Cloning and analysis of the core sequence of small subunit ribosomal RNA of Endoreticulatus bombycis 3. (Muller, 1997), ( Weiss et al.

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1 49 (3) : , 2003 A cta Zoologica S inica RNA 3 ( SSUr RNA) 33 (, ) Cloning and analysis of the core sequence of small subunit ribosomal RNA of Endoreticulatus bombycis 3 PAN Min2Hui 33 WAN Yong2Ji L U Cheng ZHOU Ze2Yang XIAN G Zhong2Huai ( Key Sericult ural L aboratory of A gricult ural Mi nist ry, Southwest A gricult ural U niversity, Chongqi ng , Chi na) Abstract Endoreticulatus bombycis is a new pathogenic microspordia isolated from the silkworm larvae. With polymerase chain reaction ( PCR) method, we amplified a fragment of the core sequence of the small subunit ribosomal RNA ( SSUr2 RNA) of Endoreticulatus bombycis. The SSUrRNA fragment was inserted into pmd182t Vector and then cloned. It had a length of nucleotides and a percentage GC content of 5113 %. The accession number in GenBank is gi A Y The secondary structure model of the SSUrRNA of Endoreticulatus bombycis was constructed both on the bases of the sequence alignment and RNAFOLD program of the PC2GEN E package. The secondary structure model re2 vealed that Endoreticulatus bombycis lacked many eukaryotic helices, such as helix10, helix 11, helix 18, helix 43 and helix 46, but it has V4 region and has more of prokaryotic character. Analyzed by Blast, Endoreticulatus bombycis, En2 doreticulatus schubergi and Pleistophora sp. ( Sd2Nu2IW8201) have a high similarity, over 98 %. Phylogeny tree of En2 doreticulatus and Pleistophora species showed that Endoreticulatus and Pleistophora sp. ( Sd2Nu2IW8201) was in a group and the other Pleistophora species were in another group [ Acta Zoologica Sinica 49 (3) : , 2003 ]. Key words Small subunit ribosomal RNA, Endoreticulatus, Silkworm, Microsporidia, Sequence analysis SSUrRNA,,, (Muller, 1997), ( Weiss et al., 1998), 80S, 5S rrna, 16S rrna ( Sogin, 1991),,,, RNA, ( Keeling et al., 1998),, , ( No ) ( This research was funded by a grant from National Key Technologies R &D Pro2 gram during the 9th five2year plan) 33 (Corresponding author). E2mail : cq. cn,, 33,, : E2mail : cq. cn ν 2003 Acta Zoologica Sinica RNA (rrna)

2 3 : RNA (SSUrRNA) D NA ( ) DNA KOH + TEK (1 mmol/ L Tris2HCl 10 mmol/ L, ED TA 0117 mol/ L KCl p H 810) (,, 2001), SSUr2 115 PCR RNA, PCR GenBank SSUrRNA V2 V4 V8, SCM7 SSUrRNA,, : P1 : 5 2CT GCA GGTAC CACCA, GGTTG A TTCT GCCTG AC23 ( Hatakeyama, P2 : 5 2GA GCT CGCA T 1997), 1 22 ( Nosem a) ( Pleistophora), Endoreticulat us schubergi ( Thelohania ) ( V ai ri morpha) ( Fujihana, 1984 a, b ;, 1995) ( En2, 012 mm dn TP, 115 mm MgCl 2, 110 doreticulat us bombycis) Ta KaRa Ex Taq TM 15 ng, DNA : 94 3 min, (, min, 55 2 min, 72 2 min, ) SSUrRNA, min, PCR Percoll Pharmacia, Glassmilk kit GIB ICO, Ta KaRa Ex Taq TM, pmd 182T Vector Ta KaRa, PCR Ta KaRa, PCR Ge2 377XL DNA Sequencer neamp PCR System 9600 ( ) Gene Tool 781, SSUrRNA , Gene Tool PCGEN E 4 5 d SSUrRNA,, 0185 %,, 4, SSUrRNA, ( Percoll) SSUrRNA ( Gatehouse et al., 1998) Blast, SSUrRNA, 0185 % ; J ukes et al. SSUrRNA, TTACC TTGTT ACGAC TT23 GCGGT PCR PCR : 0105 M PCR, Glass2 milk Kit pmd 182T Vector, DH5, AB I PRISM TM 117 (1969)

