Cloning of the phosphoenolpyruvate carboxylase gene (PEPC) from Isoetes shangrilaensis X. Liu and construction of its expression vector
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摘要: 以中国特有植物香格里拉水韭（Isoetes shangrilaensis X.Liu）为材料，通过转录组测序数据分析筛选出磷酸烯醇式丙酮酸羧化酶基因（IsPEPC），根据该基因序列，从香格里拉水韭cDNA中克隆获得磷酸烯醇式丙酮酸羧化酶（PEPCase）的编码基因IsPEPC，并将此基因插入pCAMBIA-2300-N-eGFP及pMD质粒载体上，再采用农杆菌介导的花序浸染法将2个重组载体分开转入野生型拟南芥（Arabidopsis thaliana（L.）Heynh.）中。结果显示：IsPEPC基因蛋白编码序列长度为2928 bp，编码975个氨基酸；同源性检索分析结果表明，该蛋白与其近源物种江南卷柏（Selaginella moellendorffii Hieron.）的PEPC基因蛋白序列同源性为79.8%。对转基因的T1代拟南芥通过抗性筛选并在gDNA水平上阳性鉴定，初步鉴定得到pC2300-N-eGFP-IsPEPC转基因株系26个和pMD-IsPEPC转基因株系32个。Abstract: Using Chinese Isoetes shangrilaensis X. Liu, the phosphoenolpyruvate carboxylase gene (IsPEPC) sequence information was screened by transcriptome sequencing analysis, and the IsPEPC gene encoding the phosphoenolpyruvate carboxylase (PEPCase) was cloned from cDNA based on the gene sequence. The IsPEPC gene was inserted into the pCAMBIA-2300-N-eGFP and pMD plasmid vectors, after which the recombinant vectors were transferred into wild-type Arabidopsis thaliana (L.) Heynh. by agrobacterium-mediated inflorescence infiltration. Results showed that the protein-coding sequence of the IsPEPC gene was 2928 bp in length and encoded 975 amino acids. Homologous searching showed that the protein was related to the PEPC protein sequence of the source species Selaginella moellendorffii Hieron., with 79.8% sequence homology. Transgenic T1 A. thaliana was screened for resistance and positively identified at the gDNA level. We initially identified 26 pC2300-N-eGFP-IsPEPC transgenic lines and 32 pMD-IsPEPC transgenic lines.
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[1] 李丽,申双和, 孙钢, 李永秀, 王晓东, 刘瑞娜. 土壤水分对冬小麦气孔导度及光合速率的影响与模拟[J]. 中国农业气象, 2016, 37(6):666-673. Li L, Shen SH, Sun G, Li YX, Wang XD, Liu RN. Simulation on and impact of soil moisture on stomatal conduc-tance and photosynthesis rate of winter wheat[J]. Chinese Journal of Agrometeorology, 2016, 37(6):666-673.
[2] 井忠平. CAM植物的研究概况[J]. 自然杂志, 1987, 12(6):425-429. Jing ZP. Research overview of CAM plants[J]. Journal of Nature, 1987, 12(6):425-429.
[3] Gennidakis S, Rao S, Greenham K, Uhrig RG, O'leary B, et al. Bacterial-and plant-type phosphoenolpyruvate carboxylase polypeptides interact in the hetero-oligomeric Class-2 PEPC complex of developing castor oil seeds[J]. Plant J, 2007, 52(5):839-849.
[4] Roberts K, Granum E, Leegood RC, Raven JA. C3 and C4 pathways of photosynthetic carbon assimilation in marine diatoms are under genetic, not environmental, control[J]. Plant Physiol, 2007, 145(1):230-235.
[5] Deng H, Zhang LS, Zhang GQ, Zheng BQ, Liu ZJ, Wang Y. Evolutionary history of PEPC genes in green plants:Implications for the evolution of CAM in orchids[J]. Mol Phylogenet Evol, 2016, 94:559-564.
[6] Zhao Y, Guo A, Wang Y, Hua J. Evolution of PEPC gene family in Gossypium reveals functional diversification and GhPEPC genes responding to abiotic stresses[J]. Gene, 2019, 698:61-71.
[7] Masumoto C, Miyazawa SI, Ohkawa H, Fukuda T, Ta-niguchi Y, et al. Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation[J]. Proc Natl Acad Sci, 2010, 107(11):5226-5231.
[8] Fan ZQ, Li JY, Lu MZ, Li X, Yin HF. Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum[J]. Acta Physiol Plant, 2013, 35(7):2269-2279.
[9] O'leary B, Park J, Plaxton WC. The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase):recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs[J]. Biochem J, 2011, 436(1):15-34.
[10] 张小娟. 转PEPC基因水稻苗期优势C4光合途径初探[D]. 南京:南京师范大学, 2014. [11] 邓华. 兰科植物景天酸代谢(CAM)途径研究[D]. 北京:中国林业科学研究院, 2015. [12] Pigg KB. Isoetalean lycopsid evolution:from the devonian to the present[J]. Am Fern J, 2001, 91(3):99-114.
