Abstract 1,5 ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of photosynthetic carbon assimilation in green plants, and its small structural subunits (Rubisco small subuni, RbcS) affect the function of Rubisco holoenzyme. In this study, the maize (Zea mays) inbred line B73 was material, DNA and RNA were firstly extracted from the leaves, the promoter sequence of ZmRbcS1 gene (GenBank No. 542212) was amplified and its structure was analyzed. Meanwhile, the ORF of the gene was amplified and the vector was constructed. Based on the analysis of the promoter by PlantCARE database, it was found that in addition to a large number of conservative light regulatory elements (such as G-box, GATA-motif, I-box, RbcS-CMA7c, etc.), the promoter sequence also contained a variety of conservative cis-acting elements (such as drought-induced response element (MBS) and gibberellin-responsive element (gibberellin-responsive element) in response to drought, hormones and other stress factors. P-box) and jasmonic acid response element (CGTCA-motif, TGACG-motif). According to the structure characteristics of ZmRbcS1 promoter and the distribution of regulating elements, three upstream primers and one downstream primer were designed for the amplification of promoter fragments of different lengths, with the lengths of 2 114, 1 021 and 566 bp, respectively. Subsequently, the 5' end of the promoter was deleted and the deletion expression vector of the reporter gene gap-glucuronidase (GUS) was constructed by fusing three segments of different lengths, and the promoter function of the instantaneous expression system of tobacco (Nicotiana benthamiana) leaves mediated by Agrobacterium tumefaciens was analyzed. Histochemical staining showed that the promoter activity increased first and then decreased with the reduction of promoter fragment. This study verified that the protein product encoded by ZmRbcS1 gene was located in the chloroplast, and the analysis and identification of its promoter clock-acting element provide the basis for further study on the function of ZmRbcS1 gene.
JIANG Yan-Ping,TIAN Qiu-Zhen,LI Hong-Wei, et al. The Activity Analysis of Promoter and the Subcellular Localization in Maize (Zea mays) RbcS1 Gene[J]. 农业生物技术学报, 2020, 28(8): 1352-1362.
[1] 何亚飞,李霞,谢寅峰.2017.Rubisco 与 Rubisco 活化酶的分子机理研究进展[J].分子植物育种,15(8):3295-3301.
(He Y F,Li X,Xie Y F.2017.Research progress on the molecular mechanism of Rubisco and Rubisco activase[J].Molecular Plant Breeding,15(8):3295-3301.)
[2] 黄海群,林拥军.2007.水稻rbcS基因启动子的克隆及结构功能分析[J].农业生物技术学报,15(3):451-458.
(Huang H Q,Lin Y J.2007.cloning and structural function analysis of rbcS gene promoter in rice[J].Journal of Agricultural Biotechnology,15(3):451-458.)
[3] 李杰,张福城,王文泉,等.2006.高等植物启动子的研究进展[J].生物技术通讯,017(004):658-661.
(Li J,Zhang F C,Wang W Q,et al.2006.Advance in the study of higher plant promoter[J].Letter Biology Technology,017(004):658-661.)
[4] 李尊强,王春军,杨爱国,等.2013.烟草 DXS 基因的克隆及其亚细胞定位分析[J].安徽农业科学,41(30):11957-11960.
(Li Z Q,Wang C J,Yang A G,et al.2013.Cloning and subcellular localization of tobacco DXS gene[J].Anhui Agricultural Science,41(30):11957-11960.)
[5] 李濯雪,陈信波.2015.植物诱导型启动子及相关顺式作用元件研究进展[J].生物技术通报,31(10):8-15.
(Li Z X,Chen X B.2015.Research progress on plant inducible promoter and related cis-acting elements[J].Biotechnology Bulletin,31(10):8-15.)
[6] 刘巧泉,于恒秀,张文娟,等.2005.水稻rbcS启动子控制的外源基因在转基因水稻中的特异性表达[J].植物生理与分子生物学学报,31(3):247-253.
(Liu Q Q,Yu H X,Zhang W J,et al.2005.Specific expression of the foreign gene regulated by the rice rbcS promoter in transgenic rice[J].Journal of Plant Physiology and Molecular Biology,31(3):247-253.)
[7] 吴蕊蕊.2011.盐地碱蓬 SsHKT1 基因启动子的克隆及功能分析[D].硕士学位论文,山东师范大学.导师:王宝山,pp.36-38.
