis(Rousi)Lian and Hippophae rhamnoides Linn subsp.yunnanensis Rousi and comparatively analyzed their sequences.The fulllength chloroplast genomes of H.gyantsensis and H.rhamnoides subsp.yunnanensis were 155,260 and 156,415 bp,respectively;both featured a quadripartite structure with two copies of a large inverted repeat(IR)separated by small(SSC)and large(LSC)single-copy regions.Each Hippophae chloroplast genome contained 131 genes,comprising 85 protein-coding,8 ribosomal RNA,and 38 transfer RNA genes.Of 1302 nucleotide substitutions found between these twogenomes,824(63.29%)occurred in the intergenic region or intron sequences,and 478(36.71%)were located in the coding sequences.The SSC region had the highest mutation rate,followed by the LSC region and IR regions.Among the protein-coding genes,three had a ratio of nonsynonymous to synonymous substitutions(ka/ks)>1 yet none were signifi-cant,and 66 had ka/ks<1,of which 46 were signifi cant.We found 20 and 16 optimal codons,most of which ended with A or U,for chloroplast protein-coding genes of H.gyantsensis and H.rhamnoides subsp.yunnanensis,respectively.Phylogenetic analysis of fi ve available whole chloroplast genome sequences in the family Elaeagnaceae—using one Ziziphus jujube sequence as the outgroup—revealed that all fi ve plant species formed a monophyletic clade with two subclades:one subclade consisted of three Hippophae species,while the other was formed by two Elaeagnus species,supported by 100%bootstrap values.Together,these results suggest the chloroplast genomes among Hippophae species are conserved,both in structure and gene composition,due to general purifying selection;like many other plants,a signifi cant AT preference was discerned for most proteincoding genes in the Hippophae chloroplast genome.This study provides a valuable reference tool for

关键词： Chloroplast genome Hippophae gyantsensis(Rousi)Lian Hippophae rhamnoides Linn subsp.yunnanensis ka/ks Optimal codon

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不同海拔喜马拉雅旱獭线粒体基因组比较分析

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野生动物学报 2022年第2期43卷 345-353页

作者：南新营李优李耀东青海大学西宁810016

以分布在不同海拔(2279、3273、4452 m)的喜马拉雅旱獭(Marmota himalayana)为供试材料,通过二代测序技术对线粒体基因组序列进行比较分析,挖掘喜马拉雅旱獭低氧适应相关基因。结果表明:不同海拔喜马拉雅旱獭线粒体序列长度之间并无差异... 详细信息

以分布在不同海拔(2279、3273、4452 m)的喜马拉雅旱獭(Marmota himalayana)为供试材料,通过二代测序技术对线粒体基因组序列进行比较分析,挖掘喜马拉雅旱獭低氧适应相关基因。结果表明:不同海拔喜马拉雅旱獭线粒体序列长度之间并无差异,但蛋白质编码基因突变位点个数随海拔增加而增多,ND5基因的突变位点个数和非同义突变位点个数都为最多,其ka/ks>1,表明ND5基因存在正选择效应,可作为适应高海拔低氧环境的候选基因。与玉树(海拔4452 m)相比,乐都(海拔2279 m)和黄南(海拔3273 m)的喜马拉雅旱獭亲缘关系相对较近。喜马拉雅旱獭13个蛋白质编码基因的核苷酸序列共编码氨基酸3812个,去掉起始密码子和终止密码子后共编码氨基酸3802个。在20种氨基酸中,Leu的使用率最高,达16.18%,Gys的使用率最低,仅有0.71%。RSCU值>1的密码子有32种,RSCU值<1的密码子有32种,其中AGG的RSCU为0。

关键词：喜马拉雅旱獭线粒体基因组 ND5 ka/ks

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kaks_Calculator 2.0:A Toolkit Incorporating Gamma-Series Methods and Sliding Window Strategies

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Genomics, Proteomics & Bioinformatics 2010年第1期8卷 77-80页

作者： Dapeng Wang Yubin Zhang Zhang Zhang Jiang Zhu Jun Yu AS Key Laboratory of Genome Sciences and Information Beijing Institute of Genomics Chinese Academy of Sciences Beijing 100029 China Institute of Computing Technology Chinese Academy of Sciences Beijing 100190 China Graduate University of Chinese Academy of Sciences Beijing 100049 China Plant Stress Genomics Research Center Division of Chemical and Life Sciences & Engineering King Abdullah University of Science and Technology Thuwa123955-6900 Saudi Arabia.

