Structure of the tuf gene and its upstream part genes and characteristic analysis of conserved regions and activity from related gene promoters of a phytoplasma
【Abstract】Studies on the genetic diversity, key gene regulation, and control of phytoplasma, which cause many diseases with various host plants and have a wide geographical distribution, will be conducive to facilitating concatenated disease control. The large DNA fragments including tuf gene sequences and upstream six genes from PaWB-sdyz, PaWB-fjfz and LY-fjya1 strains were amplified using long fragment PCR primers. The sequence characteristic of conserved regions of the phytoplasma gene promoter and MLSA were performed. The upstream sequences adjoining the tuf gene were recombined with promoter-probe vector pSUPV4 to analyze their promoter activity. The sequences, 12 745–12 748 bp in length, of upstream tuf genes were amplified from the three strains. The comparative analysis showed that the gene structure order of the tuf gene and its upstream six gene sequences of PaWB-sdyz, PaWB-fjfz, LY-fjya1, OY-M, AYWB, PAa, SLY, AT phytoplasma strains were identical in the arrangement of 5’-rplL-rpoB-rpoC-rps12-rps7-fusA-tuf-3’. The potential sequence pattern of conserved region of the phytoplasma promoter was deduced: T90T100G92T75G67A85 (−35 region); T90A96T92A98T73T90 (−10 region). The different phytoplasma strains were clearly divided with comparatively high bootstrap values based on MLSA of coding genes, non-coding sequences, and deduced amino acid sequences of rplL-tuf nucleotide sequences. Genetic variation was comparatively high in the non-coding nucleotide sequences. A 130-bp upstream sequence of the tuf gene in PaWB-fjfz, LY-fjya1 strains and a 129-bp upstream sequence of the tuf gene in CWB-hnsy1 strain, and three representative strains of three variation types of upstream sequences adjoining the tuf gene from 16SrI group, were tested for promoter activity.
【Keywords】 phytoplasma; promoter; gene structure; genetic diversity; multilocus sequence analysis;
Andersen MT, Liefting LW, Havukkala I, Beever RE (2013) Comparison of the complete genome sequence of two closely related isolates of ‘Candidatus phytoplasma australiense’ reveals genome plasticity. BMC Genomics, 14, 529.
Bai XD, Zhang JH, Ewing A, Miller SA, Radek AJ, Shevchenko DV, Tsukerman K, Walunas T, Lapidus A, Campbell JW, Hogenhout SA (2006) Living with genome instability: The adaptation of phytoplasma to diverse environments of their insect and plant hosts. Journal of Bacteriology, 188, 3682–3696.
Du HT, Zhu HY, Wang JM, Zhao W, Tao XL, Ba CF, Tian YM, Su YH (2014) Single-nucleotide polymorphisms and activity analysis of the promoter and enhancer of the pig lactase gene. Gene, 545, 56–60.
Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39, 783–791.
Ishii Y, Kakizawa S, Hoshi A, Maejima K, Kagiwada S, Yamaji Y, Oshima K, Namba S (2009) In the non-insect transmissible line of onion yellows phytoplasma (OY-NIM), the plasmid-encoded transmembrane protein ORF3 lacks the major promoter region. Microbiology, 155, 2058–2067.
Kube M, Schneider B, Kuhl H, Dandekar T, Heitmann K, Migdoll AM, Reinhardt R, Seemüller E (2008) The linear chromosome of the plant-pathogenic mycoplasma ‘Candidatus phytoplasma mali’. BMC Genomics, 9, 306.
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874.
Lee IM, Hammond RW, Davis RE, Gundersen DE (1993) Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma like organisms. Phytopathology, 83, 834–842.
Li MG (2004) Advanced Molecular Genetics. Science Press, Beijing (in Chinese).
Liu Z, Zhang L, Xue C, Fang H, Zhao J, Liu M (2017) Genome-wide identification and analysis of MAPK and MAPKK gene family in Chinese jujube (Ziziphus jujuba Mill.). BMC Genomics, 18, 855.
Miyata S, Furuki K, Oshima K, Sawayanagi T, Nishigawa H, Jung HY, Ugaki M, Namba S (2002a) Complete nucleotide sequence of the S10-spc operon of phytoplasma: Gene organization and genetic code resemble those of Bacillus subtilis. DNA and Cell Biology, 21, 527–534.
Miyata S, Furuki K, Sawayanagi T, Oshima K, Kuboyama T, Tsuchizaki T, Ugaki M, Namba S (2002b) Gene arrangement and sequence of str operon of phytoplasma resemble those of Bacillus more than those of Mycoplasma. Journal of General Plant Pathology, 68, 62–67.
Oshima K, Kakizawa S, Nishigawa H, Jung HY, Wei W, Suzuki S, Arashida R, Nakata D, Miyata S, Ugaki M, Namba S (2004) Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nature Genetics, 36, 27–29.
Post LE, Nomura M (1980) DNA sequences from the str operon of Escherichia coli. The Journal of Biological Chemistry, 255, 4660–4666.
Ren ZG (2014) The Qualitative and Quantitative Detection and Identification of Several Important Phytoplasmas in China. Postdoctoral Research Report, Chinese Academy of Forestry, Beijing (in Chinese with English abstract).
Salehi M, Hosseini SAE, Salehi E, Bertaccini A (2017) Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in Iran. Folia Microbiologica, 62, 99–109.
Sanangelantoni AM, Tiboni O (1993) The chromosomal location of genes for elongation factor Tu and ribosomal protein S10 in the cyanobacterium Spirulina platensis provides clues to the ancestral organization of the str and S10 operons in prokaryotes. Journal of General Microbiology, 139, 2579–2584.
Tran-Nguyen LTT, Kube M, Schneider B, Reinhardt R, Gibb KS (2008) Comparative genome analysis of ‘Candidatus phytoplasma australiense’ (subgroup tuf-Australia I; rp-A) and ‘Ca. phytoplasma asteris’ strains OY-M and AY-WB. Journal of Bacteriology, 190, 3979–3991.
Turner PC (translated by Liu JY, Liu WY) (2010) Molecular Biology, 3rd edn. Science Press, Beijing (in Chinese).
Wang SJ (2017) Establishment of Molecular Detection Technology and Genetic Diversity Analysis of Plant Phytoplasma in China. PhD dissertation, Chinese Academy of Forestry, Beijing (in Chinese with English abstract).
Yang YH (2008) Molecular Biology of the Gene. Higher Education Press, Beijing (in Chinese).
Yu SS, Xu QC, Lin CL, Wang SJ, Tian GZ (2016a) Genetic diversity of phytoplasmas: Research status and prospects. Biodiversity Science, 24, 205–215 (in Chinese with English abstract).
Yu SS, Lin CL, Pan J, Ren ZG, Piao CG, Wang LF, Guo MW, Tian GZ (2016b) Comparative analysis of structure, function and genetic variation of upstream sequences adjoining tuf gene in paulownia and jujube witches’-broom phytoplasmas. Microbiology China, 43, 1060–1069 (in Chinese with English abstract).
Zhang Y, Han Q, Li C, Li W, Fan H, Xing Q, Yan B (2014) Genetic analysis of the TBX1 gene promoter in indirect inguinal hernia. Gene, 535, 290–293.
Zhu YX, Li Y (2002) Modern Molecular Biology, 2nd edn. Higher Education Press, Beijing (in Chinese).
Zurawski G, Zurawski SM (1985) Structure of the Escherichia coli S10 ribosomal protein operon. Nucleic Acids Research, 13, 4521–4526.