低温胁迫对转SoTUA基因甘蔗生理生化特性的影响
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摘要
【目的】研究低温胁迫对转α-微管蛋白(TUA)基因甘蔗生理生化特性的影响,对甘蔗TUA基因(SoTUA)进行功能鉴定,为甘蔗抗寒品种选育提供参考依据。【方法】通过对pCAMBIA3300载体上的标记基因GFP进行PCR扩增及测序,鉴定转SoTUA基因阳性甘蔗品系。从中选择4个转基因品系(L1、L2、L3和L4),以野生型ROC22植株为对照(CK),对各处理进行低温(4℃)胁迫处理,在胁迫第0、4、7和12 d时分别测定不同处理甘蔗的生理生化指标。【结果】运用PCR筛选得到转SoTUA基因T1代甘蔗植株,对其中4个品系及CK的低温胁迫试验结果表明,在低温下各品系可溶性糖含量以不同模式变化,转基因各品系普遍高于CK;各品系中的可溶性蛋白在冷驯化前期强烈诱导,总体变化趋势为先升高后降低,转基因各品系普遍高于CK;各品系中丙二醛(MDA)含量增加,其中CK的增幅最大;过氧化物酶(POD)活性下降,除L2外,POD活性变化总体上呈先升高后降低的变化趋势;脯氨酸含量在低温胁迫过程中的变化趋势无明显规律。利用改进的极点排序法对各品系抗寒性进行评价,可知各品系抗寒性强弱排序为:L3>L1>L2>L4>CK。【结论】低温胁迫下4个转SoTUA基因甘蔗品系在生理生化指标上的表现均优于CK,即SoTUA基因在甘蔗体内的过量表达有利于甘蔗抗寒性能的提高。
【Objective】This study was conducted to investigate the effects of low temperature stress on physiological and biochemical characteristics of α-tubulin(TUA)transgenic sugarcane,and to identify the function of sugarcane TUA gene(SoTUA). It provided reference for sugarcane cold-resistant variety breeding.【Method】SoTU Atransgenic sugarcane lines were identified by PCR amplification and sequencing of GFP marker gene on vector pCAMBIA3300. Four transgenic lines(L1,L2,L3 and L4)were chosen for a low temperature(4 ℃)stress assay when the wild type of sugarcane variety ROC22 was used as control(CK). The changes of physiological and biochemical indexes were measured at 0,4,7,12 d after treatment.【Result】The SoTUA transgenic T1 sugarcane plants were screened by PCR,and the results of low temperature stress assay on four lines and the control showed that the soluble sugar content of each line varied in different patterns at low temperature,and the transgenic lines were generally higher than the CK. Soluble protein of all lines was strongly induced in the early stage of cold treatment,the overall change trend was first increased and then decreased,and the transgenic lines were generally higher than the CK. Malonaldehyde(MDA)content in each line increased,especially in the control. Peroxidase(POD)activity decreased,except for L2,POD activity generally increased and then decreased. The proline content change had no obvious regulations during the low temperature stress. The improved pole ordination method was used for the comprehensive evaluation of cold resistance. The results showed that the cold resistance of all the lines was ranked as L3>L1>L2>L4>CK. 【Conclusion】The physiological and biochemical performances of the four SoTUA transgenic sugarcane lines were better than the CK under low temperature stress. Therefore,it can be concluded that the overexpression of SoTUA gene in sugarcane is conducive to the improvement of cold resistance.
【Objective】This study was conducted to investigate the effects of low temperature stress on physiological and biochemical characteristics of α-tubulin(TUA)transgenic sugarcane,and to identify the function of sugarcane TUA gene(SoTUA). It provided reference for sugarcane cold-resistant variety breeding.【Method】SoTU Atransgenic sugarcane lines were identified by PCR amplification and sequencing of GFP marker gene on vector pCAMBIA3300. Four transgenic lines(L1,L2,L3 and L4)were chosen for a low temperature(4 ℃)stress assay when the wild type of sugarcane variety ROC22 was used as control(CK). The changes of physiological and biochemical indexes were measured at 0,4,7,12 d after treatment.【Result】The SoTUA transgenic T1 sugarcane plants were screened by PCR,and the results of low temperature stress assay on four lines and the control showed that the soluble sugar content of each line varied in different patterns at low temperature,and the transgenic lines were generally higher than the CK. Soluble protein of all lines was strongly induced in the early stage of cold treatment,the overall change trend was first increased and then decreased,and the transgenic lines were generally higher than the CK. Malonaldehyde(MDA)content in each line increased,especially in the control. Peroxidase(POD)activity decreased,except for L2,POD activity generally increased and then decreased. The proline content change had no obvious regulations during the low temperature stress. The improved pole ordination method was used for the comprehensive evaluation of cold resistance. The results showed that the cold resistance of all the lines was ranked as L3>L1>L2>L4>CK. 【Conclusion】The physiological and biochemical performances of the four SoTUA transgenic sugarcane lines were better than the CK under low temperature stress. Therefore,it can be concluded that the overexpression of SoTUA gene in sugarcane is conducive to the improvement of cold resistance.
