烟草PR3b转录后剪切元件NRSE1与GUS融合表达后的可变剪切

doi:10.3864/j.issn.0578-1752.2020.08.003

摘要/Abstract

摘要：

【目的】烟草（Nicotiana tabacum L.）碱性几丁质酶基因PR3b在低烟碱突变体（nic1和nic2）中存在转录后mRNA可变剪切现象,但其可变剪切的发生机制仍不清楚。将PR3b的可变剪切元件NRSE1（nicotine- synthesis related splicing element 1）与GUS融合表达,分析NRSE1元件的独立可变剪切特性,以揭示其作用机制。【方法】利用PCR扩增方法获得PR3b cDNA序列中的NRSE1元件片段,并利用基因重组技术构建了烟草PR3b可变剪切元件NRSE1与GUS的融合表达载体。将融合表达载体导入农杆菌LBA4404后,通过农杆菌介导的叶盘转化法培育了表达NRSE1与GUS融合子的低烟碱突变体nic1和nic2及野生型烟草转基因植株;通过RT-PCR检测及GUS染色鉴定出阳性植株后,利用RT-PCR分析NRSE1与GUS融合表达后在低烟碱突变体和野生型烟草中的可变剪切特性;对转基因植株的幼苗进行乙烯（ET）和茉莉酸（JA）处理,通过GUS染色方法分析ET和JA处理对转基因植株中GUS活性的影响,并通过RT-PCR方法分析ET和JA处理对转基因植株中NRSE1与GUS融合子的可变剪切特性影响,以及对转基因植株中NRSE1与GUS融合子表达水平的影响。【结果】通过RT-PCR检测及GUS染色鉴定出表达NRSE1元件与GUS融合子的低烟碱突变体和野生型烟草转基因植株;RT-PCR检测及测序分析证明,NRSE1元件与GUS融合表达后仍能在低烟碱突变体发生高水平的可变剪切,剪切修饰区段的序列变化与烟草中PR3b的mRNA可变剪切修饰一致;利用ET和JA处理转基因植株进行的GUS染色表明,ET和JA处理对转基因植株的GUS活性有不同程度的影响;但利用ET和JA处理转基因植株进行的RT-PCR分析表明,ET和JA处理不改变NRSE1元件原有的诱导剪切特性,也不影响转基因植株中NRSE1元件与GUS融合子的表达水平。【结论】PR3b的可变剪切元件NRSE1与GUS在烟草中融合表达后,仍能在低烟碱突变体nic1和nic2中发生高水平的可变剪切;NRSE1在烟草中的可变剪切不依赖PR3b的其他mRNA区段,是烟草PR3b发生可变剪切的独立元件;ET和JA处理对NRSE1元件与GUS融合表达植株的GUS活性具有一定影响,可能存在翻译水平的调控作用。

关键词: 烟草, PR3b, 低烟碱突变体, 可变剪切, 茉莉酸, 乙烯

Abstract:

【Objective】Previously, a post-transcriptional splicing of tobacco PR3b gene was observed in the low-nicotine mutants (nic1, nic2) of Nicotiana tabacum L. cv. Burley 21, yet the mechanism underlying this phenomenon is still unclear. In this study, we developed transgenic plants expressing the fusion of the alternative splicing element NRSE1 (nicotine-synthesis related splicing element 1) from PR3b and the GUS gene to investigate the splicing properties of the NRSE1 element after excising from PR3b mRNA, in order to reveal its regulatory mechanism. 【Method】The NRSE1 element was amplified from PR3b cDNA by PCR amplification, and the vector for expressing the fusion of NRSE1 element and the GUS gene was constructed by molecular methods. And, the vector was used to develop transgenic plants expressing the fusion of NRSE1 element and GUS gene with wild type tobacco and the low-nicotine mutants nic1 and nic2 via agrobacterium (LBA4404) mediated transformation method. The transgenic plants were identified by RT-PCR and GUS staining, and the splicing of the fusion of NRSE1 element and GUS gene in the transgenic plants of wild type tobacco and low-nicotine mutants were then analyzed by RT-PCR. Seedlings of the transgenic plants were treated with ethylene (ET) and jasmonic acid (JA), respectively. And, the effects of ET and JA treatment on the GUS activity and the splicing of the fusion of NRSE1 element and GUS gene in the transgenic plants were analyzed by GUS staining and RT-PCR, respectively. The effects of ET and JA treatment on the expression level of the fusion of NRSE1 element and GUS gene were analyzed as well. 【Result】A set of transgenic wild type tobacco and low-nicotine mutants expressing the fusion of NRSE1 element and GUS gene were identified by RT-PCR and GUS staining. Further RT-PCR and sequencing analyses showed that the NRSE1 element could be alternatively spliced at higher levels in the low-nicotine mutants after fusion with the GUS gene, in a pattern consistent with its alternative splicing in the PR3b mRNA as previously report. ET and JA treatments could alter the GUS activity in the transgenic plants, but did not affect the inducible splicing of the NRSE1 element or the expression level of the fusion of NRSE1 element and GUS gene in the transgenic plants. 【Conclusion】 A highly splicing of the NRSE1 element was observed in the low-nicotine mutants after fusion expression with GUS gene. The alternatively splicing of NRSE1 element is independent of the rest regions of PR3b mRNA. ET and JA treatments had an effect on the GUS activities of the transgenic plants expressing the fusion of NRSE1 element and GUS gene, which may result from a translational regulation.

