离子束和高压电场诱变大肠杆菌K12的实验研究
摘要
近年来,在国际上,人们对转基因物种的担忧越来越严重,因而传统的物理因子生物效应,特别是诱变育种研究被更多学者重新关注起来。电场,离子束是比较常见的物理诱变因子,经过几十年的研究,取得了令人瞩目的成就,然而,一些诱变机理性的课题还需进一步深入研究。
电场对生物体的效应是可遗传的,还是当代效应?或者说电场有没有诱变效应,一直以来科学界都不置可否,未有定论,本文以模式生物大肠杆菌K12为研究对象,首先考察了几种平板电场对野生大肠杆菌K12的诱变率。结果发现高压直流、交流、半波整流平板电场处理大肠杆菌K12,3种电场在低剂量处理时(1.5kV/cm时)都表现为对大肠杆菌K12的刺激效应,随着电场剂量增大,3种电场对大肠杆菌K12逐步变为抑制效应。其中直流平板电场的效应最为明显。交流和半波整流平板电场对大肠杆菌K12诱变率变化不大,均未达到对照组的2倍。直流平板电场在4.5kV/cm时,突变率达到极大值5.8×10-6,是对照组的2.32倍。所以,我们认为,高压直流、交流、半波整流平板电场对大肠杆菌具有一定的诱变效应,但是其诱变效应不明显。随后,我们用场强为1,2,3,4kV/cm的高压芒刺电场处理大肠杆菌10分钟,在场强为1kV/cm时,突变率为31.2×10-6,分别是自发突变(1.2×10-6)和对照(2.5×10-6)的26倍和12倍。此结果表明高压芒刺电场是一种损伤低、突变率高的很好的诱变剂。碱基置换是高压芒刺电场处理组的主要类型突变,30%碱基置换属于G:C→A:T转换,70%为A:T→T:A,G:C→T:A,A:T→C:G,G:C→C:G颠换。与自发突变相比,电场处理组中的碱基置换、碱基插入、碱基缺失的增加很明显。同时在突变热点处5'→TGGC-3'的缺失/增加从82%下降到26%。但此位点的绝对突变率有所增加。高压芒刺电场组还发现了自发突变组中没有发现的A:T→T:A的颠换和280bp的大片段缺失,同时还有G:C→A:T的增加。这表明高压芒刺静电场处理组的突变谱与SOS反应产生的突变谱有明显相似的地方,本文认为高压芒刺静电场对大肠杆菌的诱变效应是由于电场作用引起大肠杆菌SOS反应而导致的；高压芒刺静电场诱发lacI的突变谱中有一个长达280bp的大片段缺失突变,这在以前的研究中是没有报道过的,同时从单碱基插入缺失以及多碱基插入缺失的发生比例也可以看出,细菌基因组进化偏爱于删除。
低能离子束生物技术是上世纪80年代我国科学家发现的,具有损伤轻、突变率高、突变谱宽的优点,被用于植物和微生物育种,然而通过这些年的研究,也发现离子束的生物效应在某些物种上在后代遗传中丢失,即遗传不稳定。同时其诱变机制还不太明确。本文用keV氮离子重复注入诱变大肠杆菌K12,发现离子束重复注入可以增加突变菌的遗传稳定性,同时离子束多次重复注入诱变与一次诱变相比产生的突变型可能更为广泛。从中筛选了离子注入多次诱变,基于lacI基因的稳定遗传大肠杆菌K12突变菌S55,运用lllumina全基因组测序技术对S55进行测序,得到S55的精细结构图谱,与参考序列进行比对发现共有18个SNP,2个Indel,9个大片段Deletion。18个SNP中11个是A,T,C等三种碱基变成G。碱基颠换占55.6%,碱基转换占44.4%。2个Indel中,+GCCA发生在突变筛选目标基因lacI基因上。4个SNP (3SNPs发生在rlpB基因上,1个发生在ygbN基因上)所在基因与生物膜及输运有关,这些基因的突变可能有助于增强大肠杆菌对离子注入刻蚀损伤的适应能力。S55中,所有的9个结构变异都属于碱基对删除类型,每个删除片段都大于1000bp,并且均包含插入序列。其中6个是属于插入序列插入引起的非功能化的假基因,1个为含插入序列的长度为23252bp的Rac噬菌体区。本实验结果说明,大肠杆菌基因组进化中的deletion bias现象皆与基因组中非功能区的丢失有关。这些插入序列也是基因组中的非稳定因素,离子重复注入引起这些片段的删除有利于大肠杆菌突变菌S55基因组的稳定性。
Accompanying the increasing worries about transgenic species the bioeffects of traditional physical factors, especially the mutational breeding of physical factors attract more and more attentions in the international circle of scientists. Electric field and low energy ion beam as two common mutagenic factors, after decades of research, have gained outstanding achievements; however, the biological mechanisms of such two mutagenic factors remain unclear.
