|
Special Issue:
植物病理合辑Plant Protection—Plant Pathology
杂草合辑Weed
|
|
|
|
| Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene, which confers glyphosate resistance of goosegrass (Eleusine indica) |
| ZHANG Chun, YU Chao-jie, ZHANG Tai-jie, GUO Wen-lei, TIAN Xing-shan |
Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China
|
|
|
|
|
摘要
草甘膦靶标酶是5-烯醇式丙酮酸莽草酸-3-磷酸合酶(EPSPS)，EPSPS基因的过表达是杂草抗草甘膦的分子机制之一。比较抗型和敏感型牛筋草中EPSPS基因调控序列，发现抗型牛筋草EPSPS基因5’端非翻译区5’-UTR Py-rich stretch元件发生突变，且该突变与提高抗草甘膦牛筋草EPSPS基因转录水平相关。然而，与该元件及整个EPSPS基因启动子序列结合的关键转录因子尚未可知。为进一步探究抗草甘膦牛筋草中EPSPS基因的转录调控机制，本研究利用RNA-seq技术分析了抗草甘膦牛筋草EPSPS过表达相关的基因调控网络，筛选出相关转录因子；通过酵母单杂交技术体外验证相关转录因子与5’-UTR Py-rich stretch元件的结合情况。转录本差异表达分析显示，与草甘膦敏感型(GS)牛筋草相比，抗草甘膦牛筋草在草甘膦处理后有2752个unigenes的表达增加，4025个unigenes的表达减少。其中，鉴定出1373个unigenes与EPSPS基因共表达。GO和KEGG通路分析表明，上调的unigenes主要富集于叶绿体，并与莽草酸生物合成通路、叶绿素II和过氧化物酶体代谢过程相关。值得注意的是，催化莽草酸转化为莽草酸3-磷酸(S3P, EPSPS的底物)的莽草酸激酶的表达也上调。筛选到8个转录因子可能参与EPSPS表达，其中预测三个转录因子(ARF2, ARF8和BPC6)能与5 ' utr Py-rich元件结合。酵母单杂交实验表明ARF8和BPC6能与野生型5’-UTR Py-rich stretch元件结合，但不再能与其突变体结合。本研究数据表明，与草甘膦敏感型牛筋草相比，抗草甘膦牛筋草中EPSPS的表达转录调控机制发生了显著的变化。首次筛选分析出与EPSPS基因表达调控相关的转录因子，可为进一步研究基于EPSPS过表达的牛筋草抗草甘膦机理提供了新的参考。
Abstract Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds. A regulatory sequence of EPSPS gene was isolated previously, and an alteration in its 5´-untranslated region (UTR) pyrimidine (Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant (GR) Eleusine indica. However, the transcription factors involved in this regulatory sequence remain to be elucidated. In this study, we investigated the regulatory network of EPSPS overexpression associated genes in a GR E. indica population by RNA-seq. The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2 752 unigenes and a decrease in the expression of 4 025 unigenes in the GR E. indica, compared to the glyphosate-susceptible (GS) E. indica. Among them, 1 373 unigenes were identified to be co-expressed with the EPSPS gene in GR E. indica. GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway, chlorophy II and peroxisome metabolism processes. Notably, the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate (S3P, a substrate of EPSPS), was also up-regulated. Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression, and three of them (ARF2, ARF8 and BPC6) showed more binding sites because of a (CT)n insertion of the 5´-UTR Py-rich stretch element in GR. However, the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS, but could not bind to the mutated form. Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E. indica. These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.
|
|
Received: 19 January 2021
Accepted:
|
| Fund: This study was supported by the National Natural Science Foundation of China (31871984), the Guangdong Provincial National Natural Science Foundation, China (2017B030311006), the Department of Science and Technology of Guangdong Province, China (2019B121201003), and the special fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science, China (202105TD, R2020PY-JX005). |
|
Corresponding Authors:
Correspondence TIAN Xing-shan, Tel: +86-20-87594497, Fax: +86-20-87561757, E-mail: 1070470768@qq.com
|
| About author: ZHANG Chun, E-mail: zhangchun_0726@163.com; * These authors contributed equally to this study. |
Cite this article:
ZHANG Chun, YU Chao-jie, ZHANG Tai-jie, GUO Wen-lei, TIAN Xing-shan.
