枣树2-半胱氨酸氧化还原酶基因Zj2-CP在干旱和盐胁迫下的功能分析

Functional analysis of 2-Cys peroxiredoxins gene(Zj2-CP) in jujube under drought and salt stresses

【目的】研究枣树2-半胱氨酸氧化还原酶基因(2-Cys peroxiredoxins, Zj2-CP)的功能,为其在枣树抗逆基因工程改良中的利用奠定基础。【方法】以‘辣椒枣’(Ziziphus jujuba‘Lajiaozao’)组培苗为研究对象,通过实时荧光定量PCR技术分析目的基因在盐胁迫及PEG胁迫条件下的表达模式,利用农杆菌介导法将本实验室已构建的植物表达载体PEZR(K)-Zj2-CP-LNY转入拟南芥,激光共聚焦显微镜进行目的基因的亚细胞定位,同时对转基因植株进行高盐和干旱胁迫处理,验证其抗逆功能。【结果】实时荧光定量PCR分析表明‘,辣椒枣’组培苗中,Zj2-CP能够被不同浓度的PEG和盐胁迫诱导表达,暗示该基因可能对枣树抗旱性和耐盐性具有重要的作用。转Zj2-CP基因的拟南芥转化株系的茎和叶表皮细胞的细胞膜和细胞质以及根的细胞膜中均检测到Zj2-CP存在。在盐胁迫处理下,转Zj2-CP基因的拟南芥的幼苗存活率显著低于野生型;在干旱胁迫处理下,转Zj2-CP基因植株主根的长度显著低于野生型。【结论】与野生型拟南芥相比,过表达Zj2-CP基因的拟南芥增加了对干旱和盐胁迫的敏感性,我们推测Zj2-CP参与植物干旱和盐胁迫响应。

【Objective】Extreme environmental stresses can induce reactive oxygen species(ROS)that generate oxidative stress at the cellular level in plants.Plants have developed diverse defensive mechanisms for scavenging oxidative stress.2-Cys peroxiredoxins(2-CP)protein has been known as a member of peroxiredoxins(Prxs)which can remove ROS and protect the photosynthetic membrane from oxidative damage in plants,but their functions under abiotic stress are not clear.As a foundation for further studying on the application of fruit stress tolerance genetic engineering,the function of Zj2-CP gene encoding a 2-Cys peroxiredoxins from Ziziphus jujuba Mill.was analyzed under drought and salt stresses.【Methods】To test the Zj2-CP responses to abiotic stress,seedlings of jujube were treated under different PEG(0.5 MPa,0.8 MPa and 1.2 MPa)and salt(50 mmol·L-1,100 mmol·L-1 and 300 mmol·L-1)stresses.The leaves were sampled for expression analysis at different times(15 min,1 h,3 h and 6 h).The total leaf RNA was extracted by CTAB(Hexadecyl trimethyl ammonium Bromide).The primers were designed according to the gene Zj2-CP sequence.The expressions of Zj2-CP under PEG and salt stresses were tested by qRT-PCR(Quantitative real-time quantitative PCR).To detect subcellular localization of Zj2-CP protei.The fusion vector named PEZR(K)-Zj2-CP-LNY with a 35 s promoter and YFP tag constructed in our previous studies was transformed into Agrobacterium tumefaciens strain LBA4404,which then was transformed into Arabidopsis to obtain stable transgenic lines of Zj2-CP using agrobacterium-mediated method.YFP fluorescence was recorded under a confocal laser scanning microscope(Leica TCS SP5).Three stable overexpression transgenic lines were used to verify the function of Zj2-CP under stress tolerance,and the survival rate and the length of primary root with 9-dayseedlings of transgenic and wild Arabidopsis plants were analyzed under NaCl(150 mmol·L-1)and mannitol(100 mmol·L-1 and 300 mmol·L-1)stresses.【Results】qRT-PCR analysis showed that under lower concentration of PEG stress(0.5 MPa and 0.8 MPa),the expression level of the target gene increased with time.On the contrary,under higher concentration,the relative expression of the gene was the strongest and reached the maximum at 15 min,and then decreased with time because of cell membrane damage under the higher concentration stress.The relative expression of the target gene in jujube seedlings showed a similar trend,first reaching the maximum at 1 h and then decreasing with time.Moreover,with the increase of salt concentration,the expression of Zj2-CP gene in jujube gradually increased.We examined the subcellular localization of Zj2-CP in stable transgenic Arabidopsis plants under a fluorescence confocal microscope.Fluorescence was mainly detected in the cell membrane and cytoplasm in leaves and stems,but was only found in the cell membrane in root.Particularly,the signal was detected in the cell membrane and cytoplasm of stomata guard cells.The stress tolerance showed that the seedlings grew well in the MS culture medium,but under the 150 mmol·L-1 salt stress the survival rate of transgenic plants was 8%,whereas the rate of wild type was 90%.The length of primary root of transgenic plants was 1.08 cm and 0.42 cm under 100 mmol·L-1 and 300 mmol·L-1 PEG stress,respectively,while that of wild type reached 1.48 cm and 0.92 cm.Stress tolerance pointed out that the transgenic plants showed lower survival rate than the control,and the length of primary root of transgenic plants under drought stress was significantly shorter than the control.The results also indicated that the phenotype of three stable transgenic lines was different,but only one line was significantly different,and the reason may be that the signal was detected in one stable transgenic line,but the exogenous gene was silenced in other two transgenic lines.【Conclusion】The gene of Zj2-CP was located in cell membrane and cytoplasm,and the expression could be induced by PEG or salt stresses,which indicated that Zj2-CP was involved in different signal pathways responding to abiotic stress.This study exhibited Zj2-CP was likely related to drought and salt resistances,and Arabidopsis thaliana overexpressing of Zj2-CP increased the sensitivity to drought and salt stress.Overall,the gene of Zj2-CP maybe plays a negative regulatory role in resisting drought and salt stress.