枯草芽孢杆菌OKB105菌株突变体文库的构建及促生相关基因的克隆和功能研究
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摘要
枯草芽孢杆菌(Bacillus subtilis)是一类重要的植物根围促生细菌(Plant growth-promoting rhizobacteria, PGPR),能产生抗逆性强的芽孢和多种抗菌物质,还能促进植物的生长。目前,在国内外已有很多商品化的枯草芽孢杆菌生防制剂。枯草芽孢杆菌的促生和防病作用的发挥首先依赖于它们在植物根部的定殖能力,但是其定殖和促进植物生长的分子机理尚不清楚。本研究通过转座子随机诱变技术,构建了枯草芽孢杆菌突变体文库,并从中筛选促进植物生长缺陷型的突变体,克隆与枯草芽孢杆菌促生相关的基因,验证和研究这些基因的促生相关功能。
目前,转座子随机诱变技术已成为发现新基因、发掘已知基因新功能的有效途径之一。Himar1转座元件,是在细菌诱变中应用最广泛的mariner家族转座元件之一,与其它转座子相比,具有转座频率高、插入位点随机性高,以及寄主范围广泛的优点。枯草芽孢杆菌OKB105菌株,对防治辣椒病毒病和烟草花叶病均有较好的效果,能显著促进植物生长,并且能诱导植物促生相关基因的表达。因此,本研究以OKB105菌株为研究对象,利用基于Himar1转座元件所构建转座子TnYLB-1,构建了包涵2000个突变体的突变体文库,并利用PCR及southern杂交的方法对随机挑选的15个突变体进行了验证。实验结果表明：pMarA质粒已被成功地转化入OKB105菌株中,并且在高温诱导下转座子TnYLB-1能够以单拷贝、随机插入到枯草芽孢杆菌OKB105基因组中。
我们以基于Himar1转座诱变体系所构建的枯草芽孢杆菌OKB105突变体文库为基础实验材料,从中筛选得到7个促烟草根生长缺陷型的突变体,实验结果表明,OKB105菌株的发酵上清液能明显地促进烟草根的生长,根长增加约55.9%,而7个突变体的发酵上清液对烟草根的生长几乎没有促进效果。反向PCR对转座子插入位点的分析显示,7个促生相关基因分别与硫氧还原蛋白还原酶、丙酮酸羧化酶、跨膜糖蛋白、带有硫氰酸酶结构域的酶、7-甲基鸟苷转甲基酶、自溶酶LytC的修复蛋白和寡聚-1,6-葡萄糖苷酶的编码、合成相关,影响枯草芽孢杆菌的细胞膜生物功能及糖代谢等途径。
在OKB105菌株突变体文库的筛选过程中,我们得到了一个促生长缺陷型的突变体M3,其中yfnI基因被转座子TnYLB-1随机插入诱变。本研究采用构建穿梭载体pMK4-yfnI的方法使M3中yfnl基因功能得到回复,并发现该基因的功能回复转化子M3Δyfnl能显著地促进烟草根的生长,与野生型OKB105的促生效果相似,烟草主根长度都增加约65%,证明了yfnl基因与OKB105的促植物生长特性相关。通过对M3生长能力的检测发现,相同的培养条件下,M3和野生型OKB105在LB及Landy培养基中的生长情况都基本一致,证明M3的促生缺陷不是由于影响菌株生长而引起的。本研究还定量检测了M3中三个芽孢杆菌促生相关基因alsD、yhcX和ysnE相对于野生型OKB105的表达水平,Quantative RT-PCR分析结果显示,三个促生相关基因在野生型OKB105与M3中的相对表达量没有显著差异,证明yfnI基因的诱变不会引起OKB105中已知的芽孢杆菌促生相关基因表达水平的变化。因此,推测yfnI基因是通过某种未知机制来调控OKB105的促生功能。
Bacillus subtilis, as an important type of plant growth-promoting rhizobacteria (PGPR), not only has broad-spectrum antimicrobial activity and stress resistance, but also can significantly promote plant growth by root application. Although the advantages of plant disease biocontrol by B. subtilis is more and more obvious now, the molecular mechanisms of colonizing in plant rhizosphere and promoting plant growth of B. subtilis have not been understood. This research was aimed at identifying the genes involved in promoting plant growth using the Bacillus subtilis OKB105 mutant library and studying the functions of these relative genes.