[13] Pedersen O, Rich SM, Pulido C, Cawthray GR, Colmer TD. Crassulacean acid metabolism enhances underwater photosynthesis and diminishes photorespiration in the aquatic plant Isoetes australis[J]. New Phytol, 2011, 190(2):332-339.
[14] Keeley JE. Isoetes howellii:a submerged aquatic CAM plant[J]. Am J Bot, 1981, 68(3):420-424.
[15] Green WA. The function of the aerenchyma in arborescent lycopsids:evidence of an unfamiliar metabolic strategy[J]. Proc Biol Sci, 2010, 277(1692):2257-2267.
[16] Yang T, Liu X. Comparing photosynthetic characteristics of Isoetes sinensis Palmer under submerged and terrestrial conditions[J]. Sci Rep, 2015, 5:17783.
[17] 张艳, 满为群, 南相日, 李柱刚. 高粱C4型PEPC基因转入大豆可改善大豆光合特性[J]. 分子植物育种, 2015, 13(2):294-300. Zhang Y, Man WQ, Nan XR, Li ZG. Sorghum C4-specific PEPC gene transformed into soybean can improve the photosynthetic characteristics of soybean[J]. Molecular Plant Breeding, 2015, 13(2):294-300.
[18] 杜西河, 许为钢, 胡琳, 张磊, 李艳, 等. 转ZmPEPC与ZmPPDK基因拟南芥对干旱胁迫的反应[J]. 分子植物育种, 2013, 11(4):477-484. Du XH, Xu WG, Hu L, Zhang L, Li Y, et al. Response of maize C4-type PEPC and PPDK transgenic Arabidopsis plants to drought-stress[J]. Molecular Plant Breeding, 2013, 11(4):477-484.
[19] 雷明月, 许为钢, 李小博, 张庆琛, 王会伟, 等. 玉米C4光合酶基因导入对拟南芥光合特性及抗旱性的影响[J]. 麦类作物学报, 2017, 37(1):108-115. Lei MY, Xu WG, Li XB, Zhang QS, Wang HW, et al. Effect of maize C4-specific photosynthesis gene on photosynthesis and drought resistance of Arabidopsis thaliana[J]. Journal of Triticeae, 2017,37(1):108-115.
[20] 钟秀娟. 转玉米C4型PEPC基因大豆的分子特征及光合特性的研究[D]. 南京:南京农业大学, 2015. [21] 吴梅, 张边江, 陈全战, 王荣富. C4高效光合基因在C3植物中的应用研究进展[J]. 中国农学通报, 2010, 26(3):68-71. Wu M, Zhang BJ, Chen QZ, Wang RF. Research progress on introducing efficient photosynthetic C4 genes into C3 plant[J]. Chinese Agricultural Science Bulletin, 2010, 26(3):68-71.
[22] 王玉民. 玉米C4途径关键酶基因(PPDK、NADP-ME)的克隆及PPDK、PEPC在拟南芥中的表达分析[D]. 郑州:河南农业大学, 2012. [23] 张方, 迟伟, 金成哲, 王强, 张其德, 吴乃虎. 高粱C4型磷酸烯醇式丙酮酸羧化酶基因的分子克隆及其转基因水稻的培育[J]. 科学通报, 2003, 48(14):1542-1546. Zhang F, Chi W, Jin CZ, Wang Q, Zhang QD, Wu NH. Molecular cloning of sorghum C4 phosphoenolpyruvate carboxylase gene and cultivation of transgenic rice[J]. Scientific bulletin, 2003, 48(14):1542-1546.
[24] 曹路遥. 玉米磷酸烯醇式丙酮酸羧化酶基因在拟南芥和烟草中的表达分析[D]. 郑州:河南科技学院, 2018. [25] Keeley JE. Aquatic CAM photosynthesis:a brief history of its discovery[J]. Aquat Bot, 2014, 118:38-44.
[26] Qin N, Xu W, Hu L, Li Y, Wang H, et al. Drought tole-rance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene[J]. Protoplasma, 2016, 253(6):1503-1512.
[27] Deng H, Liang SZ, Guo QZ, Zheng BQ, Liu ZJ, Wang Y. Evolutionary history of PEPC genes in green plants:implications for the evolution of CAM in orchids[J]. Mol Phylogenet Evol, 2016, 94:559-564.
[28] Brautigam A, Schluter U, Eisenhut M, Gowik U. On the evolutionary origin of CAM photosynthesis[J]. Plant Phy-siol, 2017, 174(2):473-477.
[29] 王丽媛, 张玉, 徐明怡, 冷海楠,伍一宁. 植物磷酸烯醇式丙酮酸羧化酶的研究进展[J]. 国土与自然资源研究, 2017, 5:86-89. Wang LY, Zhang Y, Xu MY, Leng HN, Wu YN. The research progress of plant phosphoenolpyruvate carboxy-lase[J]. Territory and Natural Resources Study, 2017, 5:86-89.
