Journal of Integrative Agriculture ›› 2021, Vol. 20 ›› Issue (6): 1570-1584.DOI: 10.1016/S2095-3119(20)63247-6

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  • 收稿日期:2020-02-13 出版日期:2021-06-01 发布日期:2021-05-19

Identification of genes involved in regulating MnSOD2 production and root colonization in Bacillus cereus 905

GAO Tan-tan1, DING Ming-zheng2, LI Yan2, ZENG Qing-chao2, WANG Qi2    

  1. 1 Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs, Beijing University of Agriculture, Beijing 102206, P.R.China
    2 Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, P.R.China
  • Received:2020-02-13 Online:2021-06-01 Published:2021-05-19
  • Contact: Correspondence WANG Qi, Tel: +86-10-62731460, E-mail: wangqi@cau.edu.cn
  • About author:GAO Tan-tan, Tel: +86-10-80794280, E-mail: gaotantan0537 @163.com;
  • Supported by:
    This work was supported by the grants from the National Natural Science Foundation of China (31701860) and the Program of Science and Technology of Beijing, China (Z191100004019025).

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摘要：

利用转座子TnYLB-1随机插入技术，构建了蜡样芽胞杆菌905中全基因组范围内的突变体库；通过流式细胞仪和实时荧光定量PCR（qRT-PCR）筛选调控sodA2转录的基因；通过测定细胞中的SOD活性，筛选可以调控MnSOD产量的基因；利用反向PCR技术、基因测序和NCBI数据库搜索得到TnYLB-1插入的基因序列；通过盆栽试验和细菌平板回收技术检测小麦根部不同蜡样芽胞杆菌菌群的数量。【结果】本研究最终获得了7个TnYLB-1转座子插入的突变体菌株，它们在mRNA和蛋白水平上改变sodA2基因的表达。序列分析和BLAST数据比对显示，在蜡样芽胞杆菌905菌株中被TnYLB-1插入的基因与模式菌株蜡样芽胞杆菌ATCC 14579菌株中的同源基因具有高度的保守性。这些基因编码热激诱导型转录抑制因子、氯离子通道蛋白、重组酶A、亚铁离子转运蛋白、核苷二磷酸激酶和组氨酸脱氨酶。此外，本研究实验数据证明，调控sodA2表达的基因同样影响蜡样芽胞杆菌905在小麦根部的定殖能力。负调控sodA2表达的基因显著降低细菌在小麦根部的定殖能力，正调控sodA2表达的基因在一定程度上可以提高细菌在小麦根部的菌群数量。hrcA、clc和recA基因正调控MnSOD2的产量和蜡样芽胞杆菌905在小麦根部的定殖能力；feoB1、feoB2、ndk和hutH基因负调控MnSOD2的产量，并且负调控蜡样芽胞杆菌905在小麦根部的定殖能力。本研究首次鉴定了蜡样芽胞杆菌905中7个调控sodA2基因表达的新基因，并且证实了其可以影响细菌在小麦根部的定殖能力；本工作为研究蜡样芽胞杆菌905在小麦根部的定殖机制指明了研究方向，并为提高PGPR菌株的生物防治效果提供了潜在的有效策略。


Abstract:

sodA2-encoding manganese-containing superoxide dismutase (MnSOD2) in Bacillus cereus 905 plays an essential role in antioxidative stress, nutrition utilization, rhizosphere and phyllosphere colonization.  However, the genes involved in regulating the sodA2 expression have not been clearly elucidated in B. cereus.  In this study, a genome-wide random insertion mutagenesis was constructed by using transposon TnYLB-1 to identify novel genes regulating the sodA2 expression.  Seven mutants that changed the sodA2 expression at both mRNA and protein levels were finally obtained.  Sequence analysis and BLAST data showed that the genes disrupted by TnYLB-1 in B. cereus 905 shared high conservations with those in the B. cereus type strain, ATCC 14579.  These genes encode heat-inducible transcription repressor, chloride channel protein, recombinase A, ferrous iron transport protein, nucleoside diphosphate kinase, and histidine ammonia-lyase.  Besides, we also provided the evidence that the genes regulating the sodA2 expression could influence colonization ability of B. cereus 905 on wheat roots.  Specifically, those genes downregulating the sodA2 expression significantly reduced bacterial colonization on wheat roots, and mutants with increased MnSOD2 activities could enhance bacterial population densities on wheat roots to a certain degree.  Our work provided information that multiple genes are involved in MnSOD2 production and wheat root colonization.  The molecular regulatory pathways through which these genes modulate the sodA2 expression and root colonization need to be investigated extensively in the future.

Key words: Bacillus cereus ,  sodA2 ,  TnYLB-1 transposon ,  colonization