3 DNA PCR, TREECON, (Van de Peer et al., 1993) SSUr RNA DNA Sequencer PCR SSUrRNA SSUrRNA PCR bp GenBank 1 SSUrRNA PCR Fig11 PCR amplif ication of core regions of SSUrRNA of Endoreticulatus bombycis M : / Hind 1 : PCR (12Product of PCR) 2 : DNA PCR ( PCR products of Bombyr mori genomic DNA) 212 SSUr RNA AB I PRISM TM 377XL bp, 20 bp,, A Y SSUr RNA Gene Tool SSUrRNA bp, GC 5113 %, 19 Endoreticulat us bombycis En2 doreticulat us schubergi SSUrRNA, PCR Ta KaRa Ex Taq TM, SSUrRNA PCR Gene Tool PCGEN E SSUr2 RNA ( 2) RNA 50, 1, P1 P2 SSUrRNA DNA PCR, kb 46, SSUr2 DNA PCR RNA SSUrRNA, ( 1) 1 SSUrRNA Table 1 The difference of SSUrRNAπs secondary structures among Endoreticulatus bombycis, procaryote and eukaryote Helix ( Procaryote) ( Eukaryote) E8 E10 E23 (V4) P37 E ( E. bombycis) P : ( Procaryote) E : ( Eukaryote) E. bombycis : + : ( Presence)

4 3 : RNA (SSUrRNA) SSUrRNA Fig12 A secondary model for SSUrRNA of Endoreticulatus bombycis

5 Pleistophora ( Jukes2Cantor ) Table 2 Pairwise distances as calculated by the Jukes2Cantor method and levels of similarity SSUrRNA ( 100 ), [ The values on the lower left are SSUrRNA evolutionary distances as calculated by the J ukes2cantor equation, and the values on the upper right are levels of similarity expressed as percentages. tance matrix are multiplied by 100) ] ( The values in the dis2 1 : Endoreticulat us bombycis 2 : Pleistophora hippoglossoi deos 3 : Pleistophora mi randellae 4 : Pleistophora ovariae 5 : Pleistophora sp. (LS) 6 : Pleistophora sp. ( PA) 7 : Pleistophora sp. ( TB) 8 : Pleistophora sp., Sd2Nu2IW : Pleistophora sp : Pleistophora typi2 calis BLAST,,, GenBank ( Endoreticulat us schubergi) ( Pleistophora) ( : Sd2Nu2IW8201) ( Iwano et al., 1984) 98 %, Pleistophora sp. ( Sd2Nu2 IW8201) : ( Gurley et al., 1893), Sd2Nu2 IW8201 SSUrRNA ( Elizabeth et al., 1982),,, (Brooks et al., 1988) ( 2), J ukes et al., TREECON ( 3) (, 1995), Pleistophora sp. ( Sd2Nu2IW82) , RNA 98 % 9818 %,,, , 70 % 80 %, SSUrRNA (Class Dihaplophasea cl. n) ( Nose2, m a bombycis ), ( Class Haplop hasea cl. n. ), , Pleistophora sp. ( Sd2Nu2IW8201) ,, SSUr2 (Diplomonadida) ( Trichomonadida) 46 V4 V4, 5,, SSUrRNA,