We present an integrated stand-alone software package named kaks_Calculator 2.0 as an updated version. It incorporates 17 methods for the calculation of nonsynonymous and synonymous substitution rates; among them, we added our modified versions of several widely used methods as the gamma series including y-NG, y-LWL, ）,-MLWL, y-LPB, y-MLPB, y-YN and y-MYN, which have been demonstrated to perform better under certain conditions than their original forms and are not implemented in the previous version. The package is readily used for the identification of positively selected sites based on a sliding window across the sequences of interests in 5＇ to 3＇ direction of protein-coding sequences, and have improved the overall performance on sequence analysis for evolution studies. A toolbox, including C＋＋ and Java source code and executable files on both Windows and Linux platforms together with a user instruction, is downloadable from the website for academic purpose at https：//***/projects/kakscalculator2/.

关键词： ka/ks gamma-series methods sliding window positively selected sites

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Evolutionary characteristics of exons expressed at different abundance levels in mammals

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Chinese Science Bulletin 2009年第19期54卷 3546-3554页

作者： YU HaiJing FANG Lin LI Xin ZHANG XiaoYan YE Jia LI Jie HU WeiHong ZHENG BingRong XIAO ChunJie Key Laboratory of Bioresources Conservation and Utilization & Human Genetics Center of Yunnan University Kunming 650091 China Beijing Genomics Institute at Shenzhen Shenzhen 518083 China CAS-Max Planck Junior Research Group and State Key Laboratory of Genetic Resources and Evolution Kunming Institute of Zoology Chinese Academy of Sciences Kunming 650223 China

With comparative genomics approaches, we evaluated the evolutionary characteristics of conservation of exons which are expressed abundantly, moderately or lowly in mammals. Using non-coding regions and pseudogenes as controls, sequence identity, phastCons and ka/ks analyses were carried out and our results showed that as the exons of high abundance are highly conserved, the minor and low exons also showed conservative characteristics in evolution. Our findings suggested that the exons with less abundance which constitute a large proportion of distinct species in transcriptome of organisms are under functional constraint and might play certain roles in enriching biological complexity in the evolution of organisms.

关键词：哺乳动物进化特征 ka/ks

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不同海拔喜马拉雅旱獭线粒体基因组比较分析

不同海拔喜马拉雅旱獭线粒体基因组比较分析

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作者：南新营青海大学

学位级别：硕士

目的通过二代测序技术对不同海拔喜马拉雅旱獭线粒体基因组序列进行比较分析,挖掘喜马拉雅旱獭低氧适应相关基因。方法在青海省海东市乐都区引胜乡(海拔2279 m,3例)、黄南州同仁县瓜什则乡(海拔3273 m,3例)、玉树州曲麻莱县麻多乡(海拔4... 详细信息

