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

• 研究报告 • 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*()   

  1. 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
  • 通讯作者: 田国忠 E-mail:tian3691@163.com
  • 作者简介:# 共同第一作者
  • 基金项目:
    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*()   

  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:2018-07-20
  • Contact: Tian Guozhong E-mail:tian3691@163.com
  • About author:# Co-first authors

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

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

表1

长片段DNA扩增引物信息"

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

表2

tuf基因启动子扩增引物"

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

表3

植原体扩增、编码区、非编码区序列和编码氨基酸序列及参照株系对应片段长度"

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

图1

不同植原体tuf基因及其上游基因结构示意图。图中样品代号见正文“1.1”和“1.2.3”; 数字代表基因间区序列长度(bp)。"

表4

植原体rplL-tuf基因间区序列相关基因启动子保守区域特征"

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

表5

部分植原体基因启动子保守区域不同位置核苷酸种类及出现频率"

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)

表6

MLSA分析rplL-tuf核苷酸序列编码区同源比对"

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

图2

基于rplL-tuf核苷酸序列编码区整合序列构建的植原体系统发育树。图中代号见“1.1”和“1.2.3”。"

表7

MLSA分析rplL-tuf核苷酸序列非编码区同源比对"

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

图3

基于rplL-tuf核苷酸序列非编码区整合序列构建的植原体系统发育树。图中株系代号见正文“1.1”和“1.2.3”。"

表8

MLSA分析RplL-TUF蛋白氨基酸序列同源比对"

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

图4

基于RplL-TUF氨基酸整合序列构建的植原体系统发育树。图中代号见正文“1.1”和“1.2.3”。"

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