李飞
果蝇非编码RNA(miRNA)基因分析、无脊椎动物(昆虫)基因组学与生物信息学分析、乙酰胆碱酯酶基因相关研究
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- 姓名:李飞
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学术头衔:
博士生导师, 教育部“新世纪优秀人才支持计划”入选者
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学科领域:
生物化学
- 研究兴趣:果蝇非编码RNA(miRNA)基因分析、无脊椎动物(昆虫)基因组学与生物信息学分析、乙酰胆碱酯酶基因相关研究
李飞 男,1977.1,博士,南京农业大学教授、博士生导师。2003年获南京农业大学博士学位,2003年7月至2005年8月在清华大学自动化系从事生物信息学博士后研究工作。2005 年全国百篇优秀博士论文作者,入选2005 年教育部新世纪优秀人才支持计划。
率先从棉蚜中克隆发现2 个乙酰胆碱酯酶基因,为昆虫种群中可能存在多个乙酰胆碱酯酶基因的假说首次提供直接的分子生物学证据;利用直接测序法从棉蚜2 个乙酰胆碱酯酶基因中发现了A302S 和F115S 两个抗性突变位点;优化了基于普鲁卡因胺亲和层析的乙酰胆碱酯酶分离纯化方法,研究粗酶液和纯化酶液在抗性毒理分析中的差异。利用生物信息学的方法分析了极短的基因选择性剪接对蛋白二级结构的影响,向人们展示了这些以往容易被忽视的现象可能蕴含的重要生物学功能。2003 年开始在国内较早地开展非编码RNA 基因的研究工作,通过提取微RNA 基因的结构特征,结合比较基因组学分析,开发出非编码RNA 基因的识别算法,从果蝇、家蚕、按蚊和蜜蜂等基因组序列已知的昆虫中发现100多个新的微RNA 基因。相关研究结果分别发表于Trends in genetics,Bioinformatics, BMC Bioinformatics, Human molecular genetics, Genome, BBRC, Insect biochemistry and molecular biology,《2004高技术发展报告》,《动物学报》,《昆虫学报》,《棉花学报》等重要学术期刊,其中SCI收录9篇,累计影响因子达46.449(以2004 年影响因子计算),被他人引用30余次。先后主持国际科学基金、国家自然科学基金(60405001)、南京农业大学人才引进基金、中国博士后科学基金等项目。
目前主要研究方向:果蝇非编码RNA(miRNA)基因分析、无脊椎动物(昆虫)基因组学与生物信息学分析、乙酰胆碱酯酶基因相关研究、。实验室主页: http://ento.njau.edu.cn/people/~lifei/lifei.htm
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【期刊论文】棉蚜乙酰胆碱受体亚基的选择性剪接和多重转录起始位点分析*
李飞, LI Fei**, HAN Zhao-Jun
Acta Zoologica Sinica 51 (5): 867-878, 2005,-0001,():
-1年11月30日
乙酰胆碱受体在神经突触传导过程中具有重要作用,也是氯化胆碱类杀虫剂的作用靶标。采用RACE技术,成功地从棉蚜中克隆了3个nAChR亚基,其中2个为α亚型,1个为β亚型,分别命名为Agα1 、Agα2和A gβ1。通过锚定mRNA 的5'mG结构,5'RACE 结果表明Agβ1有三个不同的剪接变体,具有不同长度的5′UTR区,表明Agβ1亚基具有多重的转录起始位点。其中,最短的剪接变体A gβ1C 在蛋白编码区域也存在选择性剪接,位于D环区域的186 bp碱基缺失。3'RACE 实验结果表明,Agα1亚基虽然具有ploy (A) 和加尾信号AATAAA等完整的mRNA基因结构,但缺失了终止子和乙酰胆碱受体α亚基保守的第4个跨膜区,文中对此做了进一步分析。分子进化树的分析表明,昆虫乙酰胆碱受体亚基应当被划分为三个不同的亚类群αⅠ,αⅡandβ。本文的研究揭示了昆虫乙酰胆碱受体亚基复杂的基因结构。
棉蚜, 乙酰胆碱受体, RACE, 选择性剪接, 多重转录起始位点
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李飞, Fei Li and Zhaojun Han*
Archives of Insect Biochemistry and Physiology 51: 37-45 (2002),-0001,():
-1年11月30日
A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33, 680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDSPAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02mM 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25℃. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations. Arch.