2021.
Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene, which confers glyphosate resistance of goosegrass (Eleusine indica). Journal of Integrative Agriculture, 20(8): 2180-2194.
|
Abe H, Urao T, Seki M, Yamaguchi-Shinozaki S K. 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. The Plant Cell, 15, 63–78.
Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K. 1997. Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression. The Plant Cell, 9, 1859–1868.
Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. 1990. Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.
Anders S, Huber W. 2012. Differential Expression of RNA-Seq Data at the Gene Level - the Deseq Package. European Molecular Biology Laboratory, Heidelberg.
Baerson S R, Rodriguez D J, Tran M, Feng Y, Biest N A, Dill G M. 2002. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiology, 129, 1265–1275.
Bao J, Corke H, Sun M. 2002. Microsatellites in starch-synthesizing genes in relation to starch physicochemical properties in waxy rice (Oryza sativa L.). Theoretical and Applied Genetics, 105, 898–905.
Bevilacqua A, Fiorenza M T, Mangia F. 2000. A developmentally regulated GAGA box-binding factor and Sp1 are required for transcription of the hsp70.1 gene at the onset of mouse zygotic genome activation. Development, 127, 1541–1551.
Bilaud T, Koering C E, Binet-Brasselet E, Ancelin K, Pollice A, Gasser S M, Gilson E. 1996. The telobox, a myb-related telomeric DNA binding motif found in proteins from yeast, plants and human. Nucleic Acids Research, 24, 1294–1303.
Bolger A M, Marc L, Bjoern U. 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics, 15, 2114–2120.
Castillo-Davis C I. 2005. The evolution of noncoding DNA: How much junk, how much func? Trends in Genetics, 21, 533–536.
Croston G E, Kerrigan L A, Lira L M, Marshak D R, Kadonaga J T. 1991. Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription. Science, 251, 643–649.
Dinelli G, Marotti I, Bonetti A, Minelli M, Catizone P, Barnes J. 2006. Physiological and molecular insight on the mechanisms of resistance to glyphosate in Conyza canadensis (L.) Cronq. Biotypes. Pesticide Biochemistry and Physiology, 86, 30–41.
Duke S O, Powles S B, Sammons R D. 2018. Glyphosate - how it became a once in a hundred year herbicide and its future. Outlooks on Pest Management, 29, 247–251.
Gaines T A, Zhang W, Wang D, Bukun B, Chisholm S T, Shaner D L, Nissen S J, Patzoldt W L, Tranel P J, Culpepper A S, Grey T L, Webster T M, Vencill W K, Sammons R D, Jiang J M, Preston C, Leach J E, Westra P. 2010. Gene amplification confers glyphosate resistance in Amaranthus palmeri. Proceedings of the National Academy of Sciences of the United States of America, 107, 1029–1034.
Gilmour S J, Sebolt A M, Salazar M P, Everard J D, Thomashow M F. 2000. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology, 124, 1854–1865.
Grabherr M G, Haas B J, Yassour M, Levin J Z, Thompson D A, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q D. 2011. Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nature Biotechnology, 29, 644–652.
Heap I. 2021. The international survey of herbicide resistant weeds. [2021-01-09]. http://www.weedscience.org
Huang Z F, Wang G R, Huang H G, Wei S H, Zhou X X, Chen J Y, Zhang C X. 2015. Isolation and functional analysis of Convolvulus arvensis EPSPS promoter. Plant Molecular Biology Reporter, 33, 1–9.
Joshi-Saha A, Reddy K S. 2015. Repeat length variation in the 5´ UTR of myo-inositol monophosphatase gene is related to phytic acid content and contributes to drought tolerance in chickpea (Cicer arietinum L.). Journal of Experimental Botany, 6619, 5683–5690.
Kalluri U C, DiFazio S P, Brunner A M, Tuskan G A. 2007. Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa. BMC Plant Biology, 7, 59.
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y. 2008. KEGG for linking genomes to life and the environment. Nucleic Acids Research, 36, 480–484.
Kashi Y, King D, Soller M. 1997. Simple sequence repeats as a source of quantitative genetic variation. Trends in Genetics, 13, 74–78.