引文
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贾亚雄,李向林,袁庆华,万里强,孟芳.2008.披碱草属野生种质资源苗期耐盐性评价及相关生理机制研究[J].中国农业科学,41(10):2999-3007.[Jia Y X,Li X L,Yuan Q H,Wan L Q,Meng F.2008.Evaluation of salt-tolerance and the related physiological characteristics of wild Elymus spp.[J].Scientia Agricultura Sinica,41(10):2999-3007.]
李合生.2000.植物生理生化试验原理和技术[M].北京:高等教育出版社.[Li H S.2000.Principles and Techniques of Plant Physiological and Biochemical Experiments[M].Beijing:Higher Education Press.]
李杨瑞.2010.现代甘蔗学[M].北京:中国农业出版社.[Li Y R.2010.Modern Sugarcane Science[M].Beijing:China Agriculture Press.]
刘雪梅,尚庆茂,张志刚.2010.辣椒不同品种种子萌芽期耐低温性及评价方法研究[J].中国生态农业学报,18(3):521-527.[Liu X M,Shang Q M,Zhang Z G.2010.Lowtemperature tolerance of pepper at germination stage and its evaluation method[J].Chinese Journal of Eco-Agriculture,18(3):521-527.]
罗明珠,刘子凡,梁计南,魏延明.2005.甘蔗抗旱性与叶片某些生理、生化性状的关系[J].亚热带农业研究,1(1):14-16.[Luo M Z,Liu Z F,Liang J N,Wei Y M.2005.The relationship between drought resistance of sugarcane and some physiological biochemical properties of leaves[J].Subtropical Agricultural Research,1(1):14-16.]
欧钊荣,谭宗琨,何艳,匡昭敏,陈汇林,莫小华,黄中艳.2008.影响我国甘蔗主产区甘蔗产量的关键气象因子及其丰欠指标[J].安徽农业科学,36(24):10407-10410.[Ou Z R,Tan Z K,He Y,Kuang Z M,Chen H L,Mo XH,Huang Z Y.2008.The key meteorological factors affecting the sugarcane yield in major production areas in China and their high-low yield indices[J].Journal of Anhui Agricultural Science,36(24):10407-10410.]
邵敏.2015.甘蔗宿根矮化病病原菌膜蛋白Lxx18460基因的特性研究[D].南宁:广西大学.[Shao M.2015.Study on the characteristics of membrane protein Lxx18460 gene of sugarcane root dwarfing disease pathogens[D].Nanning:Guangxi University.]
孙波.2016.低温胁迫对甘蔗幼苗根系生长代谢的影响和相关基因α-tubulin的功能研究[D].南宁:广西大学.[Sun B.2016.Effect of low temperature stress on root growth and metabolism in sugarcane seedlings and the function analysis ofα-tubulin gene[D].Nanning:Guangxi University.]
孙波,刘光玲,杨丽涛,李杨瑞.2016.甘蔗幼苗根系形态结构及保护系统对低温胁迫的响应[J].中国农业大学学报,19(6):71-81.[Sun B,Liu G L,Yang L T,Li Y R.2016.Response of chilling stress on root morphology and protection systems of sugarcane seedlings[J].Journal of China Agricultural University,19(6):71-81.]
汤章城.1999.现代植物生理学实验指南[M].北京:科学出版社.[Tang Z C.1999.Guidelines for Modern Plant Physiology Experiments[M].Beijing:Science Press.]