Aphis gossypii Glover, acetylcholinesterase, purification, procainamide affinity column, kinetic characterization, inhibition
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【期刊论文】Two different genes encoding acetylcholinesterase existing in cotton aphid (Aphis gossypii)
李飞, Fei Li and Zhao-Jun Han
Genome 45: 1134-1141 (2002),-0001,():
-1年11月30日
Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5′-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09: A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5′-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.
Aphis gossypii Glover, acetylcholinesterase, gene clone, homology modeling, alternative splicing, phylogenetic analysis.,
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李飞, Fang Wen, *, Fei Li, Huiyu Xia, Xin Lu, , Xuegong Zhang and Yanda Li
TRENDS in Genetics Vol.20 No.5 May 2004,-0001,():
-1年11月30日
The systematic analysis of very short alternative splicing (VSAS) in the human genome showed that VSAS might contribute more to protein-function diversity than expected. More than 65% of VSAS fragments have different secondary structures from flanking regions. They tend to have a non-loop structure and have an important influence on protein functions, as shown by the predicted 3D structure of human IL-4d2. The observed VSAS events can be classified into two groups depending on whether they insert new structure domains in the proteins, and they might be of different evolutionary status.
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李飞, Fei Li, Zhaojun Han*
Insect Biochemistry and Molecular Biology 34(2004)397-405,-0001,():
-1年11月30日
Two acetylcholinesterase genes, Ace1 and Ace2, have been fully cloned and sequenced from both organophosphate-resistant and susceptible clones of cotton aphid. Comparison of both nucleic acid and deduced amino acid sequences revealed considerable nucleotide polymorphisms. Further study found that two mutations occurred consistently in all resistant aphids. The mutation F139L in Ace2 corresponding to F115S in Drosophila acetylcholinesterase might reduce the enzyme sensitivity and result in insecticide resistance. The other mutation A302S in Ace1 abutting the conserved catalytic triad might affect the activity and insecticide sensitivity of the enzyme. Phylogenetic analysis showed that insect acetylcholinesterases fall into two subgroups, of which Ace1 is the paralogous gene whereas Ace2 is the orthologous gene of Drosophila AChE. Both subgroups contain resistance-associated AChE genes. To avoid confusion in the future work, a nomenclature of insect AChE is also suggested in the paper.
Aphis gossypii Glover, Acetylcholinesterase, Single nucleotide polymorphism, Insecticide resistance
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【期刊论文】Evidence and characteristics of putative human α recombination hotspots
李飞, Jing Zhang, , Fei Li, Jun Li, Michael Q. Zhang, and Xuegong Zhang, *
Human Molecular Genetics, 2004, Vol. 13, No.22 2823-2828,-0001,():
-1年11月30日
Understanding recombination rate variation is very important for studying genome diversity and evolution, and for investigation of phenotypic association and genetic diseases. Recombination hotspots have been observed in many species and are well studied in yeast. Recent study demonstrated that recombination hotspots are also a ubiquitous feature of the human genome. But the nature of human hotspots remains largely unknown. We have developed and validated a novel computational method for testing the existence of hotspots as well as for localizing them with either unphased or phased genotyping data. To study the characteristics of hotspots within or close to genes, we scanned for unusually high levels of recombination using the European population samples in the SeattleSNPs database, and found evidence for the existence of human a hotspots similar to those of yeast. This type of hotspots, found at promoter regions, accounts for about half of the total detected and appears to depend on some specific transcription factor binding sites (such as CGCCCCCGC). These characteristics can explain the observed weak correlation between hotspots and GC-content, and their variation may contribute to the diversity of hotspot distribution among different individuals and species. These long-sought putative human a recombination hotspots should deserve further experimental investigations.
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【期刊论文】The effect of U1 snRNA binding free energy on the selection of 5'splice sites
李飞, Jianning Bi, Huiyu Xia, Fei Li, Xuegong Zhang, Yanda Li*
Biochemical and Biophysical Research Communications 333(2005)64-69,-0001,():
-1年11月30日
The importance of U1 snRNA binding free energy in the regulation of alternative splicing has been studied in some genes with site-directed mutagenesis. Here we report a large-scale analysis of its impact on 50 splice site (50ss) selection in human genome. The results show that free energy exerts different effects on alternative 50ss choice in different situations and 8.1 kcal/mol is a hreshold. When both free energies of two competing 50ss are larger than 8.1 kcal/mol, the 50ss with lower free energy is more frequently used. However, in other pairs of 50ss, lower-free-energy 50ss does not seem to be favored and even the other 50ss is used more frequently, which suggests that very low binding free energy would impair splicing. Some observations hold true only for those alternative 50 splicing with short alternative exons (<50nt), which implies a complex mechanism of 50ss selection involving both U1 snRNA binding free energy and regulatory factors.
