嗜盐芽胞杆菌冷激蛋白基因HhCsp提高玉米苗期耐旱性

doi:10.13560/j.cnki.biotech.bull.1985.2026-0371

摘要/Abstract

摘要：

目的针对商业化转基因玉米品种不足和干旱造成的减产问题，利用嗜盐芽胞杆菌（Halobacillus halophilus, H. halophilus）冷激蛋白（Cold shock protein, Csp）基因创制耐旱转基因株系，为培育耐旱玉米种质奠定基础。方法利用生物信息学方法对HhCsp序列进行分析，经密码子优化并合成，构建HhCsp双子叶植物表达载体转化拟南芥进行耐旱性初步鉴定。构建HhCsp单子叶植物表达载体，经农杆菌介导法转化玉米，并鉴定耐旱性。结果序列分析表明，HhCsp序列长198 bp，编码65个氨基酸，分子量为7.3 kD，等电点为4.47，包含Csp家族典型且高度保守的RNA结合基序RNP1和RNP2。密码子优化后，HhCsp序列GC含量由37.4%提升至50%，转化拟南芥后可增强转基因株系的耐旱性。进一步将优化的HhCsp转化玉米。PCR、反转录PCR（reverse transcript PCR, RT-PCR）、实时荧光定量PCR（real-time quantitative PCR, RT-qPCR）及蛋白免疫印迹杂交（Western Blot, WB）检测结果表明，在4个转基因玉米株系中HhCsp成功插入玉米基因组，且正常转录和翻译。表型鉴定结果证实，干旱胁迫后，与野生型（wild type, WT）相比，所有转基因玉米株系均萎蔫程度更轻。而且转基因株系主根长、生物量、根冠比及相对含水量均显著大于WT；相反，干旱后，转基因株系叶片相对电导率与丙二醛含量均显著低于WT。结论异源表达HhCsp基因可增强转基因玉米苗期的耐旱性。

关键词: 玉米, 干旱, 冷激蛋白, 基因工程

Abstract:

Objective To address the scarcity of commercialized transgenic maize varieties and maize yield losses caused by drought stress, drought-tolerant transgenic lines expressing the cold shock protein (Csp) gene from Halobacillus halophilus (H. halophilus) were developed, thereby laying a foundation for the breeding of drought-tolerant transgenic maize germplasm. Methods The HhCsp gene sequence was analyzed using bioinformatics methods. Following codon optimization and synthesis, a dicotyledonous plant expression vector harboring HhCsp was constructed and introduced into Arabidopsis thaliana for a preliminary drought tolerance assessment. Finally, a monocotyledonous plant expression vector carrying HhCsp was constructed and introduced into maize via Agrobacterium-mediated transformation, followed by an assessment of drought tolerance in the transgenic lines. Result Sequence analysis revealed that the coding sequence (CDS) of HhCsp is 198 bp in length, encoding 65 amino acids with a molecular weight of 7.3 kD and an isoelectric point (pI) of 4.47. This protein contains typical, highly conserved RNA-binding motifs RNP1 and RNP2, which are characteristic of the Csp family. Following codon optimization, the GC content of the HhCsp sequence was increased from 37.4% to 50.0%. Transgenic Arabidopsis lines expressing HhCsp showed enhanced drought tolerance. Furthermore, the codon-optimized HhCsp gene was introduced into maize. The results of PCR, reverse transcription PCR (RT-PCR), real-time quantitative PCR (RT-qPCR), and Western blot (WB) assays demonstrated that HhCsp was successfully integrated into the maize genome and was normally transcribed and translated in four transgenic maize lines. Phenotypic evaluation confirmed that under drought stress, all transgenic maize lines showed only mild wilting compared with the wild type (WT). In addition, the primary root length, biomass, root-shoot ratio, and relative water content of the transgenic lines were significantly higher than those of the WT. In contrast, the relative electrolyte leakage and malondialdehyde content in leaves of the transgenic lines were significantly lower than those of the WT after drought treatment. Conclusion Heterologous expression of the HhCsp gene enhances the drought tolerance of transgenic maize lines at the seedling stage.

Key words: maize, drought, cold-shock protein, genetic engineering

于好强, 张鑫, 段华明, 万涛, 鄢腊梅, 曹博, 杨青青, 付凤玲, 李晚忱. 嗜盐芽胞杆菌冷激蛋白基因HhCsp提高玉米苗期耐旱性[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0371.

YU Hao-qiang, ZHANG Xin, DUAN Hua-ming, WAN Tao, YAN La-mei, CAO Bo, YANG Qing-qing, FU Feng-ling, LI Wan-chen. Heterologous Expression of Cold Shock Protein Gene HhCsp from Halobacillus Halophilus Enhances Drought Tolerance of Maize Seedling[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0371.

