Structures 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 integrated 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. Sequence characteristic of conserved regions of the phytoplasma gene promoter and MLSA were performed. Upstream sequences adjoining the tuf gene was 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. 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: T 90 T 100 G 92 T 75 G 67 A 85 (-35 region); T 90 A 96 T 92 A 98 T 73 T 90 (-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

Accurate and sensitive detection of areca palm yellow leaf phytoplasma in China by droplet digital PCR targeting tuf gene sequence

“…The results showed that the established ddPCR method was reliable for quantitative detection of AYL phytoplasma, with relatively good effect. The tuf gene is a functional protein coding gene and a relatively conserved sequence in the phytoplasma which plays an important role in protein translation and evolution of phytoplasma (Koui et al, 2003; Lai et al, 2014; Lee et al, 2000; Schneider et al, 1997; Yu et al, 2016,b, 2017, 2018). In the study, the tuf gene was used as a diagnostic assay for AYL phytoplasma for the first time.…”

“…AYL1, AYL2 and AYL3 represent AYL samples; H1, H2 and H3 represent asymptomatic samples; ck1 represented TE buffer used for DNA extraction; ck2 represented ddH2O used for ddPCR reaction system. Events represented the number of droplets generated in each reaction Schneider et al, 1997;Yu et al, ,b, 2017Yu et al, , 2018. In the study, the tuf gene was used as a diagnostic assay for AYL phytoplasma for the first time.…”

“…In this study, a ddPCR detection system for AYL phytoplasma belonging to 16SrI group, and associated with AYL disease in Hainan Province of China, was developed based on the tuf gene sequence. The tuf gene is a conserved gene in phytoplasmas and has been widely used in the detection, identification and study of genetic variation of phytoplasmas (Koui, Natsuaki, & Okuda, 2003; Lee, Davis, & Gundersen‐Rindal, 2000; Schneider, Gibb, & Seemüller, 1997; Wang et al, 2010; Wang et al, 2017; Yu et al, 2016,b, 2017, 2018). Early detection of the AYL phytoplasma using developed ddPCR system can assist in managing the disease by cutting the transmission route of AYL phytoplasma from the source, effectively preventing the spread and epidemic of phytoplasma and its related diseases.…”