[30] Sánchez R, Cejudo FJ. Identification and expression ana-lysis of a gene encoding a bacterial-type phosphoenolpyruvate carboxylase from Arabidopsis and rice[J]. Plant Physiol, 2003, 132(2):949-957.
[31] Pan L, Zhang J, Chen N, Chen M, Wang M, et al. Molecular characterization and expression profiling of the phosphoenolpyruvate carboxylase genes in peanut (Arachis hypogaea L.)[J]. Russ J Plant Physl, 2017, 64(4):576-587.
[32] 马海洋, 赵秋芳, 陈曙, 石伟琦, 冼皑敏. 菠萝PEPC基因家族生物信息学分析[J]. 热带作物学报, 2020, 41(1):97-103. Ma HY, Zhao QF, Chen S, Shi WQ, Xian AM. Bioinformatics analysis of PEPC gene family in pineapple[J]. Chinese Journal of Tropical Crops, 2020, 41(1):97-103.
[33] West-Eberhard MJ, Smith JAC, Winter K. Photosynthesis, reorganized[J]. Science, 2011, 332(6027):311-312.
[34] Winter K, Holtum JAM. Facultative crassulacean acid metabolism (CAM) plants:powerful tools for unravelling the functional elements of CAM photosynthesis[J]. J Exp Bot, 2014, 65(13):3425-3441.
[35] Matiz A, Mioto PT, Mayoragg AY, Mayorga AY, Freschi L, Mercier H. CAM photosynthesis in bromeliads and agaves:what can we learn from these plants?[M]//Dubinsky Z, ed. Photosynthesis. Rijeka:InTech, 2013.
[36] Garcia TM, Arcia TM, Heyduk K, Kuzmick ER, Mayer JA. Crassulacean acid metabolism biology[J]. New Phytol, 2014, 204:738-740.
[37] 李霞, 焦德茂, 戴传超, 王守海, 吴爽, 李成荃. 转育PEPC基因的杂交水稻的光合生理特性[J]. 作物学报, 2001(2):137-143. Li X, Jiao DM, Dai CC, Wang SH, Wu S, Li CM. Photosynthetic characteristics for rice hybrids with transgenic PEPC parent HPTER-01[J]. Acta Agronomica Sinica, 2001(2):137-143.
[38] 李小博, 许为钢, 雷明月, 张庆琛, 王会伟, 等. 转玉米C4光合途径PEPC、ppdk、nadp-me基因拟南芥光合特性对强光胁迫的反应[J]. 分子植物育种, 2017, 15(3):911-919. Li XB, Xu WG, Lei MY, Zhang QC, Wang HW, et al. The response of photosynthetic characteristics of maize C4-type PEPC, ppdk and nadp-me transgenetic Arabidopsis thaliana on high light stress[J]. Molecular Plant Breeding, 2017, 15(3):911-919.
[39] 吴琼, 许为钢, 李艳, 齐学礼, 胡琳, 等. 田间条件下转玉米C4型PEPC基因小麦的光合生理特性[J]. 作物学报, 2011, 37(11):2046-2052. Wu Q, Xu WG, Li Y, Qi XL, Hu L, et al. Physiological characteristics of photosynthesis in transgenic wheat with maize C4-PEPC gene under field conditions[J]. Acta Agronomica Sinica, 2011, 37(11):2046-2052.
[40] 张庆琛, 许为钢, 胡琳, 李艳, 张磊, 齐学礼. 玉米C4型全长PEPC基因导入普通小麦的研究[J]. 麦类作物学报, 2010, 30(2):194-197. Zhang QC, Xu WG, Hu L, Li Y, Zhang L, Qi XL. Deve-lopment of transgenic wheat plants with maize C4-specific PEPC gene by particle bombardment[J]. Journal of Tri-ticeae, 2010, 30(2):194-197.
[41] 焦德茂, 李霞, 黄雪清, 匡廷云, 戈巧英, 等. 转PEPC基因水稻具有初级CO2浓缩机制的生理特点[J]. 中国科学(C辑:生命科学), 2003(1):33-39. Jiao DM, Li X, Huang XQ, Kuang TY, Ge QY, et al. Physiological characteristics of PEPC transgenic rice with primary CO2 concentration mechanism[J]. Chinese Science (C Series:Life Sciences), 2003(1):33-39.
[42] Zhao YP, Guo AH, Wang YM, Hua JP. Evolution of PEPC gene family in Gossypium reveals functional diversification and GhPEPC genes responding to abiotic stresses[J]. Gene, 2019, 698:61-71.
[43] Li YY,Xu JJ, Haq NU, Zhang H, Zhu XG. Was low CO2 a driving force of C4 evolution:Arabidopsis responses to long-term low CO2 stress[J]. J Exp Bot, 2014, 65(13):3657-3667.
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