Key words: Nicotiana tabacum L., PR3b gene, low-nicotine mutant, alternative splicing, jasmonic acid, ethylene

赵雪,王锋,王文静,刘晓峰,卞士权,刘艳华,刘新民,杜咏梅,张忠锋,张洪博. 烟草PR3b转录后剪切元件NRSE1与GUS融合表达后的可变剪切[J]. 中国农业科学, 2020, 53(8): 1524-1531.

ZHAO Xue,WANG Feng,WANG WenJing,LIU XiaoFeng,BIAN ShiQuan,LIU YanHua,LIU XinMin,DU YongMei,ZHANG ZhongFeng,ZHANG HongBo. Splicing Property Analyses of the NRSE1 Element from Tobacco PR3b mRNA After Fusion Expression with GUS Gene[J]. Scientia Agricultura Sinica, 2020, 53(8): 1524-1531.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.08.003

https://www.chinaagrisci.com/CN/Y2020/V53/I8/1524

图/表 5

图1

图2

图3

图4

图5

参考文献 34

[1]	JALALI B L, BHARGAVA S, KAMBLE A . Signal transduction and transcriptional regulation of plant defence responses. Journal of Phytopathology, 2006,154:65-74.
[2]	张玉, 杨爱国, 冯全福, 蒋彩虹, 耿锐梅, 罗成刚 . 植物病程相关蛋白及其在烟草中的研究进展. 生物技术通报, 2012(5):20-24.
	ZHANG Y, YANG A G, FENG Q F, JIANG C H, GENG R M, LUO C G . Plant pathogenesis-related proteins and research progress in tobacco. Biotechnology Bulletin, 2012(5):20-24. (in Chinese)
[3]	MARTIN G B, BOGDANOVE A J, SESSA G . Understanding the functions of plant disease resistance proteins. Annual Review of Plant Biology, 2003,54:23-61.
[4]	GLAZEBROOK J . Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology, 2005,43:205-227.
[5]	MA H R, WANG F, WANG W J . Alternative splicing of basic chitinase gene PR3b in the low-nicotine mutants of Nicotiana tabacum L. cv. Burley 21. Journal of Experimental Botany, 2016,67:5799-5809.
[6]	RUBÉN A, TIBURCIO A F . Determination of arginine and ornithine decarboxylase activities in plants. Methods in Molecular Biology (Clifton, N.J.), 2018,1694:117-122.
[7]	PATEL J, ARIYARATNE M, AHMED S, GE L, PHUNTUMART V, KALINOSKI A, MORRIS P F . Dual functioning of plant arginases provides a third route for putrescine synthesis. Plant Science, 2017,262:62-73.
[8]	SHUNSUKE I, KATSUHITO H, MAKIKO N, HISAE K, YOSHIKATSU M, TAKASHI H, YOUJI S, YASUYUKI Y, KENZO N . Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures. Plant Molecular Biology, 1998,38(6):1101-1111.
[9]	CHATTOPADHYAY M K, GHOSH B . Molecular analysis of polyamine biosynthesis in higher plants. Current Science, 1998,74:517-522.
[10]	DALTON H L, BLOMSTEDT C K, NEALE A D, GLEADOW R, DEBOER K D, HAMILL J D . Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L. Journal of Experimental Botany, 2016,67(11):3367-3381.
[11]	KAJIKAWA M, SIERRO N, HASHIMOTO T, SHOJI T . A model for evolution and regulation of nicotine biosynthesis regulon in tobacco. Plant Signaling & Behavior, 2017,12(6):e1338225.
[12]	NACONSIE M, KATO K, SHOJI T . Molecular evolution of N-methylputrescine oxidase in tobacco. Plant and Cell Physiology, 2014,55:436-444.
[13]	AKIRA K . Molecular biology of pyridine nucleotide and nicotine biosynthesis. Frontiers in Bioscience, 2004,9(1-3):1577-1586.
[14]	RALPH E D, XIE J H . Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum. Phytochemistry, 2013,94:10-27.
[15]	DEBOER K, LYE J, AITKEN C, SU A, HAMILL J . The A622 gene in Nicotiana glauca (tree tobacco): Evidence for a functional role in pyridine alkaloid synthesis. Plant Molecular Biology, 2009,69(3):299-312.
[16]	KAJIKAWA M, HIRAI N, HASHIMOTO T . A PIP-family protein is required for biosynthesis of tobacco alkaloids. Plant Molecular Biology, 2009,69:287-298.
