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

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。

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

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.