唐仕云,杨丽涛,李杨瑞.2012.不同甘蔗基因型对低温胁迫的响应及其耐寒性评价[J].热带作物学报,33(4):635-641.[Tang S Y,Yang L T,Li Y R.2012.Response of different sugarcane genotypes to low temperature and evaluation for cold tolerance of sugarcane[J].Chinese Journal of Tropical Crops,33(4):635-641.]
吴佳溶,陈志厚,徐茜,徐辰生.2018.南平烟区抗寒优质烤烟新品种选育研究[J].江西农业学报,30(6):63-66.[Wu J R,Chen Z H,Xu Q,Xu C S.2018.Research on breeding of cold-resistance and high-quality flue-cured tobacco new varieties in Nanpin garea[J].Acta Agriculturae Jiangxi,30(6):63-66.]
严寒静,谈锋.2005.桅子叶片生理特性与抗寒性的关系[J].植物资源与环境学报,14(4):21-24.[Yan H J,Tan F.2005.Relationship between physiological characteristics of gardenia leaf and cold resistance[J].Journal of Plant Resources and Environment,14(4):21-24.]
杨德光,孙玉珺,Ali Raza Irfan,刘昕萌,吕金莹,尉菊萍,宫磊,刘哲,白冰.2018.低温胁迫对玉米发芽及幼苗生理特性的影响[J].东北农业大学学报,49(5):1-8.[Yang DG,Sun Y J,Irfan A R,Liu X M,LüJ Y,Wei J P,Gong L,Liu Z,Bai B.2018.Effect of low temperature stress on germination and physiological of maize seedling[J].Journal of Northeast Agricultural University,49(5):1-8.]
张保青.2013.低温胁迫下甘蔗后期生理特性及差异蛋白质组学研究[D].南宁:广西大学.[Zhang B Q.2013.Physiological characteristics and differential proteomics of sugarcane under low temperature stress[D].Nanning:Guangxi University.]
张远兵,刘爱荣,王兵,李根.2010.铜胁迫下7个高羊茅品种耐性和铜积累能力的比较[J].热带作物学报,31(5):750-757.[Zhang Y B,Liu A R,Wang B,Li G.2010.The comparison of copper tolerance and copper accumulation activity of seven festuca arundinacea varieties under copper stress[J].Chinese Journal of Tropical Crops,31(5):750-757.]
张云.2015.华南忍冬ARF1、TUA3以及AP4-μ基因克隆与功能验证[D].武汉:华中科技大学.[Zhang Y.2015.Cloning and functional verification of ARF1,TUA3 and AP4-μgenes from Lonicerajaponicain south China[D].Wuhan:Huazhong University of Science and Technology.]
邹凯茜,商桑,田丽波,朱国鹏,周萌萌,潘琼玉,曾丽萍.2018.低温胁迫对嫁接苦瓜幼苗渗透调节物质的影响[J].热带作物学报,39(8):1533-1539.[Zou K X,Shang S,Tian L B,Zhu G P,Zhou M M,Pan Q Y,Zeng L P.2018.Effects of low temperature stress on osmotic solutes of grafted bitter gourd seedlings[J].Chinese Journal of Tropical Crops,39(8):1533-1539.]
Abdrakhamanova A,Wang Q Y,Khokhlova L,Nick P.2003.Is microtubule disassembly a trigger for cold acclimation?[J].Plant and Cell Physiology,44(7):676-686.
Apel K,Hirt H.2004.Reactive oxygen species:Metabolism,oxidative stress,and signal transduction[J].Annual Review of Plant Biology,55:373-399.
Gilroy S,Trewavas A.2001.Signal processing and transduction in plant cells:The end of the beginning?[J].Nature Reviews Molecular Cell Biology,2(4):307-314.
Kaplan F,Sung D Y,Guy C L.2010.Roles ofβ-amylase and starch breakdown during temperature stress[J].Physiologia Plantarum,126(1):120-128.
Kazemi-Shahandashti S S,Maali-Amiri R,Zeinali H,Ramezanpour S S.2013.Change in membrane fatty acid compositions and cold-induced responses in chickpea[J].Molecular Biology Reports,40:893-903.
Kerr G P,Carter J V.1990.Relationship between freezing tolerance of root-tip cells and cold stability of microtubules in rye(Secale cereal L.cv Puma)[J].Plant Physiology,93(1):77-82.
Liu H M,Wen Y H,Wang S K,You Y.2003.A review of plant cold resistance[J].Northern Horticulture,6:14-5.
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