At present, the ability to create random DNA insertions in bacterial chromosomes has been a very powerful technique for discovering new genes and discovering new functions of known genes. The transposable element Himarl, a member of the mariner family of transposons, has been extensively used to generate libraries of mutants in many bacteria. When compared to other transposons, Himarl transposon has been shown to insert randomly into the chromosomes of a number of bacterial species with high transposition frequency, and has a broad host range. B. subtilis OKB105, a derivative strain of B. subtilis 168, can protect plants against CMV and TMV, and significantly enhanced the plant height and fresh weight by enhancing the expression of plant growth-related genes, In this study, insertional mutagenesis of B. subtilis OKB105 was performed by use of a Himarl-based transposon TnYLB-1, and 2000 transposants were selected randomly. PCR and southern hybridization analyses of 15 transposants showed that the plasmid pMarA carrying the transposon TnYLB-1 could transform into OKB105 successfully, and TnYLB-1 transposed into the B. subtilis chromosome randomly under the condition of high temperature.
7 mutants defective in promoting plant growth were selected from the Bacillus subtilis OKB105 TnYLB-1 mutant library. The results of square Petri dishes experiments demonstrated that the supernatant liquid of B. subtilis OKB105 could significantly promote the growth of tobacco roots, the root growth had been increasd by 55.9%. In contrary, supernatant liquid of the 7 mutants did not have a positive effect on the root growth of tobacco. To identify the genes in these mutant loci, the inserted transposon and its flanking regions were cloned by inverse PCR and sequenced from each mutant. Transposon insertions in these seven mutants were found to be in the gene trxB, pycA, yfnl, ybfQ, rsmG, ycdG, lytB. The functions of these genes are respectively relative to encoding the proteins such as thioredoxin reductase, pyruvate carboxylase, transmembrane glycoprotein, enzyme with rhodanese domain, glucose-inhibited division protein, modifier protein of major autolysin LytC, oligo-1,6-glucosidase.These genes may have effects on biological function of cell membrane and glucose metabolism of B. subtilis.
M3, a mutant defective in promoting plant growth, was obtained by screening the TnYLB-1 transposon mutant library of B. subtilis OKB105. Inverse PCR demonstrated that the insertion site of M3 was gene yfnI. In this study, a shuttle vector pMK4-yfnI was constructed and transformed into M3 to restore the function of yfnl gene. The complementation experiment showed that the transformant M3ΔyfnI had the same effect with OKB105 on promoting the growth of tobacco roots, the root growth had been increasd by 65%. The results verified that yfnl was significantly correlated with promoting plant growth of B. subtilis OKB105. The result of growth curve determination of M3 and OKB105 showed that M3 had the same growth ability with OKB105 in both LB medium and Landy medium. It was concluded that the growth of B. subtilis OKB105 was not be affected if yfnl had been mutated. In the present study, there are there genes alsD, yhcX and ysnE relative to promoting plant growth of B. subtilis OKB105. Quantative RT-PCR analysis demonstrated that in M3 the expression level of these three genes showed no significant change compared to wild type OKB105, confirming that the disrupted gene has no effect on the transcription of the three genes. Finally, it was assumed that gene yfnl could regulate the ability of promoting plant growth of B. subtilis OKB 105 according to an unknown mechanism.
目前,转座子随机诱变技术已成为发现新基因、发掘已知基因新功能的有效途径之一。Himar1转座元件,是在细菌诱变中应用最广泛的mariner家族转座元件之一,与其它转座子相比,具有转座频率高、插入位点随机性高,以及寄主范围广泛的优点。枯草芽孢杆菌OKB105菌株,对防治辣椒病毒病和烟草花叶病均有较好的效果,能显著促进植物生长,并且能诱导植物促生相关基因的表达。因此,本研究以OKB105菌株为研究对象,利用基于Himar1转座元件所构建转座子TnYLB-1,构建了包涵2000个突变体的突变体文库,并利用PCR及southern杂交的方法对随机挑选的15个突变体进行了验证。实验结果表明：pMarA质粒已被成功地转化入OKB105菌株中,并且在高温诱导下转座子TnYLB-1能够以单拷贝、随机插入到枯草芽孢杆菌OKB105基因组中。
我们以基于Himar1转座诱变体系所构建的枯草芽孢杆菌OKB105突变体文库为基础实验材料,从中筛选得到7个促烟草根生长缺陷型的突变体,实验结果表明,OKB105菌株的发酵上清液能明显地促进烟草根的生长,根长增加约55.9%,而7个突变体的发酵上清液对烟草根的生长几乎没有促进效果。反向PCR对转座子插入位点的分析显示,7个促生相关基因分别与硫氧还原蛋白还原酶、丙酮酸羧化酶、跨膜糖蛋白、带有硫氰酸酶结构域的酶、7-甲基鸟苷转甲基酶、自溶酶LytC的修复蛋白和寡聚-1,6-葡萄糖苷酶的编码、合成相关,影响枯草芽孢杆菌的细胞膜生物功能及糖代谢等途径。
在OKB105菌株突变体文库的筛选过程中,我们得到了一个促生长缺陷型的突变体M3,其中yfnI基因被转座子TnYLB-1随机插入诱变。本研究采用构建穿梭载体pMK4-yfnI的方法使M3中yfnl基因功能得到回复,并发现该基因的功能回复转化子M3Δyfnl能显著地促进烟草根的生长,与野生型OKB105的促生效果相似,烟草主根长度都增加约65%,证明了yfnl基因与OKB105的促植物生长特性相关。通过对M3生长能力的检测发现,相同的培养条件下,M3和野生型OKB105在LB及Landy培养基中的生长情况都基本一致,证明M3的促生缺陷不是由于影响菌株生长而引起的。本研究还定量检测了M3中三个芽孢杆菌促生相关基因alsD、yhcX和ysnE相对于野生型OKB105的表达水平,Quantative RT-PCR分析结果显示,三个促生相关基因在野生型OKB105与M3中的相对表达量没有显著差异,证明yfnI基因的诱变不会引起OKB105中已知的芽孢杆菌促生相关基因表达水平的变化。因此,推测yfnI基因是通过某种未知机制来调控OKB105的促生功能。
