CaWRKY5启动子分离及其在烟草瞬间表达系统中分析
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摘要
WRKY是植物基因组中特有的一类转录因子大家族,研究表明植物WRKY家族的不同成员参与了植物生长、发育以及对逆境胁迫应答的调控并在其中起重要的作用。CaWRKY5是辣椒WRKY家族的一个成员,初步研究结果表明该基因的转录表达受高温、盐胁迫以及病原菌侵染等的影响,并在对这些逆境抗性反应中起重要的调节作用,但对其诱导表达的分子机制还不很清楚。为了进一步明确CaWRKY5受逆境胁迫诱导表达的分子基础,本研究从辣椒基因组DNA中分离获得了CaWRKY5的相应启动子,并利用农杆菌介导的烟草叶片瞬间表达系统,通过deletion缺失突变体分析进一步分析了该启动子对不同逆境的应答并分析了可能的顺式作用元件。主要研究如下:
1)利用LA-PCR技术从辣椒基因组DNA克隆得到CaWRKY5基因5’端上游-936bp的启动子调控序列。用生物信息学对序列进行分析表明,该启动子TATA框为起始密码子ATG上游-140bp至-136bp的TATATAA,同时含有多种与逆境胁迫相关的顺式作用元件,如W-box,S-box,SA应答元件和类似ABRE元件等。
2)CaWRKYK5基因-936bp启动子是一个完整的启动子区域,具有启动子表达活性。在青枯病病原菌侵染和MeJA诱导作用下,长于-886bp的启动子缺失体都可以激活GUS基因的表达,而-489bp的启动子序列却不足以激活GUS表达,说明启动子应答青枯病菌的核心元件分布在启动子-886bp至-489bp之间。-336bp以上的启动子区域在机械损伤和SA的诱导处理下均可激活GUS基因的表达,说明启动子应答机械损伤和SA的核心区域分布在启动子上游的-336bp的区域里。对启动子4个缺失体进行ET(ethylene treatment)处理应答分析,发现-936bp和-336bp区域的缺失体均可激活GUS基因表达,而其中的-886bp和-489bp区域的缺失体却不能激活GUS基因的表达,说明启动子应答乙烯的核心区域分布在-336bp至ATG以及-936bp至-886bp的区域,而-489bp至-336bp区域中可能含有对乙烯应答起着负调控的调控元件。
Proteins in WRKY superfamily include many plant-specific transcription factors, which play important roles in the regulation of plant growth, development and stresses responses. CaWRKY5 is a member of the pepper (Capsicum annuum L.) WRKY family. Our previous study found that the expression of CaWRKY5 was induced by heat, high salinity and pathogen infection, indicating the possible involvement of CaWRKY5 in plant defense reaction against biotic and abiotic stresses. However the molecular mechanism of the transcription expression of CaWRKY5 against different stresses remains unclear. In this study, to gain insight into the molecular basis and possible signaling pathway of the induction of CaWRKY5 response to environment stress, we isolated the promoter of CaWRKY5. Deletion analysis of the CaWRKY5 promoter against different stresses as well as exogenous hormones was carried out via an Agrobacterium-mediated transient expression assay in tobacco leaves. The results are as followings:
(1) The 936bp upstream promoter sequence of the CaWRKY5 was isolated from the genomic DNA of pepper by LA-PCR. Bioinformatics analysis showed that the TATA-box of the promoter locates in -140bp to -136bp, and several cis-acting elements including W-box, S-box, SA-responsive element, ABRE responsive to different stresses or hormones were found in the promoter.
(2)The -936bp region upstream of translation start codon in CaWRKY5 seemed to be an intact promoter. The fragment from translation start codon to -936bp and from translation start codon to -886bp in the promoter were enough for GUS expression against pathogen infection and MeJA induction, However the fragment from translation start codon to -489bp abolished the response of GUS to pathogen infection and MeJA induction. These results suggested that within region from -886bp to -489bp in the promoter of CaWRKY5 there may exist the pathogen and MeJA responsive cis-element. The result also showed that in promoter of CaWRKY5, the fragment from translation start codon to -336bp were sufficient for GUS expression in response to mechanical wounding and SA treatment, suggesting that wounding and SA responsive cis-element may exist in this region. The deletion analysis of CaWRKY5 promoter against exogenous ethylene treatment suggested the ethylene responsive cis-element may locate within the region from translation start codon to -336bp region and from -936bp to -886bp of the promoter. Whereas, the promoter region from -886bp to -336bp were showed to inhibit the expression of GUS, indicating that some ethylene-related negative cis-element appeared to be located within the region from -489bp to -336bp of the promoter.
