CAJ | 학술논문

设计引物并 PCR扩增河北平山株系 PaWBPs 和江西吉安株系 PaWBJan 的 tmk 基因，直接和克隆测定tmk序列，利用DNAMAN，MEGA等软件进行序列多样性、序列变异、蛋白功能域、系统进化等比较分析。结果表明：从 PaWBPs和 PaWBJan中克隆测序的93条和41条 tmk-a中，分别有52和24种不同的序列，tmk-a ORF 可被划分为2类，tmk-a-1为639 bp，tmk-a-2为627 bp，PaWBPs株系tmk-a-1与tmk-a-2序列条数的比值约为2.5，而PaWBJan株系为3.3； PaWBPs有5个相同的 tmk-a-1序列与 PaWBJan的一个 tmk-a-1序列及枣疯植原体 tmk-Y 序列完全一致。因多位点突变，PaWBPs含假基因比例达48.1%，PaWBJan的为41.7%； tmk-b基因为单一序列，两株系的 tmk-b核酸序列相似性为99.8%，编码蛋白仅有一个氨基酸差异。TMK-a和 TMK-b中都具有胸苷酸激酶的3个保守功能域。系统进化分析显示，泡桐丛枝植原体的2个株系的所有tmk-a序列都与枣疯植原体tmk-X和tmk-Y等聚为一个进化枝Ⅰ中；而tmk-b与小麦蓝矮tmk-2等聚为另一进化枝Ⅲ，基于tmk-b序列和基于16S rDNA序列构建的系统进化树一致，tmk-b有助于植原体16Sr组水平的分类，而 tmk-a可用于植原体株系变异和遗传多样性分析。
설계인물병 PCR확증하북평산주계 PaWBPs 화강서길안주계 PaWBJan 적 tmk 기인，직접화극륭측정tmk서렬，이용DNAMAN，MEGA등연건진행서렬다양성、서렬변이、단백공능역、계통진화등비교분석。결과표명：종 PaWBPs화 PaWBJan중극륭측서적93조화41조 tmk-a중，분별유52화24충불동적서렬，tmk-a ORF 가피화분위2류，tmk-a-1위639 bp，tmk-a-2위627 bp，PaWBPs주계tmk-a-1여tmk-a-2서렬조수적비치약위2.5，이PaWBJan주계위3.3； PaWBPs유5개상동적 tmk-a-1서렬여 PaWBJan적일개 tmk-a-1서렬급조풍식원체 tmk-Y 서렬완전일치。인다위점돌변，PaWBPs함가기인비례체48.1%，PaWBJan적위41.7%； tmk-b기인위단일서렬，량주계적 tmk-b핵산서렬상사성위99.8%，편마단백부유일개안기산차이。TMK-a화 TMK-b중도구유흉감산격매적3개보수공능역。계통진화분석현시，포동총지식원체적2개주계적소유tmk-a서렬도여조풍식원체tmk-X화tmk-Y등취위일개진화지Ⅰ중；이tmk-b여소맥람왜tmk-2등취위령일진화지Ⅲ，기우tmk-b서렬화기우16S rDNA서렬구건적계통진화수일치，tmk-b유조우식원체16Sr조수평적분류，이 tmk-a가용우식원체주계변이화유전다양성분석。
The diversity,variation,predicted protein function domains and evolution of thymidylate kinase gene ( tmk) ,which was obtained by cloning DNA sequencing of PCR products with designed primer from paulownia witches’-broom phytoplasmas Pingshan strain ( PaWBPs) in Hebei Province and Ji’an strain ( PaWBJan) in Jiangxi Province were comparatively analyzed by DNAMAN and MEGA software. The 52 and 24 different tmk-a homologues respectively from the cloned 93 sequences of PaWBPs and 41 of PaWBJan in total were identified,whereas the tmk-b gene sequence was relatively conserved,with 99. 8% similarity and only one amino acid variation of the predicted proteins between the two strains. All tmk-a ORFs were classified into tmk-a-1 (639 bp) and tmk-a-2 (627 bp) . The ratio of tmk-a-1/tmk-a-2 in PaWBPs and PaWBJan was 2. 5 and 3. 3,respectively. Five cloned tmk-a-1 sequences of PaWBPs were the same as one in PaWBJan as well as tmk-Y of jujube witches ’ broom phytoplasma. In addition,PaWBPs and PaWBJan respectivety contained up to 48. 1% and 41. 7% of predicted tmk-a pesudogenes in total tmk-a due to the multiple locus mutation. Rich sequence variability was found in tmk-a homologues of PaWBPs and PaWBJan and there existed three functional domains in amino acid sequences of both tmk-a and b which are necessary for thymidylate kinase activity. The phylogenetic analysis showed that all the tmk-a homologues of both strains with tmk-X and Y were clustered into clade I,while tmk-b with wheat blue dwarf tmk-2 into clade III,suggesting that tmk-b nucleotide sequences consistent with highly conserved 16 S rDNA could be used for the classification of phytoplasmas on 16 Sr group levels whereas diverse tmk-a might be helpful for analyzing the genetic variation and diversity of different phytoplasma strains.