6 3 : RNA (SSUrRNA) Fig13 Phylogeny tree of Endoreticulatus species and Pleistophora species, [ The bar ( = 011) on the upper left is evolutionary distances ; The number on the branch is bootstrap values] Pleistophra Pleistophora sp. ( Sd2Nu2IW8201) ( 3) , 3 SSUrRNA Table 3 Comparison of the secondary structure of SSUr RNA of Microsporidia and some other organisms ( Microspori dia) Helix ( ) V4 ( N. bomycis) + ( V. necat ri x) + + ( Endoreticulat us bombycis) ( Pleistophora sp. Sd2Nu2IW8201) + + ( Endoreticulat us schubergi) + ( Encephalitozoon cuniculi) (Diplomonadida) ( Tritrichomonus) : ( Presence) : (Absence) Pleistophora sp. ( Sd2Nu2IW82) 9818 %,, 70 % 80 %, Pleistophora sp. ( Sd2 Nu2IW82), Pleistophora,,,,, Pleistophora sp. ( Sd2Nu2IW82), ( References) Brooks, W. M. and J. J. Becnel 1988 Establishment of En2 doreticulat us n. g. for Pleistophoridae. J. Protozool. 35 : Elizabeth, U. and I. Edwin 1982 Genus Pleistophora Gurley, 1893 : an assemblage of at least three genera. J. Protozool. 29 (1) : Fujihana, K1 1984a Pleistophora Isolated from the silkworm, Bombyx mori. J. Seric. Sci. J pn. 53 (5) : [ 1984a Pleistophora. 53 (5) : ]

7 Fujihana, K1 1984b Thelohania sp. isolated from the silkworm, Bombyx mori. J. Seric. Sci. Jpn. 53 (5) : [ 1984b Thelohania SP. ods for preparing DNA of different species of microsporidia. Jour2 53 (5) : ] Gatehouse, H. S, and L. A. Malone 1998 The ribosomal RNA gene region of N. apis ( Microspora) : DNA sequence for small and large subunit rrna genes and evidence of a large tandem repeat unit size. J. Invertebr. Pathol. 77 : Gurley, R. R On the classification of the Myxospora, a group of protozoan parasites infecting fishes. B ull. US Fish. Com m. 11 : Hatakeyama, Y Analyses and taxonomic inferences of small subunit ribosomal RNA sequences of five microsporidia pathogenic to the silkworm, Bombyx mori. J. Seric. Sci. Jpn. 66 (4) : Iwano, H., N. Shimizu and Y. Kawakami 1994 Spore dimorphism and some other biological features of a Nosema sp. isolated from the lawn grass cutworm, S podoptera depravata. A ppl. Ento2 mol. Zool. 29 (2) : J ukes, T. H. and C. R. Cantor 1969 Evolution of protein molecules. In : Munro, H. H. ed. Mammalian Protein Metabolism. New York : Academic Press, Keeling, P. J. and G. I. McFadden 1998 Origins of mi2 crosporidia. Trends Microbio. 6 (1) : Muller, M What are microsporidia? Parasitology Today 13 (12) : Pan, M. H., Y. J. Wan and C. Lu 2001 Studies on the meth2 nal of Southwest A gricult ural U niversity 23 ( 2) : [,, 2001 DNA. 23 (2) : ] Sogin, M. L Early evolution and the origin of eukaryotes. Curr. Opi n. Genet. Dev. 1 (4) : Van de Peer, Y., J. M. Neefs and P. De Rijk 1993 Evolution of eukaryotes as deduced from small ribosomal subunit RNA se2 quences. Biochemical Systematics and Ecology 21 (1) : Wan, Y. J., L. Zhang and Z. P. Chen 1995 Study of a pathogenic microspordia SCM7 ( Endoreticulat us sp. ) isolated from the larva of silkworm, Bombyx mori. Acta Sericologica Si nica 21 (3) : [,, 1995 SCM 7 ( Endoreticulat us sp. ). 21 (3) : ] Weiss, L. M. and C. R. Vossbrinck 1998 Microsporidiosis : molecular and diagnostic aspects. J. A dv. Parasitol. 40 : Zhang, L., Y. Du and Y. J. Wan 1995 A new species of En2 doreticulat us ( Protozoa, Microspore) from the larva of silkworm, Bombyx. L. Journal of Southwest A gricult ural U niversity 17 (4) : [,, (4) : ]