目的通过二代测序技术对不同海拔喜马拉雅旱獭线粒体基因组序列进行比较分析,挖掘喜马拉雅旱獭低氧适应相关基因。方法在青海省海东市乐都区引胜乡(海拔2279 m,3例)、黄南州同仁县瓜什则乡(海拔3273 m,3例)、玉树州曲麻莱县麻多乡(海拔4452 m,2例)3个地区采集喜马拉雅旱獭肝脏组织共8例,于液氮中保存并带回,储存于-80℃冰箱。用痕量样品基因组DNA提取试剂盒提取喜马拉雅旱獭基因组DNA。采用Illumina Hiseq X测序技术平台对8例样品进行paired-end双末端测序,然后使用fast QC软件(http://www.***.babraham.***.uk/projects/fastqc/)观测测序质量,并使用NGSQC软件对测序数据进行质控。测序由百迈客生物科技有限公司完成。对质控过的clean data进行初步拼接,使用的软件为SPAdes,经过同源比对和蛋白序列比对后,利用PRICE(Paired-Read Iterative Contig Extension)进行迭代延伸、gap补洞及重拼接,直至获得完整的线粒体基因组序列。获得完整的线粒体基因组序列以后,使用MEGAX软件分析不同海拔喜马拉雅旱獭线粒体基因组大小和碱基组成。采用mafft软件进行序列比对,用MEGAX软件基于组装好的线粒体基因组采用最大似然法(maximum likelihood,ML)构建系统发育树。用SPASS 19.0软件对三个海拔喜马拉雅旱獭线粒体基因组中13个蛋白编码基因的突变位点个数进行统计学分析,统计学方法采用单因素方差分析。使用MEGAX软件做基于密码子的比对分析,使用kaks＿calculator2.0软件(https://***/projects/kakscalculator2/)计算ka ks,计算方法选择γ-YN。密码子偏好性分析使用codon W软件。基因共线性分析采用Mauve v2.3.1软件。结果1、不同海拔喜马拉雅旱獭线粒体序列长度均为16 442 bp,A、T、G、C平均含量分别为32.1%、31.4%、12.8%、23.7%。每个样品由37个片段拼接而成,最大片段为1827 bp,最小片段为59 bp,GC含量为36.5%。2、基于线粒体基因组组装好的序列,以温哥华旱獭(序列号:MK859897.1)为外群,用MEGAX软件采用最大似然法构建系统发育树,从系统树可看出,黄南的2、3号聚为一支,再与乐都的3号聚为一支,乐都的1、2号聚为一支后又同黄南1号聚为一支,最后与玉树的1、2号聚为一支。3、不同海拔喜马拉雅旱獭线粒体基因组中13个蛋白编码基因共有65个突变位点,包括同义突变位点41个,如YS-1、YS-2的COX1基因第1380位G→A,COX2基因第648位T→A,ND4L基因的第513位T→C,ATP6基因第324位A→G,ATP8基因第6位C→T;非同义突变位点24个,如HN-2、HN-3的ND4基因第335位C→T,导致ND4氨基酸序列的第112位A→V(丙氨酸→缬氨酸),ND5基因第22位T→C,导致ND5氨基酸序列的第8位S→P(丝氨酸→脯氨酸)。其中COX3基因最为保守,没有发生变异,而ND5基因有18个突变位点,比例最高(27.7%)。且玉树的突变位点个数显著高于乐都和黄南(Pka/ks>1。4、喜马拉雅旱獭13个蛋白质编码基因的核苷酸序列共编码氨基酸3812个,去掉起始密码子和终止密码子后共编码氨基酸3802个,在20种氨基酸中,Leu的使用率最高,达16.18%,Cys的使用率最低,仅有0.71%。5、不同海拔喜马拉雅旱獭在整个线粒体基因组范围内没有发生基因片段的丢失、复制、重排等大规模的变化。结论1、不同海拔喜马拉雅旱獭线粒体序列长度之间并无差异,其核苷酸组成也无差异,有明显的AT偏倚,8个样品之间没有差异。2、跟玉树相比,乐都和黄南的喜马拉雅旱獭亲缘关系相对较近。3、蛋白质编码基因突变位点个数随海拔高度增加而增多,ND5基因存在正选择效应,可作为适应高海拔低氧环境的候选基因。4、喜马拉雅旱獭中RSCU>1的密码子有32种,RSCU<1的密码子有32种,其中AGG的RSCU为0,即在编码过程中没有使用该密码子。5、不同海拔喜马拉雅旱獭在整个线粒体基因组范围内均具有很好的共线性。

关键词：喜马拉雅旱獭线粒体基因组 ND5 ka/ks RSCU

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拟石莲属植物对低温胁迫的生理响应及转录组分析

拟石莲属植物对低温胁迫的生理响应及转录组分析

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作者：韦朝妹贵州大学

学位级别：硕士

拟石莲属是一类常用于园林绿化和室内装饰的多肉植物,具有种类繁多、形态奇特、适应性强和观赏周期长等优点,其还在制药或食品行业具有可持续开发的潜在用途,从而得以火热发展。拟石莲属植物主要分布在美洲的墨西哥,引种我国后多为设施... 详细信息