Can the bioeffects of electric field be hereditable? In other words, does electric field have mutagenic effects? There was a noncommittal answer on this question in scientists. This paper uses the model organism E.coli as a research object. Firstly, we studied the mutation rate of several plate electric field. Results indicated that both the high voltage DC, AC, and half-wave rectification electric fields all induced stimulative effects at low dosage, and with the dosage increasing these three plate electric fields induced inhibitory effects. Among three electric fields, the most evident effects were induced by DC plate electric field. At the field intensity of4.5kV/cm, the mutation rate reaches maximum.5.8×10-6, which is2.32times more than the controls. So, although these three plate electric field have mutagenic effects on E.coli, but the mutagenic effects seem not obvious. However, the case is different for prickle electrostatic field. Escherichia coli K12W3110was treated by high voltage prickle electrostatic field(HVPEF)10min with the field intensity of1,2,3and4kV/cm respectively. The mutant frequency reached31.2×10-6at field intensity of1kV/cm, which was about26-fold over the background (1.2×10-6) and12-fold over the controls (2.5×10-6). The result indicated that the HVPEF could act as an effective mutagen. It was observed that the base pair (bp) substitutions were the main type of HVPEF induced mutations. All types of base pair substitutions were observed,30%of the bp substitutions were G:C→A:T transitions, and70%of the bp substitutions were A:T→T:A, G:C→T:A, A:T→C:G, G:C→C:G transversions. As compared with the spontaneous group, the base-pair substitutions, deletions and additions of the HVPEF exposure group were significantly increased. Although the deletion/addition of a5'-TGGC-3'sequence at hot spot was decreased from82%to26%, the remarkable increase in absolute Mutation Frequency (MF) of the deletion or the addition of a5'-TGGC-3'sequence at hot spot in the HVPEF exposure group suggested that HVPEF did induce the mutations of this type. The spectra of our HVPEF exposure group with increasing G:C to A:T transitions and A:T to T:A transversions show their similarity with SOS response and indicate that the HVPEF exposure may induce SOS response. On the other hand, from the occurrence of large deletion fragment of of280bp and the high base pair deletion/insertion ratio, we find that the bacterial genome evolution is biased to deletion.
Low-energy ion beam biotechnology was developed in the1980s by Chinese scientists. Low-energy ion beam can cause strong mutagenesis in microbes and plants seeds and has been introduced as a useful tool for microbial and plant breeding. However the specificity and mechanism on the biological effect of low-energy ion beam remains unclear. Wild type Escherichia coli K12strain W3110was irradiated by10keV nitrogen ions. It was found that the multiple ion irradiations can increase genetic stability of mutants and generate a wider mutational spectrum. Through irradiation by N+ions six times and selection of lac constitutive mutants after each time a stable mutant S55was obtained. By sequencing the whole genome the fine map of S55was completed. Compared with reference sequences, a total of eighteen single nucleotide polymorphisms (SNPs), two insertions and deletions (Indels) and nine structural variations (SVs) were found in S55genome. Among18SNPs,11are transversional from A, T, or C to G, accounted for55.6%of point mutations, and7transitional accounted for44.4%. GCCA insertion occurs in the target gene lacI. Four SNPs (three SNPs in rlpB and one in ygbN) are connected with cell envelope and transport. The mutation of these genes possibly makes Escherichia coli genome more adapted to the etch and damage caused by ion irradiation. All nine structural variations of S55are of deletion type. Each of the deleted regions is longer than1000bp and contains insertion sequence (IS) elements. Six deleted SVs contain IS disrupted, nonfunctional pseudogenes and another one of length23252bp is the Rac prophage region. The result shows that the deletion bias observed in the Escherichia coli K12genome evolution is generally related to the deletion of some non-functional regions. Furthermore, since ISs are instable factors in a genome, the multiple ion irradiation that causes these fragments deleted in S55is beneficial to the genome stability.