1)利用LA-PCR技术从辣椒基因组DNA克隆得到CaWRKY5基因5’端上游-936bp的启动子调控序列。用生物信息学对序列进行分析表明,该启动子TATA框为起始密码子ATG上游-140bp至-136bp的TATATAA,同时含有多种与逆境胁迫相关的顺式作用元件,如W-box,S-box,SA应答元件和类似ABRE元件等。
2)CaWRKYK5基因-936bp启动子是一个完整的启动子区域,具有启动子表达活性。在青枯病病原菌侵染和MeJA诱导作用下,长于-886bp的启动子缺失体都可以激活GUS基因的表达,而-489bp的启动子序列却不足以激活GUS表达,说明启动子应答青枯病菌的核心元件分布在启动子-886bp至-489bp之间。-336bp以上的启动子区域在机械损伤和SA的诱导处理下均可激活GUS基因的表达,说明启动子应答机械损伤和SA的核心区域分布在启动子上游的-336bp的区域里。对启动子4个缺失体进行ET(ethylene treatment)处理应答分析,发现-936bp和-336bp区域的缺失体均可激活GUS基因表达,而其中的-886bp和-489bp区域的缺失体却不能激活GUS基因的表达,说明启动子应答乙烯的核心区域分布在-336bp至ATG以及-936bp至-886bp的区域,而-489bp至-336bp区域中可能含有对乙烯应答起着负调控的调控元件。
Proteins in WRKY superfamily include many plant-specific transcription factors, which play important roles in the regulation of plant growth, development and stresses responses. CaWRKY5 is a member of the pepper (Capsicum annuum L.) WRKY family. Our previous study found that the expression of CaWRKY5 was induced by heat, high salinity and pathogen infection, indicating the possible involvement of CaWRKY5 in plant defense reaction against biotic and abiotic stresses. However the molecular mechanism of the transcription expression of CaWRKY5 against different stresses remains unclear. In this study, to gain insight into the molecular basis and possible signaling pathway of the induction of CaWRKY5 response to environment stress, we isolated the promoter of CaWRKY5. Deletion analysis of the CaWRKY5 promoter against different stresses as well as exogenous hormones was carried out via an Agrobacterium-mediated transient expression assay in tobacco leaves. The results are as followings:
(1) The 936bp upstream promoter sequence of the CaWRKY5 was isolated from the genomic DNA of pepper by LA-PCR. Bioinformatics analysis showed that the TATA-box of the promoter locates in -140bp to -136bp, and several cis-acting elements including W-box, S-box, SA-responsive element, ABRE responsive to different stresses or hormones were found in the promoter.
(2)The -936bp region upstream of translation start codon in CaWRKY5 seemed to be an intact promoter. The fragment from translation start codon to -936bp and from translation start codon to -886bp in the promoter were enough for GUS expression against pathogen infection and MeJA induction, However the fragment from translation start codon to -489bp abolished the response of GUS to pathogen infection and MeJA induction. These results suggested that within region from -886bp to -489bp in the promoter of CaWRKY5 there may exist the pathogen and MeJA responsive cis-element. The result also showed that in promoter of CaWRKY5, the fragment from translation start codon to -336bp were sufficient for GUS expression in response to mechanical wounding and SA treatment, suggesting that wounding and SA responsive cis-element may exist in this region. The deletion analysis of CaWRKY5 promoter against exogenous ethylene treatment suggested the ethylene responsive cis-element may locate within the region from translation start codon to -336bp region and from -936bp to -886bp of the promoter. Whereas, the promoter region from -886bp to -336bp were showed to inhibit the expression of GUS, indicating that some ethylene-related negative cis-element appeared to be located within the region from -489bp to -336bp of the promoter.
引文
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[2] Narusaka Y, Nakashima K, Shinwari Z K, Sakuma Y, Furihata T, Abe H, Narusaka M, Shinozaki K, Yamaguchi-Shi-nozaki K. Interaction between two cis-acting elements, ABRE and DRE in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high salinity stresses[J]. Plant J, 2003, 34(2): 137-148.
[3] Rushton P J, Macdonald H, Huttly A K, et a1. Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of a-Amy2 genes [J].Plant Mol Biol, 1995, 29(4): 691-702.
[4] Rushton P J, Torres J T, Parniske M, et a1.Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J].EMBO J, 1996, 15(20): 5690-5700.
[5] dePater S, Greco V, Pham K, et a1. Characterization of a zinc-dependent transcriptional activator from Arabidopsis [J]. Nucleic Acids Res, 1996, 24(3): 4624-4631.
[6] Kalde M, Barth M, Somssich I E, et al. Members of the Arabidopsis WRKY Group III transcription factors are part of different plant defense signaling pathways [J]. Mol Plant Microbe Interact, 2003, 16(4): 295-305.
[7] Wu K L, Guo Z J, Wang H H, et al. The WRKY family of transcription factors in rice and Arabidopsis and their origins [J]. DNA Res, 2005, 12(1): 19-26.
[8] Zhang Y, Wang L. The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants [J]. BMC Evol Biol, 2005, 5(1): 1-12.
[9] Eulgem T, Rushton P J, Robatzek S, el a1.The WRKY superfamily of plant transcription factors [J]. Trends Plant Sci, 2000, 5(5): 199-206.
[10] Turck F,Zhou A,Somssich I E.Stimulus-dependent.promoter-specific binding of transcription factor WRKY1 to its native promoter and the defense-related gene PcPR1-1 in parsley[J]. Plant Cell, 2004, 16(10): 2573-2585.
[11] Wang Z, Zhu Y, et at. A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter [J]. Planta, 2009, 230(6): 1155-1166.
[12] Ichimura K, Tena G, Henry Y, Zhang S, Hirt H, Ellis B, E, Morris P.C, Wilson C, C Champion A, Innes R.W, Sheen J., Ecker J R. Scheel D., Heberle-Bors E., Walker J C., and Schinozaki K. Mitogen-activated protein kinase cascades in plants: a new nomenclature[J]. Trends Plant Sci, 2002, 7: 301-308.
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