49 (3) :414 420, 2003 A cta Zoologica S inica RNA 3 ( SSUr RNA) 33 (, 400716) 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 400716, 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 1 230 nucleotides and a percentage GC content of 5113 %. The accession number in GenBank is gi11181769 A Y009115. 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) : 414-420, 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),, 2002207218, 2003202220 3 ( No1962616202203) ( This research was funded by a grant from National Key Technologies R &D Pro2 gram during the 9th five2year plan) 33 (Corresponding author). E2mail : pmh @swau. cq. cn,, 33,, : E2mail : pmh @swau. cq. cn ν 2003 Acta Zoologica Sinica RNA (rrna)
3 : RNA (SSUrRNA) 415-114 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, 11511 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 ri2 1 119 1 221 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, (, 94 115 min, 55 2 min, 72 2 min, 30 1995) SSUrRNA, 72 10 min, 4 116 PCR 1 111 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 ( ) 112 11711 Gene Tool 781, SSUrRNA 113 10 8, 11712 Gene Tool PCGEN E 4 5 d SSUrRNA,, 0185 %,, 4, SSUrRNA, ( Percoll) SSUrRNA ( Gatehouse et al., 1998) 11713 Blast, SSUrRNA, 0185 % ; J ukes et al. SSUrRNA, TTACC TTGTT ACGAC TT23 GCGGT 11512 PCR PCR : 0105 M PCR, Glass2 milk Kit pmd 182T Vector, DH5, AB I PRISM TM 117 (1969)
16 4 49 DNA PCR, TREECON, (Van de Peer et al., 1993) 2 211 SSUr RNA DNA Sequencer PCR SSUrRNA SSUrRNA PCR 1 1 230 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 1 250 bp, 20 bp,, A Y009115 213 SSUr RNA 21311 Gene Tool SSUrRNA 1 230 bp, GC 5113 %, 19 Endoreticulat us bombycis En2 doreticulat us schubergi SSUrRNA, 6 1 1 PCR Ta KaRa Ex Taq TM, SSUrRNA PCR 21312 Gene Tool PCGEN E SSUr2 RNA ( 2) RNA 50, 1, P1 P2 SSUrRNA DNA PCR, 10 11 18 43 112 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 E45 1 1 2 1 2 3 4 5 6 7 8 9 10 11 12 1 2 1 + + + + + + + + + + + + + + + + + + + + + + + ( E. bombycis) P : ( Procaryote) E : ( Eukaryote) E. bombycis : + : ( Presence)
3 : RNA (SSUrRNA) 417 2 SSUrRNA Fig12 A secondary model for SSUrRNA of Endoreticulatus bombycis
18 4 49 2 Pleistophora ( Jukes2Cantor ) Table 2 Pairwise distances as calculated by the Jukes2Cantor method and levels of similarity 1 2 3 4 5 6 7 8 9 10 1 7917 7616 7615 7114 7613 7916 9818 7916 7819 2 221464 9312 9219 8114 9111 9916 7914 9911 9911 3 251346 51602 9718 8013 9014 9312 7616 9215 9211 4 251439 51777 11754 8011 9015 9219 7613 9213 9211 5 271412 161445 171601 171483 7819 8115 7115 8018 8012 6 251392 61960 71787 71852 181244 9111 7610 9017 9010 7 221577 01292 51604 51772 161346 71114 7913 9819 9819 8 01493 221856 251595 251804 271393 251815 221969 7819 7812 9 221281 01588 61026 60197 171078 71226 01735 231352 9914 10 221365 01517 61064 61393 161812 71344 01665 231353 01074 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., Sd2Nu2IW8201 9 : Pleistophora sp. 2 10 : Pleistophora typi2 calis 21313 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) 0100493, RNA 98 % 9818 %,,, 0122856, 70 % 80 %, SSUrRNA (Class Dihaplophasea cl. n) ( Nose2, m a bombycis ), ( Class Haplop hasea cl. n. ), 10 11 18 43, Pleistophora sp. ( Sd2Nu2IW8201) 3 1982,, SSUr2 (Diplomonadida) ( Trichomonadida) 46 V4 V4, 5,, SSUrRNA,
3 : RNA (SSUrRNA) 419 3 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) 0100493, 3 SSUrRNA Table 3 Comparison of the secondary structure of SSUr RNA of Microsporidia and some other organisms ( Microspori dia) Helix ( ) 10 11 18 43 46 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 : 481 488. Elizabeth, U. and I. Edwin 1982 Genus Pleistophora Gurley, 1893 : an assemblage of at least three genera. J. Protozool. 29 (1) : 39 49. Fujihana, K1 1984a Pleistophora Isolated from the silkworm, Bombyx mori. J. Seric. Sci. J pn. 53 (5) : 398 402. [ 1984a Pleistophora. 53 (5) : 398 402. ]
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