电场对生物体的效应是可遗传的,还是当代效应?或者说电场有没有诱变效应,一直以来科学界都不置可否,未有定论,本文以模式生物大肠杆菌K12为研究对象,首先考察了几种平板电场对野生大肠杆菌K12的诱变率。结果发现高压直流、交流、半波整流平板电场处理大肠杆菌K12,3种电场在低剂量处理时(1.5kV/cm时)都表现为对大肠杆菌K12的刺激效应,随着电场剂量增大,3种电场对大肠杆菌K12逐步变为抑制效应。其中直流平板电场的效应最为明显。交流和半波整流平板电场对大肠杆菌K12诱变率变化不大,均未达到对照组的2倍。直流平板电场在4.5kV/cm时,突变率达到极大值5.8×10-6,是对照组的2.32倍。所以,我们认为,高压直流、交流、半波整流平板电场对大肠杆菌具有一定的诱变效应,但是其诱变效应不明显。随后,我们用场强为1,2,3,4kV/cm的高压芒刺电场处理大肠杆菌10分钟,在场强为1kV/cm时,突变率为31.2×10-6,分别是自发突变(1.2×10-6)和对照(2.5×10-6)的26倍和12倍。此结果表明高压芒刺电场是一种损伤低、突变率高的很好的诱变剂。碱基置换是高压芒刺电场处理组的主要类型突变,30%碱基置换属于G:C→A:T转换,70%为A:T→T:A,G:C→T:A,A:T→C:G,G:C→C:G颠换。与自发突变相比,电场处理组中的碱基置换、碱基插入、碱基缺失的增加很明显。同时在突变热点处5'→TGGC-3'的缺失/增加从82%下降到26%。但此位点的绝对突变率有所增加。高压芒刺电场组还发现了自发突变组中没有发现的A:T→T:A的颠换和280bp的大片段缺失,同时还有G:C→A:T的增加。这表明高压芒刺静电场处理组的突变谱与SOS反应产生的突变谱有明显相似的地方,本文认为高压芒刺静电场对大肠杆菌的诱变效应是由于电场作用引起大肠杆菌SOS反应而导致的；高压芒刺静电场诱发lacI的突变谱中有一个长达280bp的大片段缺失突变,这在以前的研究中是没有报道过的,同时从单碱基插入缺失以及多碱基插入缺失的发生比例也可以看出,细菌基因组进化偏爱于删除。
低能离子束生物技术是上世纪80年代我国科学家发现的,具有损伤轻、突变率高、突变谱宽的优点,被用于植物和微生物育种,然而通过这些年的研究,也发现离子束的生物效应在某些物种上在后代遗传中丢失,即遗传不稳定。同时其诱变机制还不太明确。本文用keV氮离子重复注入诱变大肠杆菌K12,发现离子束重复注入可以增加突变菌的遗传稳定性,同时离子束多次重复注入诱变与一次诱变相比产生的突变型可能更为广泛。从中筛选了离子注入多次诱变,基于lacI基因的稳定遗传大肠杆菌K12突变菌S55,运用lllumina全基因组测序技术对S55进行测序,得到S55的精细结构图谱,与参考序列进行比对发现共有18个SNP,2个Indel,9个大片段Deletion。18个SNP中11个是A,T,C等三种碱基变成G。碱基颠换占55.6%,碱基转换占44.4%。2个Indel中,+GCCA发生在突变筛选目标基因lacI基因上。4个SNP (3SNPs发生在rlpB基因上,1个发生在ygbN基因上)所在基因与生物膜及输运有关,这些基因的突变可能有助于增强大肠杆菌对离子注入刻蚀损伤的适应能力。S55中,所有的9个结构变异都属于碱基对删除类型,每个删除片段都大于1000bp,并且均包含插入序列。其中6个是属于插入序列插入引起的非功能化的假基因,1个为含插入序列的长度为23252bp的Rac噬菌体区。本实验结果说明,大肠杆菌基因组进化中的deletion bias现象皆与基因组中非功能区的丢失有关。这些插入序列也是基因组中的非稳定因素,离子重复注入引起这些片段的删除有利于大肠杆菌突变菌S55基因组的稳定性。
Accompanying the increasing worries about transgenic species the bioeffects of traditional physical factors, especially the mutational breeding of physical factors attract more and more attentions in the international circle of scientists. Electric field and low energy ion beam as two common mutagenic factors, after decades of research, have gained outstanding achievements; however, the biological mechanisms of such two mutagenic factors remain unclear.
