Biodiversity Science ›› 2018, Vol. 26 ›› Issue (7): 738-748.doi: 10.17520/biods.2018017

• Original Papers • Previous Article     Next Article

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

Shaoshuai Yu, Caili Lin, Shengjie Wang, Wenxin Zhang, Guozhong Tian*()   

  1. Key Laboratory of Forest Protection of State Forestry Administration, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091
  • Received:2018-01-16 Accepted:2018-03-26 Online:2018-09-11
  • Tian Guozhong E-mail:tian3691@163.com
  • About author:# Co-first authors

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: 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.

Key words: phytoplasma, promoter, gene structure, genetic diversity, multilocus sequence analysis

Table 1

Information of the primers used to amplify the large DNA fragments"

引物 Primer 序列 Sequence (5’-3’) 长度 Length (nt) Tm (℃) G+C (%) 扩增长度 Length amplified (bp)
op1 5'-GATTGACATGGCTAAGTTAACG-3' 22 54.9 40.9 3,676
op2 5'-TACACCTTTGTTTCCGTGGC-3' 20 58.4 50.0
op3 5'-AGGTAGCGGTCAAGAAGAAAT-3' 21 55.4 42.9 6,965
op4 5'-GAACCGCAAAGAACTGGG-3' 18 56.0 55.6
op5 5'-AATATTATTGACACTCCCGGAC-3' 22 55.5 40.9 2,661
op6 5'-ACTCTACCAGTAACAACAGTTCCTC-3' 25 56.1 44.0
op7 5'-CACATTTTATTAGCGCGCC-3' 19 57.2 47.4 1,519
op8 5'-AAAACCTAACGCAATCATGG-3' 20 55.5 40.0

Table 2

Primers used to amplify tuf gene promoters"

引物 Primer 引物序列 Primer sequences (5’-3’) 引物大小 Primer size (bp) Tm (℃) 引入酶切位点 Restriction site added
TPf cccaagcttACAACCTTACACTAAAAAAC 29 45 Hind III
TP4f cccaagcttACAACCTTAAACTAAAAAAC 29 45 Hind III
TPr cgcggatcCATTTTTCAAAGGCCTC 25 45 BamH I

Table 3

Length of sequence amplified, coding region, non-coding region and amino acid sequence of phytoplasmas as well as reference strains"

株系
Strain
级别
Group
扩增序列长度
Sequence amplified
length (bp)
编码区长度
Coding region
length (bp)
非编码区长度
Non-coding region
length (bp)
氨基酸长度/个
Amino acid sequence
length
PaWB-sdyz 16SrI-D 12,746 12,307 439 4,096
PaWB-fjfz 16SrI-D 12,745 12,307 438 4,096
LY-fjya1 16SrI-B 12,748 12,307 441 4,096
OY-M 16SrI-B 12,745 12,307 438 4,096
AYWB 16SrI-A 12,735 12,343 392 4,107
PAa 16SrXII 12,611 12,211 400 4,063
SLY 16SrXII 12,611 12,211 400 4,063
AT 16SrX 12,835 12,301 534 4,093
PG-8A 4,587 4,158 429 1,382

Fig. 1

Gene structure diagram of tuf gene and its upstream genes from different phytoplasmas. The codes of samples in the figure were shown in “1.1” and “1.2.3”. Numbers represented the length of intergenic sequences (bp)."

Table 4

Characteristics of conserved region of relevant gene promoter of phytoplasma rplL-tuf intergenic sequences"

株系
Strains
rplL-rpoB rpoC-rps12 rps12-rps7 fusA-tuf
-35 -10 -35 -10 -35 -10 -35 -10
PaWB-sdyz TTGCAT TATACC - - ATAAAA AAAAAT TTGTGA TATATT
PaWB-fjfz TTGCAT TATACC - - ATAAAA AAAAAT TTGTGA TATATT
LY-fjya1 TTGCAT TATACC - - ATAAAA AAAAAT TTGTAA TATATT
OY-M TTGAAT TATAAC - - ATAAAA AAAAAT TTGTGA TATATT
AYWB TTGCAT TATACC - - - - TTGTGA TATTAT
PAa TTGTAT TTTAAT - - - - TTGATA TATATT
SLY TTGTAT TTTAAT - - - - TTGATA TATATT
AT ATGATA AATAAT TTGACT TATAAT - - - -
PG-8A - - TTGACA TATAAT - - ATGATA TATTGT

