牛支原体NM 2012株全基因组测序缺口修补及序列分析

发布时间：2018-05-01 23:15

本文选题：牛支原体 + 克隆　；参考：《内蒙古农业大学》2015年硕士论文

【摘要】：牛支原体是能够导致牛肺炎、关节炎、流产、乳腺炎等多种疾病的一种病原体。给养牛业造成了巨大的经济损失,严重影响了养牛业的健康发展。由于王艳杰测序的M. bovis NM 2012菌株的全基因组序列存在序列缺口和多余的重复序列,为了得到M.bovis NM 2012基因组完整序列,本试验对王艳杰测序的M.bovis NM 2012进行基因组补缺口(补洞)工作。本试验选择M.bovis HB0801为参考基因序列,经过将王艳杰测序的M.bovis NM 2012与M.bovis HB0801比对后发现,共有34处未完成的基因序列,在未完成的序列两端各增加200～1,000bp设计引物,进行PCR扩增和克隆测序,并利用生物信息学软件进行拼接,从而完成M.bovisNM 2012菌株的全基因组补缺口(补洞)工作,并应用生物信息学软件对M.bovisNM 2012菌株的ORFs、脂蛋白、IS元件等进行预测,并对相关的基因进行功能注释。比对结果显示,在34处未完成的基因序列中,有21处由字母N代替,字母N的长度是在10～2,359bp之间,字母N的总长度为13,131bp；34处未完成的序列中,有23处是缺口序列,总共缺失的序列是24,763bp,11处多余重复的序列长度为14,765bp。克隆测序结果表明,在23处缺口序列中,总共缺失的序列是20,222bp；11处多余重复序列的总长度为14,765bp,实际有10,226bp为无效的重复序列,4,539bp确为M.bovis NM 2012本身的序列；本试验中,去除由未知序列N代替的13,131bp和无效的重复序列10,226bp,补齐缺失的序列20,222bp。将王艳杰测序的全基因组大小为993,483bp的M.bovis NM 2012,经过补缺口工作,最终拼接得到M. bovis NM 2012全基因组大小为990,348bp。M.bovis NM 2012菌株全基因组大小为990,348bp, GC%含量为29.29%;与M.bovis HB0801进行比对后,预测编码区含839个ORFs,含有34个tRNA,6个rRNA,预测含有103个脂蛋白和2个Vsp。通过对5株牛支原体全基因组进行基因组共线性分析比较,M. bovis NM 2012基因组与M.bovis PG45基因组的相似性最小,与M.bovis HB0801基因组的相似性最高。
[Abstract]:Mycoplasma bovis is a pathogen that can cause many diseases, such as pneumonia, arthritis, abortion, mastitis and so on. It has caused enormous economic losses to the cattle industry and seriously affected the healthy development of the cattle industry. In order to obtain the complete genome sequence of M.bovis NM 2012, there is a gap in the whole genome sequence and redundant repeat sequence in the whole genome sequence of M. bovis NM 2012, which was sequenced by Wang Yanjie. M.bovis NM 2012, which was sequenced by Wang Yanjie, was studied in this experiment. In this experiment, M.bovis HB0801 was selected as the reference gene sequence. After comparing the M.bovis NM 2012 and M.bovis HB0801 sequenced by Wang Yanjie, it was found that there were 34 uncompleted gene sequences, and the primers designed by 200~1000bp were added at each end of the unfinished sequence. PCR amplification, cloning and sequencing were carried out, and bioinformatics software was used to assemble the whole genome gap (hole) of M.bovisNM 2012 strain. Bioinformatics software was used to predict ORFs, lipoprotein is elements of M.bovisNM 2012 strain, and to annotate the related genes. The results showed that of the 34 uncompleted sequences, 21 were replaced by the letter N, the length of the letter N was between 10~2359bp, and the total length of the letter N was 13131bpng34, of which 23 were gap sequences. The total missing sequence was 24763bpmpn11, and the length of the redundant repeats was 14765bp. The results of cloning and sequencing showed that the total missing sequence of 23 gap sequences was 20222bp-1, the total length of redundant repeats was 14765 BP, and the 4539bp repeats with 10226bp as invalid were indeed the sequences of M.bovis NM 2012. The 13131bp replaced by the unknown sequence N and the invalid repeat sequence 10226bpwere removed, and the missing sequence 20222bpwas added. The whole genome size of M.bovis NM2012, which was 993483bp, was sequenced by Wang Yanjie. After filling the gap, the whole genome size of M. bovis NM 2012 strain 990348bp.M.bovis NM 2012 was 990348bpand the content of GC% was 29.290.After comparing with M.bovis HB0801, the whole genome size of M. bovis NM 2012 was 990348bpand the content of GC% was 29.29. The predicted coding region contained 839 ORFs, 34 tRNAs, 6 rRNAs, 103 lipoproteins and 2 Vsps. The genomic collinear analysis of 5 strains of Mycoplasma bovis was conducted to compare the similarity between M. bovis NM 2012 genome and M.bovis PG45 genome, and the highest similarity with M.bovis HB0801 genome.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.62

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