微孢子虫基因组分析.ppt

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1、Preliminary analysis of genome of nosema bombycis潘国庆 周泽扬 向仲怀It is just the beginning of the big job-The key lab of Sericulture of Agricultural Ministry,Southwest Agricultural University,Chongqing,P.R.China,400716.1/21/20231microsporidiaFirst recognized in 1857,till now,has been 147 years.The first g

2、enome was published 2001,which is Encephalitozoon cuniculi,with 2.9 Mb.Found in from protists to inverbrates and all class of vertebrate.More than 140 genera and almost 1200 species recognized.1/21/20232Characteristic of the phylumExist only as spores outside the host cellInvasion of host cell invol

3、ves eversion of polar tube.No mitochondria and typical Golgi apparatus,and their ribosome are not of typical eukaryotic type but resemble the ribosome of prokaryotic organisms.1/21/20233The big question for evolutionary biologistsAn important problem to resolve in order to determine the phylogenetic

4、 position of microsporidia is whether they are primitive amitochondrial,having split from other eukaryotes before the origin of the mitochondria,or secondarily amitochondrial,being derived from ancestors with mitochondria.1/21/20234But we are more than itMore researcher invest their energy into the

5、microsporidia,especially the genus Encephalitozoon,which is related to human diseases.Our purpose is divided into two parts,one is for basic research,to discover the relationship between the Nb and silkworm,Nb and Ec;the other is that we are eager to find some key genes useful to prevent the Nb dise

6、ase.1/21/20235backgroundNosema bombycis is the first important pathogen in silkworm feeding.Nosema bombycis test is time&energy-consuming in silkworm feeding.1/21/20236Nosema bombycisLineage(full)cellular organisms;Eukaryota;Fungi/Metazoa group;Fungi;Microsporidia;Apansporoblastina;Nosematidae;Nosem

7、a 1/21/202371/21/202381/21/20239Little information avaliable1/21/202310Proteinsentriesin NCBI 1/21/202311Nb purification with Percoll 1/21/202312N.bN.b全全基基因因组组shotgunshotgun测测序序文文库库构构建建 N.b complete genome DNAhydroshearKit extraction1.5-2kb2-2.5kbPUC18 vectorPCR ReactionForward sequenceReverse seque

8、nce1/21/202313 1.Bascalling (Phred and Phd2fasta)2.Vector Mark(crossmatch)3.Assemble (Phrap)4.Finishing (consed)5.Repeat Mark,ORF Prediction,Gene Annotation (Glimmer,Blastx,Blastn,Clastalw,tRNAscan)6.GenBank(nr)测序和数据处理测序和数据处理1/21/202314Data source0.26 million reactions from 2.5kb genome library,the

9、length of per reads:more than 400bp.4000 reactions from 8kb genome library.The total length of whole reads:6.0 X equivalent of genomeContigs num:3000(1kb),with N50=8.0kbScaffold num:N50=10kb1/21/202315Preliminary resultpredicted gene num:around 6,000.The hits numbers with nr database(1e-6)is around

10、3,700.The anno num:3,770 The predicted genes including intron is about 200.And the intron is small size.The new version of the anno genes is about 5,000.1/21/202316comment They estimated that gene num is similar with yeast.And the 2nd-version annotation was just over yesterday.And I have not get the

11、 updated data.We still need to construct a BAC library of Nb to obtain bigger scaffold,we hope we can get help from institute and university at domestic/abroad.1/21/202317Comparison Nb with other speciesspeciesHits num.e1e-6programmeE.c2,385blastpPseudomonas syringae pv.tomato假单孢菌489blastpTwo strain

12、s of yeast sequencedabout 1,600blastpsilkworm164tblastn1/21/202318Analysis of anno 3 anno from Nosema bombycis.(p30.4,elongation factor I,DNA repair protein);one anno from Nosema locustae.2148 anno from E.c，57.0%.97 anno from Plasmodium falciparum(疟原虫)3D7;63 anno from other plasmodium strains.9 anno

13、 from Bombyx mori,one is eIF2 ALPHA,the other are related to transposon.56 anno from Salmonella enterica subsp.enterica serovar Typhi(沙门氏菌属);49 anno Salmonella typhimurium LT2;36 anno from E.coli52 anno from oryza sativa171 anno form Anopheles gambiae str.PEST疟蚊1/21/202319Some key genes1.1.tRNA synt

14、hetasetRNA synthetase(合成酶合成酶):20202.2.Two Two tRNA synthetasetRNA synthetase not found not found:asp andasp and tyr tyr3.3.TransposonTransposon(转位子转位子)and)and retrotransposon retrotransposon related related gene:gene:4974974.4.Ribosomal protein:Ribosomal protein:93935.5.KinaseKinase(激酶):):33336.6.un