Kumar R, Tyagi A K, Sharma A K. 2011. Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development. Molecular Genetics and Genomics, 285, 245–260．
Kumar S, Bhatia S. 2016. A polymorphic (GA/CT)n-SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don. Scientific Reports, 6, 33280.
Langmead B, Salzberg S L. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods, 9, 357–366.
Lu Q, Wallrath L L, Granok H, Elgin S C. 1993. (CT)n (GA)n
repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene. Molecular and Cellular Biology, 13, 2802–2814.
Maeda H, Dudareva N. 2012. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annual Review of Plant Biology, 63, 73–105.
Meister R J, Williams L A, Monfared M M, Gallagher T L, Kraft E A, Nelson C G, Gasser C S. 2004. Definition and interactions of a positive regulatory element of the Arabidopsis inner no outer promoter. Plant Journal, 37, 426–438.
Melfi R, Palla F, Di Simone P, Alessandro C, Cali L, Anello L, Spinelli G. 2000. Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences. Journal of Molecular Biology, 304, 753–763.
Mohammadparast S, Bayat H, Biglarian A, Ohadi M. 2014. Exceptional expansion and conservation of a CT-repeat complex in the core promoter of PAXBP1 in primates. American Journal of Primatology, 76, 747–756.
Ni M, James M, Tepperman J M, Quail P H. 1999. Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. Nature, 400, 781–784.
Ni M, Tepperman J M, Quail P H. 1998. PIF3, a phytochrome interacting factor necessary for normal photo induced signal transduction is a novel basic helix-lop-helix protein. Cell, 95, 657–667.
Nole-Wilson S, Krizek B A. 2000. DNA binding properties of the Arabidopsis floral development protein AINTTGUMENTA. Nucleic Acids Research, 28, 4076–4082.
Ogo Y, Itai R N, Nakanishi H, Kobayashi T, Takahashi M, Mori S, Nishizawa N K. 2007. The rice bHLH protein OsIRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions. The Plant Journal, 51, 366–377.
Quattrocchio F, Wing J, Woude K V D, Souer E, Vetten N D, Mol J, Koes R. 1999. Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. The Plant Cell, 11, 1433–1444.
Roberts A, Pachter L. 2013. Streaming fragment assignment for real-time analysis of sequencing experiments. Nature Methods, 10, 71–73.
Sammons D R, Gaines T A. 2014. Glyphosate resistance: State of knowledge. Pest Management Science, 70, 1367–1377.
Sangwan I, O’Brian M R. 2002. Identification of a soybean protein that interacts with GAGA element dinucleotide repeat DNA. Plant Physiology, 129, 1788–1794.
Santi L, Wang Y, Stile M R, Berendzen K, Wanke D, Roig C, Pozzi C, Müller K, Müller J, Rohde W, Salamini F. 2003. The GA octodinucleotide repeat binding factor BBR participates in the transcriptional regulation of the homeobox gene Bkn3. Plant Journal, 34, 813–826.
Shang Y, Yan L, Liu Z Q, Cao Z, Mei C, Xin Q, Wu F Q, Wang X F, Du S Y, Jiang T, Zhang X F, Zhao R, Sun H L, Liu R, Yu Y T, Zhang D P. 2010. The Mg-chelatase H subunit of Arabidopsis anta gonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition. The Plant Cell, 22, 1909–1935.
Shanks C M, Hecker A, Cheng C Y, Brand L, Collani S, Schmid M, Schaller G, Wanke D, Harter K, Kieber J J. 2018. Role of BASIC PENTACYSTEINE transcription factors in a subset of cytokinin signaling responses. Plant Journal, 95, 458–473.
Sharkhuu A, Narasimhan M L, Merzaban J S, Bressan R A, Weller S, Gehring C. 2014. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate. The Plant Journal, 78, 916–926.
Silva D R O, Vargas L, Agostinetto D, Santos F M. 2017. Photosynthetic performance of glyphosate resistant and glyphosate susceptible hairy fleabane under light intensity. Planta Daninha, 35, e017166275.
Simar-Blanchet A E, Legraverend C, Thissen J P, Cam A L. 1998. Transcription of the rat serine protease inhibitor 2.1 gene in vivo: Correlation with GAGA box promoter occupancy and mechanism of cytokinemediated down-regulation. Molecular Endocrinology, 12, 391–404.
