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

doi:10.17520/biods.2018017

Abstract

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: 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.

http://jtp.cnki.net/bilingual/detail/html/SWDY201807009

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

Shaoshuai Yu, Caili Lin, Shengjie Wang, Wenxin Zhang, Guozhong Tian. 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[J]. Biodiv Sci, 2018, 26(7): 738-748.

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URL: https://www.biodiversity-science.net/EN/10.17520/biods.2018017

https://www.biodiversity-science.net/EN/Y2018/V26/I7/738

Figures/Tables 12

References 26

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引物 Primer	序列 Sequence (5’-3’)	长度 Length (nt)	T_m(℃)	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	3,676
op3	5'-AGGTAGCGGTCAAGAAGAAAT-3'	21	55.4	42.9	6,965
op4	5'-GAACCGCAAAGAACTGGG-3'	18	56.0	55.6	6,965
op5	5'-AATATTATTGACACTCCCGGAC-3'	22	55.5	40.9	2,661
op6	5'-ACTCTACCAGTAACAACAGTTCCTC-3'	25	56.1	44.0	2,661
op7	5'-CACATTTTATTAGCGCGCC-3'	19	57.2	47.4	1,519
op8	5'-AAAACCTAACGCAATCATGG-3'	20	55.5	40.0	1,519

引物 Primer	序列 Sequence (5’-3’)	长度 Length (nt)	T_m(℃)	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	3,676
op3	5'-AGGTAGCGGTCAAGAAGAAAT-3'	21	55.4	42.9	6,965
op4	5'-GAACCGCAAAGAACTGGG-3'	18	56.0	55.6	6,965
op5	5'-AATATTATTGACACTCCCGGAC-3'	22	55.5	40.9	2,661
op6	5'-ACTCTACCAGTAACAACAGTTCCTC-3'	25	56.1	44.0	2,661
op7	5'-CACATTTTATTAGCGCGCC-3'	19	57.2	47.4	1,519
op8	5'-AAAACCTAACGCAATCATGG-3'	20	55.5	40.0	1,519

引物 Primer	引物序列 Primer sequences (5’-3’)	引物大小 Primer size (bp)	T_m(℃)	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

引物 Primer	引物序列 Primer sequences (5’-3’)	引物大小 Primer size (bp)	T_m(℃)	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

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