图/表 7

图1 不同来源的Csp氨基酸序列分析A：多序列比对；B：系统进化树分析。BsCsp，枯草芽胞杆菌冷激蛋白（GenBank号：NP_388791.1）；DeCsp，恩克莱恩屋芽胞杆菌冷激蛋白（GenBank号：WP_045851116.1）；FmCsp，澳门假芽胞杆菌（GenBank号：WP_007201157.1）；HhCsp嗜盐芽胞杆菌（GenBank号：CCG43925.1）；PhCsp，嗜盐海芽胞杆菌（GenBank号：WP_026800953.1）； PlCsp，海滨海芽胞杆菌（GenBank号：WP_036833629.1）；PyCsp，盐城海芽胞杆菌（GenBank号：WP_036818395.1）；TpCsp，沼泽毛球菌（GenBank号：CZQ94952.1）

Fig. 1 Amino acid sequence analysis of different Csp proteinsA: Multiple-sequence alignment; B: phylogenetic tree analysis; BsCsp, cold shock protein from Bacillus subtilis (GenBank No. NP_388791.1); DeCsp, cold shock protein from Domibacillus enclensis (GenBank No. WP_045851116.1); FmCsp, cold shock protein from Fictibacillus macauensis (WP_007201157.1); HhCsp, cold shock protein from Halobacillus halophilus (GenBank No. CCG43925.1); PhCsp, cold shock protein from Pontibacillus halophilus (GenBank No. WP_026800953.1); PlCsp, cold shock protein from Pontibacillus litoralis (GenBank No. WP_036833629.1); PyCsp, cold shock protein from Pontibacillus yanchengensis (GenBank No. WP_036818395.1); TpCsp, cold shock protein from Trichococcus palustris (GenBank No. CZQ94952.1)

图2 密码子优化前后HhCsp序列比对HhCsp：优化前的序列；HhCsp-o：优化后的序列

Fig. 2 Sequence alignment of HhCsp before and after codon optimizationHhCsp: sequence before codon optimization. HhCsp-o: codon optimized sequence

图3 转HhCsp拟南芥株系鉴定A：PCR检测；B：RT-PCR检测。+阳性对照（35S-HhCsp质粒作模板）；-阴性对照（WT的DNA和cDNA分别作模板）；WT，野生型；OE1、OE2、OE3分别表示转HhCsp基因拟南芥株系

Fig. 3 Identification of transgenic Arabidopsis line expressing HhCspA: PCR detection of transgenic lines; B: RT-PCR detection of transgenic lines. +positive control (35S-HhCsp plasmid served as template); -negative control (DNA and cDNA from WT used as template, respectively); WT, wild type; OE1, OE2, and OE3 represent transgenic Arabidopsis lines with HhCsp, respectively

图4 转HhCsp基因增强拟南芥株系耐旱性A：转基因株系在渗透胁迫培养基上的表型；B：转基因株系自然干旱后表型；C：根长统计结果；D：自然干旱后各株系存活率。WT，野生型；OE1、OE2与OE3，分别表示转HhCsp基因拟南芥株系。*和**，分别表示在P<0.05和P<0.01水平差异显著。n=36，误差线代表标准差，下同

Fig. 4 Expression of the HhCsp gene enhances drought tolerance in transgenic Arabidopsis linesA: Phenotype of transgenic lines on the medium with mannitol. B: Drought phenotype of transgenic lines after natural drought stress. C: Statistical data of root length. D: Survival rate of each line after drought stress. WT, wild type; OE1, OE2, and OE3 represent transgenic Arabidopsis lines carrying HhCsp, respectively. * and ** indicate significance at P<0.05 and P<0.01, respectively. n=36. Error bars indicate standard derivation (SD), the same below

图5 转HhCsp基因玉米株系鉴定A：喷施草铵膦后的表型，左侧为阴性苗，右侧为阳性苗；B：PCR检测结果；C：RT-qPCR检测结果；D：RT-PCR结果；E：WB检测结果。WT：野生型；L1、L2、L3与L4分别表示转HhCsp基因玉米株系。+阳性对照（Ubi-HhCsp质粒作模板）

Fig. 5 Identification of transgenic maize lines expressing the HhCsp geneA: Phenotype of transgenic maize after spraying glufosinate; Negative (left) and positive (right) transgenic seedlings were shown; B: PCR detection result; C: RT-qPCR detection result; D: RT-PCR detection result; E: WB result. WT: wild type; L1, L2, L3, and L4 represent transgenic maizelines carrying the HhCsp gene, respectively. + positive control (35S-HhCsp plasmid served as template)

