ZmLTP3基因对玉米的遗传转化及耐盐性鉴定
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摘要
为探讨转脂蛋白在非生物逆境中的作用,将Zm LTP3基因利用花粉管通道法转入玉米自交系京2416中,在PCR和EPSPS速测试纸鉴定基础上连续自交获得转基因阳性株系。以转基因株系OE6、OE10、OE18作为试验组,以自交系京2416为对照(WT),进行盐胁迫处理,比较二者之间在形态指标和生理指标方面的差异。结果显示,在正常生长条件下,转基因株系与野生型的各项指标均无显著差异。在盐胁迫条件下,与野生型相比,转基因株系的株高、茎基宽、根长、鲜质量、干质量及叶绿素含量均显著增加,表现出较好的生长状态;超氧化物歧化酶(SOD)、过氧化物酶(POD)及过氧化氢酶(CAT)活性也显著升高,显示出较高的活性氧清除能力;同时,丙二醛(MDA)含量及相对外渗电导率水平则显著下降,暗示其体内较低程度的细胞伤害水平。以上结果表明,Zm LTP3基因的过量表达可提高玉米的耐盐胁迫能力。
To study the function of lipid transfer proteins( LTP) in abiotic stress,the Zm LTP3 gene was transferred into maize Jing 2416 inbred lines by pollen tube pathway method. Positive transgenic lines were obtained by continuous selfing based on the screening of PCR and EPSPS. Three transgenic lines OE6,OE10 and OE18 were selected as the experimental group,and Jing 2416 inbred lines( WT) were used as CK. Both of them were subjected to salt stress. Morphological and physiological indexes were compared between OE and WT lines. The results showed that OE and WT plants showed no significant differences in both morphological and physiological indexes under normal condition. Under the salt stress condition,compared with WT plants,the OE plants had significantly higher height,stem diameter,root length,fresh weight,dry weight and chlorophyll content,significantly higher activities of superoxide dismutase( SOD),peroxidase( POD) and catalase( CAT),significantly lower malondialdehyde( MDA) content and relative conductivity ratio level,showing their better growth status,higher active oxygen radicals scavenging capacity and less cell injury. These results indicated that overexpression of Zm LTP3 gene could improve the salt tolerance in transgenic maize.
To study the function of lipid transfer proteins( LTP) in abiotic stress,the Zm LTP3 gene was transferred into maize Jing 2416 inbred lines by pollen tube pathway method. Positive transgenic lines were obtained by continuous selfing based on the screening of PCR and EPSPS. Three transgenic lines OE6,OE10 and OE18 were selected as the experimental group,and Jing 2416 inbred lines( WT) were used as CK. Both of them were subjected to salt stress. Morphological and physiological indexes were compared between OE and WT lines. The results showed that OE and WT plants showed no significant differences in both morphological and physiological indexes under normal condition. Under the salt stress condition,compared with WT plants,the OE plants had significantly higher height,stem diameter,root length,fresh weight,dry weight and chlorophyll content,significantly higher activities of superoxide dismutase( SOD),peroxidase( POD) and catalase( CAT),significantly lower malondialdehyde( MDA) content and relative conductivity ratio level,showing their better growth status,higher active oxygen radicals scavenging capacity and less cell injury. These results indicated that overexpression of Zm LTP3 gene could improve the salt tolerance in transgenic maize.
引文
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[13]李杰,郭欣慰,张中保,等.将拟南芥ATNCED3基因导入玉米自交系的研究[J].作物杂志,2014(1):58-62.
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[15]Kumar P,Tewari R K,Sharma P N.Modulation of copper toxicity-induced oxidative damage by excess supply of Iron in maize plants[J].Plant Cell Reports,2008,27(2):399-409.
[16]Zhou Y,Tang N,Huang L,et al.Effects of salt stress on plant growth,antioxidant capacity,glandular trichome density,and volatile exudates of schizonepeta tenuifolia Briq[J].International Journal of Molecular Sciences,2018,19(1):252.