Singh K B. 1998. Transcriptional regulation in plants: The importance of combinatorial control. Plant Physiology, 118, 1111–1120.
Sreenivasulu M, Ramesh P, Damodharam T. 2015. Effect of glyphosate on chlorophyll and carotenoids in weed species. International Journal of Advanced Scientific and Technical Research, 5, 2249–2254.
Sweeney M T, Thomson M J, Pfeil B E, McCouch S. 2006. Caught red-Handed: Rc encodes a basic helix-lop-helix protein conditioning red pericarp in rice. The Plant Cell, 18, 283–294．
Trapnell C, Williams B A, Petea G, Mortazavi A, Kwan G, Baren M J V, Salzberg S L, Wold B J, Pachter L. 2010. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology, 28, 511–515.
Trifonov E N. 2004. Tuning function of tandemly repeating sequences: A molecular device for fast adaptation. In: Wasser S P, ed., Evolutionary Theory and Processes: Modern Horizons. Springer, Dordrecht. pp. 115–138.
Tsukiyama T, Becker P B, Wu C. 1994. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature, 367, 525–532.
Tsukiyama T, Wu C. 1995. Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell, 83, 1011–1020.
Tzin V, Galili G. 2010. New insights into the shikimate and aromatic amino acids biosynthesis pathways in plants. Molecular Plant, 3, 956–972.
Viana A A, Fragoso R R, Guimarães L M, Pontes N, Oliveira-neto O B, Artico S Nardeli S M, Alves-Ferreira M, Batista J A N, Silva M C M, Grossi-de-Sa M F. 2011. Isolation and functional characterization of a cotton ubiquitination-related promoter and 5´UTR that drives high levels of expression in root and flower tissues. BMC Biotechnology, 11, 115.
Vinces M D, Legendre M, Caldara M, Hagihara M, Verstreoen K J. 2009. UnsTable tandem repeats in promoters center transcriptional evolvability. Science, 324, 1213–1216.
Wiersma A T, Gaines T A, Preston C, Hamilton J P, Giacomini D, Buell C R, Leach J E, Westra P. 2015. Gene amplification of 5-enol-pyruvylshikimate-3-phosphate synthase in glyphosate-resistant Kochia scoparia. Planta, 41, 463–474.
Wyse B D, Linas S L, Thekkumkara T J. 2000. Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription. Journal of Molecular Endocrinology, 25, 97–108.
Ye J, Zhang Y, Cui H, Liu J, Wu Y, Cheng Y, Xu H, Huang X, Li S, Zhou A, Zhang X, Bolund L, Chen Q, Wang J, Yang H, Fang L, Shi C. 2018. WEGO 2.0: A web tool for analyzing and plotting GO annotations. Nucleic Acids Research, 46, W71–W75.
Yu Q, Jalaludin A, Han H, Chen M, Sammons R D, Powles S B. 2015. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance. Plant Physiology, 167, 1440–1447.
Yuan C I, Chaing M Y, Chen Y M. 2002. Triple mechanisms of glyphosate-resistance in a naturally occurring glyphosate-resistant plant Dicliptera chinensis. Plant Science, 163, 543–554.
Zhang C, Feng L, He T T, Yang C H, Chen G Q, Tian X S. 2015. Investigating the mechanisms of glyphosate resistance in goosegrass (Eleusine indica) population from South China. Journal of Integrative Agriculture, 14, 714–723.
Zhang C, Feng L, Tian X S. 2018. Alterations in the 5´ untranslated region of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene influence EPSPS overexpression in glyphosate-resistant Eleusine indica. Pest Management Science, 74, 2561–2568.
Zhang T J, Feng L, Tian X S, Yang C H, Gao J D. 2015. Use of chlorophyll fluorescence and P700 absorbance to rapidly detect glyphosate resistance in goosegrass (Eleusine indica). Journal of Integrative Agriculture, 14, 714–723.
Zhou J, Li F, Wang J L, Ma Y, Chong K, Xu Y Y. 2009. Basic helix-loop-helix transcription factor from wild rice (OrbHLH2) improves tolerance to salt- and osmotic stress in Arabidopsis. Journal of Plant Physiology, 166, 1296–1306.
|
| No Suggested Reading articles found! |
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