拟石莲属是一类常用于园林绿化和室内装饰的多肉植物,具有种类繁多、形态奇特、适应性强和观赏周期长等优点,其还在制药或食品行业具有可持续开发的潜在用途,从而得以火热发展。拟石莲属植物主要分布在美洲的墨西哥,引种我国后多为设施栽培,也有露地栽培,如遇冬季持续的低温,会导致植株生长不良,甚至某些种类不能越冬,生产效益低下,推广应用受限。迄今为止,未见关于该属植物抗寒机制的系统报道。鉴于拟石莲属植物在我国园林绿化应用的需要,亟待对其主栽品种的耐寒性进行科学评价。本文通过观测六种拟石莲属植物叶片表型和生理指标在低温胁迫下的动态变化,结合相关性、PCA和OPLS-DA分析,系统评价它们的耐寒性,筛选耐寒和冷敏感物种。并结合转录组测序技术,比较低温胁迫下耐寒和冷敏感物种的抗性差异,挖掘与其抗性有关的重要功能蛋白,初步揭示其适应低温的分子机制,为多肉植物耐寒种质选择及推广应用、抗寒新品种的培育奠定理论基础。主要研究结果如下:1.低温胁迫下,不同耐寒物种的表型变化和生理特性存在显著性差异。低温胁迫使拟石莲属植物叶片的Chl含量降低,光合作用下降,并通过积累SP来降低渗透势,POD为六种拟石莲属植物应答低温胁迫的主要防御酶。GA能增强植株细胞膜的稳定性,提高叶片的保水能力。各生理指标间均具有一定的相关性,通过主成分分析将12个单项指标转换为4个相互独立的综合指标,代表了全部数据88.01%的信息量。六种拟石莲属植物抗寒性强弱排序为:杜里万莲>大和锦>吉娃莲>玉蝶>花月夜>大瑞蝶,其中杜里万莲可作为抗寒种源的优选材料;OPLS-DA分析显示Chl、SS含量和CAT、POD活性的VIP值均大于1,可作为区分冷敏感物种大瑞蝶和耐寒物种杜里万莲的生理物质特性。2.结合转录组测序技术,对拟石莲属植物与低温响应的差异基因进行分析。研究表明大瑞蝶在低温胁迫3d、6d和9d与对照相比分别有9566、8285和8055个DEGs上调,6855、8000和7894个DEGs下调,共同表达的DEGs有6453个。大瑞蝶在低温胁迫后DEGs数量呈逐渐下降趋势,在9d达到最低值;而杜里万莲在低温胁迫3d、6d和9d与对照相比分别有8590、8585和9463个DEGs上调,5883、6604和6370个DEGs下调,共同表达的DEGs有6413个,杜里万莲在低温胁迫后DEG数量逐渐升高,当9d时达最高峰。说明冷敏感物种大瑞蝶为尽快适应低温环境,其响应低温相关基因表达的时间比耐寒物种杜里万莲要早。3.基于转录组测序数据、ABM和WGCNA分析将筛选出的耐寒物种杜里万莲和冷敏感物种大瑞蝶转录组的差异蛋白编码基因进行深入分析,挖掘拟石莲属中耐低温候选基因。结果表明低温胁迫通过相关代谢物调整,激活泛素-蛋白酶体系统降解受损或折叠错误的蛋白质,减少氧化应激损伤和提高不饱和脂肪酸以及胆固醇含量来提高拟石莲属植物的耐寒性。而受损蛋白降解和离子平衡维持等相关基因的适应性进化可能是拟石莲属植物耐寒性较强的原因。

关键词：拟石莲属低温胁迫耐寒评价转录组测序 WGCNA ka/ks

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Transcriptomic comparison to identify rapidly evolving genes in Braya humilis