Bacillus subtilis, as an important type of plant growth-promoting rhizobacteria (PGPR), not only has broad-spectrum antimicrobial activity and stress resistance, but also can significantly promote plant growth by root application. Although the advantages of plant disease biocontrol by B. subtilis is more and more obvious now, the molecular mechanisms of colonizing in plant rhizosphere and promoting plant growth of B. subtilis have not been understood. This research was aimed at identifying the genes involved in promoting plant growth using the Bacillus subtilis OKB105 mutant library and studying the functions of these relative genes.
At present, the ability to create random DNA insertions in bacterial chromosomes has been a very powerful technique for discovering new genes and discovering new functions of known genes. The transposable element Himarl, a member of the mariner family of transposons, has been extensively used to generate libraries of mutants in many bacteria. When compared to other transposons, Himarl transposon has been shown to insert randomly into the chromosomes of a number of bacterial species with high transposition frequency, and has a broad host range. B. subtilis OKB105, a derivative strain of B. subtilis 168, can protect plants against CMV and TMV, and significantly enhanced the plant height and fresh weight by enhancing the expression of plant growth-related genes, In this study, insertional mutagenesis of B. subtilis OKB105 was performed by use of a Himarl-based transposon TnYLB-1, and 2000 transposants were selected randomly. PCR and southern hybridization analyses of 15 transposants showed that the plasmid pMarA carrying the transposon TnYLB-1 could transform into OKB105 successfully, and TnYLB-1 transposed into the B. subtilis chromosome randomly under the condition of high temperature.
7 mutants defective in promoting plant growth were selected from the Bacillus subtilis OKB105 TnYLB-1 mutant library. The results of square Petri dishes experiments demonstrated that the supernatant liquid of B. subtilis OKB105 could significantly promote the growth of tobacco roots, the root growth had been increasd by 55.9%. In contrary, supernatant liquid of the 7 mutants did not have a positive effect on the root growth of tobacco. To identify the genes in these mutant loci, the inserted transposon and its flanking regions were cloned by inverse PCR and sequenced from each mutant. Transposon insertions in these seven mutants were found to be in the gene trxB, pycA, yfnl, ybfQ, rsmG, ycdG, lytB. The functions of these genes are respectively relative to encoding the proteins such as thioredoxin reductase, pyruvate carboxylase, transmembrane glycoprotein, enzyme with rhodanese domain, glucose-inhibited division protein, modifier protein of major autolysin LytC, oligo-1,6-glucosidase.These genes may have effects on biological function of cell membrane and glucose metabolism of B. subtilis.
M3, a mutant defective in promoting plant growth, was obtained by screening the TnYLB-1 transposon mutant library of B. subtilis OKB105. Inverse PCR demonstrated that the insertion site of M3 was gene yfnI. In this study, a shuttle vector pMK4-yfnI was constructed and transformed into M3 to restore the function of yfnl gene. The complementation experiment showed that the transformant M3ΔyfnI had the same effect with OKB105 on promoting the growth of tobacco roots, the root growth had been increasd by 65%. The results verified that yfnl was significantly correlated with promoting plant growth of B. subtilis OKB105. The result of growth curve determination of M3 and OKB105 showed that M3 had the same growth ability with OKB105 in both LB medium and Landy medium. It was concluded that the growth of B. subtilis OKB105 was not be affected if yfnl had been mutated. In the present study, there are there genes alsD, yhcX and ysnE relative to promoting plant growth of B. subtilis OKB105. Quantative RT-PCR analysis demonstrated that in M3 the expression level of these three genes showed no significant change compared to wild type OKB105, confirming that the disrupted gene has no effect on the transcription of the three genes. Finally, it was assumed that gene yfnl could regulate the ability of promoting plant growth of B. subtilis OKB 105 according to an unknown mechanism.
引文
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