[17]	KAJIKAWA M, SHOJI T, KATO A . Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco. Plant Physiology, 2011,155:2010-2022.
[18]	SHOJI T, HASHIMOTO T . Tobacco MYC2 regulates jasmonate inducible nicotine biosynthesis genes directly and by way of the NIC2 locus ERF genes. Plant & Cell Physiology, 2011,52(6):1117-1130.
[19]	LEGG P D, CHAPLIN J F, COLLINS G B . Inheritance of percent total alkaloids in Nicotiana tabacum L.: Populations derived from crosses of low alkaloid lines with burley and flue-cured varieties. Journal of Heredity, 1969,60:213-217.
[20]	HIBI N, HIGASHIGUCHI S, HASHIMOTO T, YAMADA Y . Gene expression in tobacco low-nicotine mutants. The Plant Cell, 1994,6(5):723-735.
[21]	WASTERNACK C, SONG S . Jasmonates: Biosynthesis, metabolism, and signaling by proteins activating and repressing transcription. Journal of Experimental Botany, 2017,68(6):1303-1321.
[22]	MEMELINK , JOHAN . Regulation of gene expression by jasmonate hormones. Phytochemistry, 2009,70(13):1560-1570.
[23]	ZHANG H B, BOKOWIEC M T, RUSHTON P J . Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis. Molecular Plant, 2012,5(1):73-84.
[24]	SHOJI T, KAJIKAWA M, HASHIMOTO T . Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. The Plant Cell, 2010,22(10):3390-3409.
[25]	董娜, 张增艳, 辛志勇 . 病原诱导的小麦转录因子TaERF1b基因的分离和表达. 中国农业科学, 2008,41(4):946-953.
	DONG N, ZHANG Z Y, XIN Z Y . Isolation and expression analysis of a pathogen-induced ERF gene in Triticum aestivum L. Scientia Agricultura Sinica, 2008,41(4):946-953. (in Chinese)
[26]	BOER K D, TILLEMAN S, PAUWELS L . APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis. The Plant Journal: for Cell and Molecular Biology, 2011,66(6):1053-1065.
[27]	GILMOUR S J, SEBOLT A M, SALAZAR M P . Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology, 2000,124(4):1854-1865.
[28]	SHARABI S M, SAMACH A, PORAT R . Overexpression of the CBF2 transcriptional activator in Arabidopsis suppresses the responsiveness of leaf tissue to the stress hormone ethylene. Plant Biology, 2010,12(4):630-638.
[29]	SHOJI T, HASHIMOTO T . Expression of a tobacco nicotine biosynthesis gene depends on the JRE4 transcription factor in heterogenous tomato. Journal of Plant Research, 2019,132(2):173-180.
[30]	赵云祥, 徐兆师, 陈明, 李连城, 陈耀锋, 邱志刚, 熊祥进, 马有志 . 小麦ERF类转录因子W17的结合特异性及亚细胞定位分析. 中国农业科学, 2008,41(6):1575-1582.
	ZHAO Y X, XU Z S, CHEN M, LI L C, CHEN Y F, QIU Z G, XIONG X J, MA Y Z . Analysis of specific binding and subcellular localization of wheat ERF transcription factor W17. Scientia Agricultura Sinica, 2008,41(6):1575-1582. (in Chinese)
[31]	ZHANG H B, ZHANG D B, CHEN J, YANG Y H, HUANG Z J, HUANG D F, WANG X C, HUANG R F . Tomato stress-responsive factor TSRF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum. Plant Molecular Biology, 2004,55(6):825-834.
[32]	CHAKRAVARTHY S, TUORI R P, D'ASCENZO M D, FOBERT P R, DESPRES C, MARTIN G B . The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements. The Plant Cell, 2003,15(12):3033-3050.
[33]	SHOJI T, HASHIMOTO T . Stress-induced expression of NICOTINE2- locus genes and their homologs encoding ethylene response factor transcription factors in tobacco. Phytochemistry, 2015,113:41-49.
[34]	LEGG P D, COLLINS G B, LITTON C C . Registration of La Burley 21 tobacco germplasm1 registration No. (GP 8). Crop Science, 1970,10(2):212.