[17]Jacq A,Pernot C,Martinez Y,et al.The Arabidopsis lipid transfer protein 2(At LTP2)is involved in Cuticle-Cell wall interface integrity and in etiolated hypocotyl permeability[J].Frontiers in Plant Science,2017,8:263.
[18]Chalbi N,Martínez-Ballesta M C,Youssef N B,et al.Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity[J].Journal of Plant Physiology,2015,175:148-156.
[19]Liu F,Zhang X,Lu C,et al.Non-specific lipid transfer proteins in plants:presenting new advances and an integrated functional analysis[J].Journal of Experimental Botany,2015,66(19):5663-5681.
[2]Edstam M M,Viitanen L,Salminen T A.Evolutionary history of the Non-Specific lipid transfer proteins[J].Molecular Plant,2011,4(6):947-964.
[3]Smith L J,Roby Y,Allison J R,et al.Molecular dynamics simulations of barley and maize lipid transfer proteins show different ligand binding preferences in agreement with experimental data[J].Biochemistry,2013,52(30):5029-5038.
[4]Edstam M M,Laurila M,Hoglund A,et al.Characterization of the GPI-anchored lipid transfer proteins in the moss Physcomitrella patens[J].Plant Physiology and Biochemistry,2014,75:55-69.
[5]Salminen T A,Blomqvist K,Edqvist J.Lipid transfer proteins:classification,nomenclature,structure,and function[J].Planta,2016,244(5):971-997.
[6]Yeats T H,Rose J K.The biochemistry and biology of extracellular plant lipid-transfer proteins(LTPs)[J].Protein Science,2008,17(2):191-198.
[7]Deeken R,Saupe S,Klinkenberg J,et al.The nonspecific lipid transfer protein At Ltp I-4 is involved in suberin formation of Arabidopsis thaliana crown galls[J].Plant Physiology,2016,172(3):1911-1927.
[8]Cameron K D,Teece M A,Smart L B.Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco[J].Plant Physiology,2006,140(1):176-183.
[9]Jiang Y,Deyholos M K.Functional characterization of Arabidopsis Na Cl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses[J].Plant Molecular Biology,2009,69(1/2):91-105.
[10]Wang N J,Lee C C,Cheng C S,et al.Construction and analysis of a plant non-specific lipid transfer protein database(ns LTPDB)[J].BMC Genomics,2012,13(S1):S9.
[11]Yang Z B,Eticha D,Rotter B,et al.Physiological and molecular analysis of polyethylene glycol-induced reduction of aluminium accumulation in the root tips of common bean(Phaseolus vulgaris)[J].New Phytologist,2011,192(1):99-113.
[12]孙小艳,朱泳,赵明敏,等.玉米转脂蛋白基因ZmLTP3的克隆及表达特性分析[J].玉米科学,2014,22(1):62-66.
[13]李杰,郭欣慰,张中保,等.将拟南芥ATNCED3基因导入玉米自交系的研究[J].作物杂志,2014(1):58-62.
[14]张宪政.作物生理研究法[M].北京:农业出版社,1992.
[15]Kumar P,Tewari R K,Sharma P N.Modulation of copper toxicity-induced oxidative damage by excess supply of Iron in maize plants[J].Plant Cell Reports,2008,27(2):399-409.
[16]Zhou Y,Tang N,Huang L,et al.Effects of salt stress on plant growth,antioxidant capacity,glandular trichome density,and volatile exudates of schizonepeta tenuifolia Briq[J].International Journal of Molecular Sciences,2018,19(1):252.
[17]Jacq A,Pernot C,Martinez Y,et al.The Arabidopsis lipid transfer protein 2(At LTP2)is involved in Cuticle-Cell wall interface integrity and in etiolated hypocotyl permeability[J].Frontiers in Plant Science,2017,8:263.
[18]Chalbi N,Martínez-Ballesta M C,Youssef N B,et al.Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity[J].Journal of Plant Physiology,2015,175:148-156.
[19]Liu F,Zhang X,Lu C,et al.Non-specific lipid transfer proteins in plants:presenting new advances and an integrated functional analysis[J].Journal of Experimental Botany,2015,66(19):5663-5681.