15、known gene:unknown gene:77777.7.ZINC FINGER:34ZINC FINGER:348.8.ENDOCHITINASE:3 items ENDOCHITINASE:3 items 9.9.obviously transcription related:39obviously transcription related:3910.10.obviously translation related:18obviously translation related:1811.11.obviously obviously ubiquitinubiquitin relat

16、ed:28 related:2812.12.obviously related to obviously related to mitochondrialmitochondrial:9:9 1/21/202320Mitochondrial related genes1/21/202321糖糖 酵酵 解解 代代 谢谢 途途 径径 Trehalose海藻海藻糖糖GlucoseTrehalase海藻糖酶海藻糖酶HexokinaseGlucose-6Pfructose-6P果糖果糖GLUCOSE 6-PHOSPHATE ISOMERASEFDP6-PHOSPHO-FRUCTOKINASE磷酸果糖激酶磷

17、酸果糖激酶GLYCERALDEHYDE-3-PHOSPHATEAldolase醛醛缩酶缩酶GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE1.3-二磷酸甘油酸二磷酸甘油酸3-磷酸甘油酸磷酸甘油酸PhospoglycerlKinase?磷酸二羟丙酮TRIOSE PHOSPHATE ISOMERASE2-磷酸甘油酸磷酸甘油酸PhosphoglyceroMutase?磷酸甘油变位酶磷酸甘油变位酶Enolase烯醇酶磷酸烯醇式丙酮酸磷酸烯醇式丙酮酸PYRUVATE KINASE丙酮酸激酶烯醇式丙酮酸烯醇式丙酮酸丙酮酸丙酮酸1/21/202322三羧酸循环代谢相关酶三羧酸

18、循环代谢相关酶(E.c中未发现中未发现)succinylsuccinyl-CoA synthetaseCoA synthetase beta chain beta chain（琥珀酸琥珀酸-CoA合成酶）合成酶）malate:quinone（苯醌）苯醌）oxidoreductase(氧化还原氧化还原酶酶)2-isopropylmalate synthase3-isopropylmalate isomerase(dehydratase脱水酶脱水酶)subunit2-oxoglutarate dehydrogenase(脱氢酶脱氢酶)E1 component3-HYDROXY-3-METHYLGL

19、UTARYL CoA REDUCTASE3-HYDROXY-3-METHYLGLUTARYL-CoA SYNTHASE 21/21/202323PPP途径ADP-L-glycero(甘油)-D-mannoheptose(甘露庚糖)-6-epimerase(差向异构酶)D-RIBULOSE 5 PHOSPHATE 3 EPIMERASE（D-核酮糖磷酸差向异构酶）RIBOSE 5-PHOSPHATE ISOMERASE5-核酮糖磷酸核糖异构酶1/21/202324Enzyme in lipid metabolism脂类代谢Lipase(脂肪酶）3-3-ketoacylketoacyl-CoA t

20、hiolaseCoA thiolase(3-(3-酮酰基辅酶酮酰基辅酶A A硫解酶）硫解酶）STEROL O-ACYLTRANSFERASESTEROL O-ACYLTRANSFERASE（固固醇醇 O-O-酰基转移酶）酰基转移酶）ACYLGLYCEROL-3-PHOSPHATE ACYLTRANSFERASEACYLGLYCEROL-3-PHOSPHATE ACYLTRANSFERASE（3-3-磷磷酸甘油乙酰基转移酶）酸甘油乙酰基转移酶）1-1-ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASEACYL-SN-GLYCEROL-3-PHOSPHATE

21、ACYLTRANSFERASEACYLCOENZYME A THIOESTERASE IIACYLCOENZYME A THIOESTERASE II（脂酰辅酶硫酯酶脂酰辅酶硫酯酶II)II)1/21/202325Nucleotide metabolism核苷代谢DIMETHYLADENOSINE TRANSFERASE（二甲基腺苷转移酶）N6-adenine methyltransferase signature（腺嘌呤甲基转移酶标签1/21/202326线粒体cytochrome b5 reductase(还原酶)PP36THIOREDOXIN family（硫氧还蛋白）1/21/2023

22、27 Our interesting aimsThe mechanisms of the polar tube extrusion.The reproduction process(developmental expression of genes)of Nb in cell.Energy metabolism of Nb.The new proof of the Nb in evolution.Relationship between the Nb and silkwormNew approach to prevent the disease.1/21/202328Collaborations are expected and welcomeWe have established collaboration with a Lab of France funded by MOST.We hope that new collaborations would be reached with other labs and institutes at domestic/abroad.1/21/202329Thanks1/21/202330