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1、2018/9/12,1,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.,2018/9/12,2,microsporidia,First recognized in 1857,till now, has bee
2、n 147 years. The first genome 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.,2018/9/12,3,Characteristic of the phylum,Exist only as spores outside the host
3、 cell Invasion of host cell involves 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.,2018/9/12,4,The big question for evolutionary biologists,An important problem to resolve
4、in order to determine the phylogenetic 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.,2018/9/12,5,But we are more than it
5、,More researcher invest their energy into the 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
6、 to find some key genes useful to prevent the Nb disease.,2018/9/12,6,background,Nosema bombycis is the first important pathogen in silkworm feeding. Nosema bombycis test is time & energy-consuming in silkworm feeding.,2018/9/12,7,Nosema bombycisLineage( full ) cellular organisms; Eukaryota; Fungi/M
7、etazoa group; Fungi; Microsporidia; Apansporoblastina; Nosematidae; Nosema,2018/9/12,8,2018/9/12,9,2018/9/12,10,Little information avaliable,2018/9/12,11,Proteins entries in NCBI,2018/9/12,12,Nb purification with Percoll,2018/9/12,13,N.b 全 基 因 组 shotgun 测 序 文 库 构 建,N.b complete genome DNA,hydroshear
8、,Kit extraction,1.5-2kb 2-2.5kb,PUC18 vector,PCR Reaction,Forward sequence,Reverse sequence,2018/9/12,14,1. 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 , tR
9、NAscan) 6. GenBank(nr),测序和数据处理,2018/9/12,15,Data source,0.26 million reactions from 2.5kb genome library, the length of per reads: more than 400bp. 4000 reactions from 8kb genome library. The total length of whole reads: 6.0 X equivalent of genome Contigs num:3000(1kb), with N50=8.0kb Scaffold num:
10、N50=10kb,2018/9/12,16,Preliminary result,predicted gene num: around 6,000. The hits numbers with nr database (1e-6) is around 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.,2018/9/12,17,comm
11、ent,They estimated that gene num is similar with yeast. And the 2nd-version annotation was just over yesterday. And I have not get the 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.,20
12、18/9/12,18,Comparison Nb with other species,2018/9/12,19,Analysis 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 ann
13、o 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.coli 52 anno from oryza sativa 171 anno form Anopheles gambiae str. PEST疟蚊,2018/9/12,20,Some key genes
14、,tRNA synthetase(合成酶): 20 Two tRNA synthetase not found: asp and tyr Transposon(转位子) and retrotransposon related gene: 497 Ribosomal protein: 93 Kinase(激酶): 33 unknown gene: 77 ZINC FINGER: 34 ENDOCHITINASE: 3 items obviously transcription related:39 obviously translation related:18 obviously ubiqui
15、tin related: 28 obviously related to mitochondrial:9,2018/9/12,21,Mitochondrial related genes,2018/9/12,22,糖 酵 解 代 谢 途 径,Trehalose 海藻糖,Glucose,Trehalase海藻糖酶,Hexokinase,Glucose-6P,fructose-6P 果糖,GLUCOSE 6-PHOSPHATEISOMERASE,FDP,6-PHOSPHO-FRUCTOKINASE 磷酸果糖激酶,GLYCERALDEHYDE -3-PHOSPHATE,Aldolase 醛缩酶,GL
16、YCERALDEHYDE-3- PHOSPHATE DEHYDROGENASE,1.3-二磷酸甘油酸,3-磷酸甘油酸,Phospoglycerl Kinase ?,磷酸二羟丙酮,TRIOSE PHOSPHATE ISOMERASE,2-磷酸甘油酸,PhosphoglyceroMutase? 磷酸甘油变位酶,Enolase 烯醇酶,磷酸烯醇式丙酮酸,PYRUVATE KINASE 丙酮酸激酶,烯醇式丙酮酸,丙酮酸,2018/9/12,23,三羧酸循环代谢相关酶(E.c中未发现),succinyl-CoA synthetase beta chain （琥珀酸-CoA合成酶） malate:quinone（苯醌）oxidoreductase(氧化还原酶)2-isopropylmalate synthase 3-isopropylmalate isomerase (dehydratase脱水酶) subunit 2-oxoglutarate dehydrogenase(脱氢酶)E1 component 3-HYDROXY-3-METHYLGLUTARYL CoA REDUCTASE 3-HYDROXY-3-METHYLGLUTARYL-CoA SYNTHASE 2,