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Research in Cold and Arid Regions 2018年第5期10卷 428-435页

作者： YuMing Wei XiaoFei Ma PengShan Zhao Animal Husbandry Pasture and Green Agriculture Institute of Gansu Academy of Agricultural Sciences LanzhouGansu 730070China Key Laboratory of Stress Physiology and Ecology in Cold and Arid Regions Northwest Institute of Eco-Environment and ResourcesChinese Academy of SciencesLanzhouGansu 730000China

The Brassicaceae species Braya humilis shows broad adaptation to different climatic zones and latitudes. However, the molecular adaptation mechanism of B. humilis is poorly understood. In China, B. humilis is mainly distributed on the QinghaiTibetan Plateau(QTP) and in the adjacent arid region. Previous transcriptome analysis of B. humilis has revealed that 39 salt and osmotic stress response genes are subjected to purifying selection during its speciation. To further explore the adaptation mechanism of B. humilis to an arid environment, OrthoMCL program was employed in this study and 6,268 pairs of orthologous gene pairs with high confidence were obtained between B. humilis and Arabidopsis thaliana. A comparative evolutionary analysis based on nonsynonymous to synonymous substitution ratio(ka/ks) was then conducted. There were 64 pairs exhibiting a ka/ks ratio more than 0.5 and among which, three instrumental candidate genes, T2_20487,T2_22576, and T2_13757, were identified with strong selection signatures(ka/ks >1). The corresponding A. thaliana orthologs are double-stranded RNA-binding domain protein, MADS-box family protein, and NADH-dehydrogenase subunit6, which is encoded by mitochondria genome. This report not only demonstrates the adaptation contribution of fast evolving nuclear genes, but also highlights the potential adaptive value of mitochondria gene to the speciation and adaptation of B. humilis toward the extreme environment in an arid region.

关键词： Braya humilis positive selection ka/ks mitochondria gene variation NADH-dehydrogenase subunit 6

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基于线粒体基因组的东方鲀属系统发育学及群体遗传学

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水产学报 2020年第11期44卷 1792-1803页

作者：周志雄刘波宫杰白玉麟阳俊逸徐鹏厦门大学海洋与地球学院福建省海洋生物遗传育种重点实验室福建省水产研究所

为了探究东方鲀属内系统发育关系,本研究首次基于群体尺度对东方鲀属物种进行系统发育分析。利用线粒体基因组,在包含10个种的124尾东方鲀中,挖掘了1 488个单核苷酸多态性(SNP)位点和4个插入缺失(INDEL),使用这些遗传变异位点进行了系... 详细信息

为了探究东方鲀属内系统发育关系,本研究首次基于群体尺度对东方鲀属物种进行系统发育分析。利用线粒体基因组,在包含10个种的124尾东方鲀中,挖掘了1 488个单核苷酸多态性(SNP)位点和4个插入缺失(INDEL),使用这些遗传变异位点进行了系统发育及群体遗传学分析。发现网纹东方鲀与铅点东方鲀可能是同种异名,且二者形成的姊妹群位于东方鲀属基部;暗纹东方鲀与红鳍东方鲀、菊黄东方鲀与双斑东方鲀各自亲缘关系较近。此外,实验发现了4尾可疑的双斑东方鲀和菊黄东方鲀杂交个体,以及2尾可疑的横纹东方鲀杂交个体。遗传多样性方面,弓斑东方鲀遗传多样性最低。此外,实验在10种东方鲀中共找到595个种间特异性SNP位点。ka/ks分析表明,nd6基因的ka/ks最高(平均为1.19),说明在东方鲀属中nd6可能经历了正选择,而ATP合成酶基因可能在东方鲀适应淡水环境中受到选择。研究表明,东方鲀属内系统发育关系较为复杂,且在野生环境下东方鲀物种间广泛存在自然杂交现象,该研究为东方鲀物种快速鉴定提供可靠的分子标记,加速东方鲀属系统发育研究进展,并为东方鲀野生种质资源保护提供了理论依据。

关键词：东方鲀线粒体基因组单核苷酸多态性标记系统发育 ka/ks

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