(Wu R R.2011.The Cloning and function analysis of Suaeda salsa SsHKT1 promoter[D].Thesis for M.S.,Shandong Normal University,Supervisor,Wang B S,pp.36-38.)
[8] 薛元夏,邓西平,杨淑慎.2016.参与逆境应答的小麦 RBCS11 基因启动子功能分析[J].农业生物技术学报,24(7):946-956.
(Xue Y X,Deng X P,Yang S S,et al.2016.Functional analysis of wheat RBCS11 gene promoter involved in adverse response[J].Journal of Agricultural Biotechnology,24(7):946-956.)
[9] 赵莉,钟鸣,马慧,等.2011.农杆菌介导的烟草高效遗传转化体系的建立[J].江苏农业科学,39(3):67-68.
(Zhao L,zhong M,Ma H,et al.,2011.Establishment of agrobacterium-mediated efficient genetic transformation system in tobacco[J].Jiangsu Agricultural Sciences,39(3):67-68.)
[10] Bracher A,Whitney S M,Hartl F U,et al.2017.Biogenesis and metabolic maintenance of rubisco[J].Annual Review of Plant Biology,68(1):29-60.
[11] Ewing R M,Jenkins G I,Langdale J A.1998.Transcripts of maize RbcS genes accumulate differentially in C-3 and C-4 tissues[J].Plant Molecular Biology,36(4):593-599.
[12] Feller U,Anders I,Mae T.2007.Rubiscolytics:Fate of rubisco after its enzymatic function in a cell is terminated[J].Journal of Experimental Botany,59(7):1615-1624.
[13] Feiz L,Williams-Carrier R,Wostrikoff K,et al.2012.Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize[J].The Plant Cell,24(8):3435-3446.
[14] Genkov T,Meyer M,Griffiths H,et al.2010 Functional hybrid rubisco enzymes with plant small subunits and algal large subunits engineered rbcS cDNA for expression in Chlamydomonas[J].Journal of Biological Chemistry,285(26):19833-19841.
[15] Gudkov S V,Grinberg M A,Sukhov V,et al.2019.Effect of ionizing radiation on physiological and molecular processes in plants[J].Journal of Environmental Radioactivity,202:8-24.
[16] Jefferson R A.1987.GUS function:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J].The EMBO Journal,6:901-907.
[17] Lescot M.2002.PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,30(1):325-327.
[18] Li R Q,Jiang M,Liu Y H,et al.2017.The xantha marker trait is associated with altered tetrapyrrole biosynthesis and deregulated transcription of PhANGs in rice[J].Frontiers in Plant Science,8:901.
[19] Lin M T,Occhialini A,Andralojc P J,et al.2014.A faster Rubisco with potential to increase photosynthesis in crops[J].Nature,513(7519):547-550.
[20] Marmorstein R.2003.Structure of SET domain proteins:A new twist on histone methylation[J].Trends in Biochemical Sciences,28(2):59-62.
[21] Ni J,Ma X,Feng Y,et al.2019.Updating and interaction of polycomb repressive complex 2 components in maize (Zea mays)[J].Planta,250:1-16.
[22] Outchkourov N S,Peters J,Jong J D,et al.2003.The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants[J].Planta,216(6):1003-1012.
[23] Rolland V,Badger M R,Price G D.2016.Redirecting the cyanobacterial bicarbonate transporters BicA and SbtA to the chloroplast envelope:Soluble and membrane cargos need different chloroplast targeting signals in plants[J].Frontiers in Plant Science,7:185.
[24] Shao R,Zheng H,Yang J,et al.2018.Proteomics analysis reveals that nitric oxide regulates photosynthesis of maize seedlings under water deficiency[J].Nitric Oxide,81:46-56.
[25] Sekhon R S,Lin H,Childs K L,et al.2011.Genome‐wide atlas of transcription during maize development[J].The Plant Journal,66(4):553-563.
[26] Suzuki Y,Nakabayashi K,Yoshizawa R,et al.2009.Differences in expression of the RBCS multigene family and Rubisco protein content in various Rice plant tissues at different growth stages[J].Plant and Cell Physiology,50(10):1851-1855.
[27] Wostrikoff K,Clark A,Sato S,et al.2012.Ectopic expression of rubisco subunits in maize mesophyll cells does not overcome barriers to cell type-specific accumulation[J].Plant Physiology,160(1):419-432.
[28] Wang L,Li L,Xu L,et al.2013.Isolation and functional analysis of the poplar RbcS gene promoter[J].Plant Molecular Biology Reporter,31(1):120-127.