Can the bioeffects of electric field be hereditable? In other words, does electric field have mutagenic effects? There was a noncommittal answer on this question in scientists. This paper uses the model organism E.coli as a research object. Firstly, we studied the mutation rate of several plate electric field. Results indicated that both the high voltage DC, AC, and half-wave rectification electric fields all induced stimulative effects at low dosage, and with the dosage increasing these three plate electric fields induced inhibitory effects. Among three electric fields, the most evident effects were induced by DC plate electric field. At the field intensity of4.5kV/cm, the mutation rate reaches maximum.5.8×10-6, which is2.32times more than the controls. So, although these three plate electric field have mutagenic effects on E.coli, but the mutagenic effects seem not obvious. However, the case is different for prickle electrostatic field. Escherichia coli K12W3110was treated by high voltage prickle electrostatic field(HVPEF)10min with the field intensity of1,2,3and4kV/cm respectively. The mutant frequency reached31.2×10-6at field intensity of1kV/cm, which was about26-fold over the background (1.2×10-6) and12-fold over the controls (2.5×10-6). The result indicated that the HVPEF could act as an effective mutagen. It was observed that the base pair (bp) substitutions were the main type of HVPEF induced mutations. All types of base pair substitutions were observed,30%of the bp substitutions were G:C→A:T transitions, and70%of the bp substitutions were A:T→T:A, G:C→T:A, A:T→C:G, G:C→C:G transversions. As compared with the spontaneous group, the base-pair substitutions, deletions and additions of the HVPEF exposure group were significantly increased. Although the deletion/addition of a5'-TGGC-3'sequence at hot spot was decreased from82%to26%, the remarkable increase in absolute Mutation Frequency (MF) of the deletion or the addition of a5'-TGGC-3'sequence at hot spot in the HVPEF exposure group suggested that HVPEF did induce the mutations of this type. The spectra of our HVPEF exposure group with increasing G:C to A:T transitions and A:T to T:A transversions show their similarity with SOS response and indicate that the HVPEF exposure may induce SOS response. On the other hand, from the occurrence of large deletion fragment of of280bp and the high base pair deletion/insertion ratio, we find that the bacterial genome evolution is biased to deletion.
Low-energy ion beam biotechnology was developed in the1980s by Chinese scientists. Low-energy ion beam can cause strong mutagenesis in microbes and plants seeds and has been introduced as a useful tool for microbial and plant breeding. However the specificity and mechanism on the biological effect of low-energy ion beam remains unclear. Wild type Escherichia coli K12strain W3110was irradiated by10keV nitrogen ions. It was found that the multiple ion irradiations can increase genetic stability of mutants and generate a wider mutational spectrum. Through irradiation by N+ions six times and selection of lac constitutive mutants after each time a stable mutant S55was obtained. By sequencing the whole genome the fine map of S55was completed. Compared with reference sequences, a total of eighteen single nucleotide polymorphisms (SNPs), two insertions and deletions (Indels) and nine structural variations (SVs) were found in S55genome. Among18SNPs,11are transversional from A, T, or C to G, accounted for55.6%of point mutations, and7transitional accounted for44.4%. GCCA insertion occurs in the target gene lacI. Four SNPs (three SNPs in rlpB and one in ygbN) are connected with cell envelope and transport. The mutation of these genes possibly makes Escherichia coli genome more adapted to the etch and damage caused by ion irradiation. All nine structural variations of S55are of deletion type. Each of the deleted regions is longer than1000bp and contains insertion sequence (IS) elements. Six deleted SVs contain IS disrupted, nonfunctional pseudogenes and another one of length23252bp is the Rac prophage region. The result shows that the deletion bias observed in the Escherichia coli K12genome evolution is generally related to the deletion of some non-functional regions. Furthermore, since ISs are instable factors in a genome, the multiple ion irradiation that causes these fragments deleted in S55is beneficial to the genome stability.
引文
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