图6 转HhCsp基因增强玉米耐旱性A：转基因株系自然干旱后表型；B：转基因株系自然干旱后根系表型；C：根长统计结果；D：各株系生物量；E：各株系的根冠比。WT，野生型；L1、L2、L3与L4，分别表示转HhCsp基因玉米株系

Fig. 6 Expression of the HhCsp gene enhances drought tolerance in maizeA: Phenotype of transgenic lines after natural drought stress. B: Root phenotype of transgenic lines after natural drought stress. C: Statistical data of root length. D: Biomass of each line. E: Root/shoot ratio of every line. WT, wild type; L1, L2, L3, and L4 represent transgenic maizelines with the HhCsp gene, respectively

图7 各株系生理指标A：相对含水量；B：相对电导率；C：丙二醛含量。WT，野生型；L1、L2、L3与L4，分别表示转HhCsp基因玉米株系

Fig. 7 Physiological indicators of each lineA: Relative water content (RWC). B: Relative electrical conductivity (REC). C: Malondialdehyde (MDA) content. WT, wild type; L1, L2, L3, and L4 represent transgenic maizelines carrying the HhCsp gene, respectively

参考文献 36

[1]	Yang ZR, Cao YB, Shi YT, et al. Genetic and molecular exploration of maize environmental stress resilience: Toward sustainable agriculture [J]. Mol Plant, 2023, 16(10): 1496-1517.
[2]	Kim KH, Lee BM. Effects of climate change and drought tolerance on maize growth [J]. Plants, 2023, 12(20): 3548.
[3]	Gupta A, Rico-Medina A, Caño-Delgado AI. The physiology of plant responses to drought [J]. Science, 2020, 368(6488): 266-269.
[4]	Abdul Aziz M, Brini F, Rouached H, et al. Genetically engineered crops for sustainably enhanced food production systems [J]. Front Plant Sci, 2022, 13: 1027828.
[5]	Klee HJ. Ripening physiology of fruit from transgenic tomato (Lycopersicon esculentum) plants with reduced ethylene synthesis [J]. Plant Physiol, 1993, 102(3): 911-916.
[6]	Cheng XR, Li HH, Tang QL, et al. Trends in the global commercialization of genetically modified crops in 2023 [J]. J Integr Agric, 2024, 23(12): 3943-3952.
[7]	Yu HQ, Wang YG, Fu FL, et al. Transgenic improvement for biotic resistance of crops [J]. Int J Mol Sci, 2022, 23(22): 14370.
[8]	Li ZX, Zhang JR, Song XY. Breeding maize hybrids with improved drought tolerance using genetic transformation [J]. Int J Mol Sci, 2024, 25(19): 10630.
[9]	Yang XX, Cheng JK, Wang FE, et al. ZmMPK5-mediated ZmOCL1 phosphorylation positively regulates drought tolerance by promoting the induction of ZmDHN2 in maize [J]. New Phytol, 2026, 250(1): 347-365.
[10]	Yang SP, Wang YJ, Huang Q, et al. A pangenome of maize provides genetic insights into drought resistance [J]. Nat Genet, 2025, 57(11): 2831-2841.
[11]	Castiglioni P, Warner D, Bensen RJ, et al. Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions [J]. Plant Physiol, 2008, 147(2): 446-455.
[12]	Tollefson J. Drought-tolerant maize gets US debut [J]. Nature, 2011, 469(7329): 144.
[13]	丁风鹅. 海洋微生物CSP基因提高转基因玉米的抗旱和耐寒性 [D]. 大连: 大连理工大学, 2018.
	Ding FE. The cold shock protein gene CSP from marine microorganism improves drought and cold tolerance of the transgenic maize [D]. Dalian: Dalian University of Technology, 2018.
[14]	Kim SY, Kim JS, Cho W, et al. A cold-shock protein from the south pole-dwelling soil bacterium Arthrobacter sp. confers cold tolerance to rice [J]. Genes, 2021, 12(10): 1589.
[15]	王梦姣, 曹钰雪, 徐永盛, 等. 过表达海洋微生物宏基因组MbCSP提高转基因拟南芥的抗旱和耐寒性 [J]. 植物研究, 2022, 42(2): 243-251.
	Wang MJ, Cao YX, Xu YS, et al. Overexpression of marine microbial metagenomic MbCSP enhanced drought and cold tolerance of transgenic Arabidopsis thaliana [J]. Bull Bot Res, 2022, 42(2): 243-251.
[16]	Yu TF, Xu ZS, Guo JK, et al. Improved drought tolerance in wheat plants overexpressing a synthetic bacterial cold shock protein gene SeCspA [J]. Sci Rep, 2017, 7: 44050.