Alternative splicing, Free energy, U1 snRNA
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【期刊论文】MicroRNA identification based on sequence and structure alignment
李飞, Xiaowo Wang†, Jing Zhang†, Fei Li, Jin Gu, Tao He, Xuegong Zhang and Yanda Li∗
,-0001,():
-1年11月30日
Motivation: MicroRNAs (miRNA) are 22 nt long non-coding RNAs that are derived from larger hairpin RNA precursors and play important regulatory roles in both animals and plants. The short length of the miRNA sequences and relatively low conservation of pre-miRNA sequences restrict the conventional sequence-alignment-based methods to finding only relatively close homologs. On the other hand, it has been reported that miRNA genes are more conserved in the secondary structure rather than in primary sequences. Therefore, secondary structural features should be more fully exploited in the homologue search for new miRNA genes. Results: In this paper, we present a novel genome-wide computational approach to detect miRNAs in animals based on both sequence and structure alignment. Experiments show this approach has higher sensitivity and comparable specificity than other reported homologue searching methods. We applied this method on Anopheles gambiae and detected 59 new miRNA genes. Availability: This programis available at http://bioinfo.au.tsinghua.edu.
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李飞, Chenghai Xue†, , Fei Li†, Tao He, Guo-Ping Liu, Yanda Li and Xuegong Zhang*
BMC Bioinformatics 2005, 6: 310,-0001,():
-1年11月30日
Background: MicroRNAs (miRNAs) are a group of short (~22 nt) non-coding RNAs that play important regulatory roles. MiRNA precursors (pre-miRNAs) are characterized by their hairpin structures. However, a large amount of similar hairpins can be folded in many genomes. Almost all current methods for computational prediction of miRNAs use comparative genomic approaches to identify putative pre-miRNAs from candidate hairpins. Ab initio method for distinguishing premiRNAs from sequence segments with pre-miRNA-like hairpin structures is lacking. Being able to classify real vs. pseudo pre-miRNAs is important both for understanding of the nature of miRNAs and for developing ab initio prediction methods that can discovery new miRNAs without known homology. Results: A set of novel features of local contiguous structure-sequence information is proposed for distinguishing the hairpins of real pre-miRNAs and pseudo pre-miRNAs. Support vector machine (SVM) is applied on these features to classify real vs. pseudo pre-miRNAs, achieving about 90% accuracy on human data. Remarkably, the SVM classifier built on human data can correctly identify up to 90% of the pre-miRNAs from other species, including plants and virus, without utilizing any comparative genomics information. Conclusion: The local structure-sequence features reflect discriminative and conserved characteristics of miRNAs, and the successful ab initio classification of real and pseudo pre-miRNAs opens a new approach for discovering new miRNAs.
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李飞, Jun Cai, *, Ying Huang, Fei Li, and Yanda Li
Structure, Function, and Bioinformatics 62: 793-799 (2006),-0001,():
-1年11月30日
Alternative translation is an important cellular mechanism contributing to the generation of proteins and the diversity of protein functions. Instead of studying individual cases, we systematically analyzed the alteration of protein subcellular location and domain formation by alternative translational initiation in eukaryotes. The results revealed that 85.7% of alternative translation events generated biological diversity, attributed to different subcellular localizations and distinct domain contents in alternative isoforms. Analysis of isoelectric point values revealed that most N-terminal truncated isoforms significantly lowered their isoelectric point values targeted at different subcellular localizations, whereas they had conserved domain contents the same as the fulllength isoforms. Furthermore, Fisher's exact test indicated that the two ways-targeting at different cellular compartments and changing domain contents-were negatively associated. The N-term truncated isoforms should have only one way to diversify their functions distinct from the full-length ones. The peculiar consequence of subcellular relocation as well as change of domain contents re-flected the very high level of biological complexity as alternative usage of initiation codons. Proteins.
alternative translation initiation events, nucleotide context, subcellular relocalization, domain contents
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