【摘要】：低能離子注入生物體技術(shù)是近些年來發(fā)展起來的物理誘變育種技術(shù),該技術(shù)一般采用活性氮離子,注入生物樣品后摻入靶分子形成新物質(zhì),引起生物體的變異,從而引發(fā)生物學效應(yīng)。為了進一步理解低能N+注入介導外源基因轉(zhuǎn)化沙漠寡營養(yǎng)細菌(DOB)引發(fā)的rRNA基因的突變情況,本研究在DOB基因組De novo測序的基礎(chǔ)上應(yīng)用生物信息學方法分析了3株離子束重組DOB菌株DOB073、DOB113、DOB981與原始菌株DOB150的rRNA基因序列,并對其rRNA基因的突變進行了研究。通過從DOB基因組De novo測序數(shù)據(jù)中獲取這些DOB菌株的5srRNA、16S rRNA、23srRNA的基因序列,研究其基因突變的基本規(guī)律,分析基因突變的具體位點,統(tǒng)計其拷貝數(shù)、GC含量、計算進化距離和分子鐘,進而構(gòu)建進化樹,并分析基因的進化程度,尋找并分析比對結(jié)果中的保守區(qū)域。rRNA基因拷貝數(shù)分析結(jié)果表明:重組突變菌DOB073的5srRNA和16s rRNA基因拷貝數(shù)分別比原始菌株DOB150增加了14和5個;重組突變菌DOB113的16s rRNA基因拷貝數(shù)比原始菌株DOB150增加了5個;重組突變菌DOB981的5srRNA基因拷貝數(shù)比原始菌株DOB150減少了1個,而其16s rRNA基因拷貝數(shù)增加了5個;3株重組突變菌株的23s rRNA基因拷貝數(shù)與對照菌株DOB150相同。rRNA基因堿基突變位點的研究結(jié)果顯示,3株重組突變菌的5srRNA基因均在第4、107、112、114處發(fā)生了堿基突變,DOB073和DOB113還在第104處發(fā)生了堿基突變;3株重組突變菌的16srRNA基因的5-6個堿基突變分別分布于C1區(qū)、V1區(qū)、V2區(qū)、V4區(qū)、V6區(qū)、V7和V9區(qū);3株重組突變菌的23srRNA基因的保守性較強,只有2-3個堿基位點發(fā)生了突變�；蜻M化分析結(jié)果顯示,重組突變菌株DOB073和DOB113的5srRNA基因和23srRNA基因的進化速度均快于重組突變菌株DOB981。而DOB981的16srRNA基因的進化速度快于DOB073和DOB113。rRNA基因序列的保守二級結(jié)構(gòu)的研究結(jié)果表明:三株重組突變菌株的16srRNA基因和原始菌株的16srRNA基因均具有9個保守二級結(jié)構(gòu),其堿基位置分別為:80-120,120-240,240-360,360-480,400-520,640-760,760-880,800-920和1420-1540。而三株重組突變菌株的23srRNA基因和原始菌株的23srRNA基因都均具有13個保守二級結(jié)構(gòu),其處堿基位置分別為:40-160,80-200,120-240,720-840,1200-1320,1480-1600,1520-1640,2120-2240,2200-2320,2240-2360,2280-2400,2360-2480,2520-2640。通過對3株重組突變DOB菌株的3種rRNA基因的研究,從分子水平上揭示了離子注入介導DOB菌株rRNA基因突變與進化的分子機制,也為離子注入介導獲得的DOB的多樣性提供了理論和實踐依據(jù),特別是是為低能離子注入介導的原核微生物的進化提供了直接的分子證據(jù)。
[Abstract]:Low energy ion implantation is a physical mutagenic breeding technique developed in recent years. Thus triggering biological effects. In order to understand the mutation of rRNA gene induced by oligonutrient desert bacteria (DOB) mediated by low energy N injection, On the basis of DOB genomic De novo sequencing, the rRNA gene sequences of three ion-beam recombinant DOB strains DOB073,DOB113,DOB981 and the original DOB150 were analyzed by bioinformatics, and their rRNA gene mutations were studied. The gene sequence of 5srRNA-16s rRNA,23srRNA of these DOB strains was obtained from DOB genomic De novo sequencing data, the basic rules of gene mutation were studied, the specific sites of gene mutation were analyzed, and the copy number and GC content were counted. Calculate the evolutionary distance and molecular clock, and then build the evolutionary tree, and analyze the degree of evolution of the gene. The copy number analysis of rRNA gene showed that the copy number of 5srRNA gene and 16s rRNA gene of recombinant mutant DOB073 were 14 and 5 more than that of the original strain DOB150, respectively. The copy number of 16s rRNA gene of recombinant mutant DOB113 was 5 more than that of original strain DOB150, the copy number of 5srRNA gene of recombinant mutant DOB981 was 1 less than that of original strain DOB150, and the copy number of 16s rRNA gene increased by 5. The copy number of 23s rRNA gene of three recombinant mutant strains was the same as that of control strain DOB150. The results of the study on the base mutation site of rRNA gene showed that the 5srRNA gene of the three recombinant mutants had a base mutation at 4107112114. DOB073 and DOB113 also had base mutation at 104th. The 5-6 base mutations of 16srRNA gene of the three recombinant mutant strains were located in C1 region, V1 region, V2 region, V4 region, V6 region, V7 and V9 region, respectively. The 23srRNA gene of the three recombinant mutants was highly conserved, and only 2-3 base loci were mutated. The results of gene evolution analysis showed that the 5srRNA gene and 23srRNA gene of the recombinant mutant DOB073 and DOB113 were both faster than that of the recombinant mutant DOB981.. The results showed that the 16srRNA gene of the three recombinant mutant strains and the 16srRNA gene of the original strain had 9 conserved secondary structures, while the evolution rate of the 16srRNA gene of DOB981 was faster than that of the conserved secondary structure of the DOB073 and DOB113.rRNA gene sequences. Their base positions are 80-120120-240240-360360-480400-520640-760760-880800-920 and 1420-1540 respectively. The 23srRNA gene of the three recombinant mutants and the 23srRNA gene of the original strain all had 13 conserved secondary structures, and their base positions were as follows: 40-160 (80-200120-240720-84040) 1200-1320 (1480-1600) 1520-164040 (2120-2240) 2200-2320 (2240-23602280-2400) 2360-2480 (2520-2640). Through the study of three rRNA genes of three recombinant mutant DOB strains, the molecular mechanism of rRNA gene mutation and evolution mediated by ion implantation was revealed at the molecular level. It also provides a theoretical and practical basis for the diversity of DOB mediated by ion implantation, especially provides direct molecular evidence for the evolution of prokaryotic microorganisms mediated by low energy ion implantation.
【學位授予單位】：新疆大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78

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