植原体tuf基因与其上游部分基因结构和相关基因启动子保守区域特征及活性分析

doi:10.17520/biods.2018017

生物多样性 ›› 2018, Vol. 26 ›› Issue (7): 738-748. DOI: 10.17520/biods.2018017

• 研究报告: 微生物多样性 • 上一篇下一篇

植原体tuf基因与其上游部分基因结构和相关基因启动子保守区域特征及活性分析

于少帅, 林彩丽, 王圣洁, 张文鑫, 田国忠^*()

中国林业科学研究院森林生态环境与保护研究所, 国家林业局森林保护学重点实验室, 北京 100091

收稿日期:2018-01-16 接受日期:2018-03-26 出版日期:2018-07-20 发布日期:2018-09-11
通讯作者: 田国忠
作者简介:# 共同第一作者
基金资助:
国家自然科学基金(31370644)

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^*()

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:2018-07-20 Published:2018-09-11
Contact: Tian Guozhong
About author:# Co-first authors

摘要/Abstract

摘要：

植原体寄主种类多, 危害范围广, 开展其遗传多样性、关键基因调控等方面研究有助于提高该病害综合防治水平。通过长片段PCR引物扩增我国PaWB-sdyz、PaWB-fjfz和LY-fjya1植原体株系tuf基因及其上游6个基因的片段, 进行植原体基因启动子保守区域序列特征和多位点序列分析。利用启动子探针载体pSUPV4检测植原体tuf基因上游序列的启动子活性。扩增获得PaWB-sdyz、PaWB-fjfz、LY-fjya1株系tuf基因上游12,745-12,748 bp序列, 比较分析发现PaWB-sdyz、PaWB-fjfz、LY-fjya1、OY-M、AYWB、PAa、SLY、AT植原体株系tuf与其上游6个基因的结构顺序皆为5’-rplL-rpoB-rpoC-rps12-rps7-fusA-tuf-3’。推测出可能的植原体启动子保守区域模式序列: T₉₀T₁₀₀G₉₂T₇₅G₆₇A₈₅(-35区); T₉₀A₉₆T₉₂A₉₈T₇₃T₉₀(-10区)。基于8个植原体株系的rplL-tuf核苷酸序列编码基因、非编码序列、氨基酸序列的多位点序列分析可将不同植原体株系以较高的支持率清晰地区分, 不同植原体株系rplL-tuf核苷酸非编码区变异水平更高。16SrI组植原体tuf基因上游序列存在3种变异类型, 其代表株系PaWB-fjfz、LY-fjya1 tuf基因上游130 bp片段和CWB-hnsy1 tuf基因上游129 bp片段皆具有启动子活性。

关键词: 植原体, 启动子, 基因结构, 遗传多样性, 多位点序列分析

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.

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

于少帅, 林彩丽, 王圣洁, 张文鑫, 田国忠 (2018) 植原体tuf基因与其上游部分基因结构和相关基因启动子保守区域特征及活性分析. 生物多样性, 26, 738-748. DOI: 10.17520/biods.2018017.

Shaoshuai Yu, Caili Lin, Shengjie Wang, Wenxin Zhang, Guozhong Tian (2018) 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. Biodiversity Science, 26, 738-748. DOI: 10.17520/biods.2018017.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2018017

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

图/表 12

参考文献 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

植原体tuf基因与其上游部分基因结构和相关基因启动子保守区域特征及活性分析

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

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株系 Strains	rplL-rpoB		rpoC-rps12		rps12-rps7		fusA-tuf
株系 Strains	-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

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)

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

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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

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