[17]	Jones PG, VanBogelen RA, Neidhardt FC. Induction of proteins in response to low temperature in Escherichia coli [J]. J Bacteriol, 1987, 169(5): 2092-2095.
[18]	Li HX, Giuliodori AM, Wang X, et al. Cold shock proteins mediate transcription of ribosomal RNA in Escherichia coli under cold-stress conditions [J]. Biomolecules, 2025, 15(10): 1387.
[19]	Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from the international DNA sequence databases [J]. Nucleic Acids Res, 1997, 25(1): 244-245.
[20]	Daniel E, Onwukwe GU, Wierenga RK, et al. ATGme: Open-source web application for rare codon identification and custom DNA sequence optimization [J]. BMC Bioinform, 2015, 16: 303.
[21]	Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [J]. Plant J, 1998, 16(6): 735-743.
[22]	Springer NM. Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB [J]. Cold Spring Harb Protoc, 2010, 2010(11): pdb.prot5515.
[23]	Mishra M, Tiwari S, Gomes AV. Protein purification and analysis: next generation Western blotting techniques [J]. Expert Rev Proteom, 2017, 14(11): 1037-1053.
[24]	Gaxiola RA, Li JS, Undurraga S, et al. Drought- and salt-tolerant plants result from overexpression of the AVP1 H⁺-pump [J]. Proc Natl Acad Sci U S A, 2001, 98(20): 11444-11449.
[25]	魏湜, 张翯, 顾万荣, 等. DCPTA对盐胁迫下玉米叶片渗透调节生理生化特征影响 [J]. 东北农业大学学报, 2015, 46(9): 1-8.
	Wei S, Zhang H, Gu WR, et al. Effect of DCPTA on the physiological and biochemical characteristics of osmotic adjustment in maize seedling leaves under salt stress [J]. J Northeast Agric Univ, 2015, 46(9): 1-8.
[26]	邹琦. 植物生理学实验指导 [M]. 北京: 中国农业出版社, 2000.
	Zou Q. Experimental instruction of plant physiology [M]. Beijing: China Agriculture Press, 2000.
[27]	Desale P, Patel B, Singh S, et al. Plant growth promoting properties of Halobacillus sp. and Halomonas sp. in presence of salinity and heavy metals [J]. J Basic Microbiol, 2014, 54(8): 781-791.
[28]	Köcher S, Müller V. The nature and function of carotenoids in the moderately halophilic bacterium Halobacillus halophilus [M]//Ventosa A, Oren A, Ma YH. Halophiles and Hypersaline Environments: Current Research and Future Trends. Berlin, Heidelberg: Springer, 2011: 303-317.
[29]	刘庆坡, 冯英, 董辉. 20个物种同义密码子偏性的比较分析 [J]. 西北农林科技大学学报: 自然科学版, 2004, 32(7): 67-71.
	Liu QP, Feng Y, Dong H. Comparative studies on synonymous codon usage bias in twenty species [J]. J Northwest Sci Tech Univ Agric For Nat Sci Ed, 2004, 32(7): 67-71.
[30]	Chen H, Tang W, Xu CG, et al. Transgenic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against lepidopteran rice pests [J]. Theor Appl Genet, 2005, 111(7): 1330-1337.
[31]	Jabeen R, Khan MS, Zafar Y, et al. Codon optimization of cry1Ab gene for hyper expression in plant organelles [J]. Mol Biol Rep, 2010, 37(2): 1011-1017.
[32]	Meyer P. Variation of transgene expression in plants [J]. Euphytica, 1995, 85(1-3): 359-366.
[33]	Vaucheret H, Béclin C, Elmayan T, et al. Transgene-induced gene silencing in plants [J]. Plant J, 1998, 16(6): 651-659.
[34]	Bae W, Xia B, Inouye M, et al. Escherichia coli CspA-family RNA chaperones are transcription antiterminators [J]. Proc Natl Acad Sci U S A, 2000, 97(14): 7784-7789.
[35]	Graumann PL, Marahiel MA. Cold shock proteins CspB and CspC are major stationary-phase-induced proteins in Bacillus subtilis [J]. Arch Microbiol, 1999, 171(2): 135-138.
[36]	Yu HQ, Lv H, Lu FZ, et al. Expression evaluation of exogenous and endogenous alcohol dehydrogenase genes in transgenic Arabidopsis [J]. Front Plant Sci, 2025, 15: 1476754.