Table 5

Nucleotide and its frequency in different sites of conserved regions of some phytoplasma gene promoters"

保守区域 Conserved region -35区 -35 region -10区 -10 region
序列特征
Sequence characteristic
A(10) A(0) A(8) A(17) A(25) A(85) A(10) A(96) A(8) A(98) A(19) A(0)
T(90) T(100) T(0) T(75) T(6) T(15) T(90) T(4) T(92) T(2) T(73) T(90)
G(0) G(0) G(92) G(0) G(67) G(0) G(0) G(0) G(0) G(0) G(0) G(0)
C(0) C(0) C(0) C(8) C(2) C(0) C(0) C(0) C(0) C(0) C(8) C(10)

Table 6

Homology matrix of rplL-tuf nucleotide sequence coding region in MLSA analysis"

株系 Strain PaWB-sdyz PaWB-fjfz LY-fjya1 OY-M AYWB PAa SLY AT PG-8A
PaWB-sdyz 100
PaWB-fjfz 99.7 100
LY-fjya1 99.3 99.3 100
OY-M 99.6 99.6 99.4 100
AYWB 95.9 95.9 95.9 96.0 100
PAa 81.1 81.1 81.2 81.1 81.1 100
SLY 81.1 81.1 81.2 81.1 81.1 99.9 100
AT 72.7 72.8 72.9 72.8 73.3 73.6 73.6 100
PG-8A 71.2 71.3 71.4 71.3 71.4 71.3 71.3 70.9 100

Fig. 2

Phylogenetic tree of the phytoplasma strains reconstructed based on the concatenated gene sequences data set of rplL-tuf nucleotide sequence coding region. The codes in the figure were shown in “1.1” and “1.2.3”."

Table 7

Homology matrix of rplL-tuf nucleotide sequence non-coding region in MLSA analysis"

株系 Strain PaWB-sdyz PaWB-fjfz LY-fjya1 OY-M AYWB PAa SLY AT PG-8A
PaWB-sdyz 100
PaWB-fjfz 99.3 100
LY-fjya1 99.3 98.6 100
OY-M 99.3 98.6 98.6 100
AYWB 94.6 94.3 94.1 94.1 100
PAa 71.9 71.8 71.3 72.3 73.2 100
SLY 71.9 71.8 71.3 72.3 73.2 100 100
AT 50.7 51.3 51.4 51.3 51.1 51.4 51.4 100
PG-8A 41.8 41.3 41.5 41.8 41.3 40.2 40.2 44.8 100

Fig. 3

Phylogenetic tree of the phytoplasma strains reconstructed based on the concatenated gene sequences data set of rplL-tuf nucleotide sequence non-coding region. The codes in the figure are shown in “1.1” and “1.2.3”."

Table 8

Homology matrix of RplL-TUF amino acid sequence in MLSA analysis"

株系 Strain PaWB-sdyz PaWB-fjfz LY-fjya1 OY-M AYWB PAa SLY AT PG-8A
PaWB-sdyz 100
PaWB-fjfz 99.6 100
LY-fjya1 98.9 99.0 100
OY-M 99.4 99.5 99.2 100
AYWB 97.8 97.9 98.0 98.0 100
PAa 83.8 83.9 84.1 83.9 83.7 100
SLY 83.8 83.9 84.1 83.9 83.7 99.8 100
AT 69.8 69.9 70.0 69.9 69.8 69.0 69.0 100
PG-8A 74.9 75.1 74.9 75.0 74.8 74.1 73.9 70.4 100

Fig. 4

Phylogenetic tree of the phytoplasma strains reconstructed based on the concatenated sequences data set of RplL-TUF amino acid sequence. The codes in the figure are shown in “1.1” and “1.2.3”."

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