两个小麦LEA基因的特征及其对非生物胁迫的响应

doi:10.3864/j.issn.0578-1752.2014.19.002

摘要/Abstract

摘要： 【目的】分析小麦LEA基因TaLEA4和TaLEA5及其编码蛋白的特征，比较它们在干旱、高盐、热和冷胁迫过程中的表达模式，探讨这两个LEA基因在小麦抗逆调控过程中的生物学功能，为其在小麦抗逆分子育种中的应用提供理论依据。【方法】利用RT-PCR技术克隆小麦的LEA基因，通过生物信息学方法分析克隆基因及其编码蛋白的结构特性，采用qRT-PCR技术检测克隆基因对ABA及非生物胁迫的响应模式。【结果】克隆了2个包含完整编码框的小麦LEA基因TaLEA4和TaLEA5，分别编码180和163个氨基酸，推断其分子量分别为18.8和16.9 kD，理论等电点分别为5.6和7.2。基因组序列分析发现，2个LEA基因中均包含1个100 bp的内含子。氨基酸序列分析发现，这两个LEA基因编码蛋白均富含极性氨基酸（约占整个氨基酸序列的71%），具有较强的亲水性。结构域分析显示，TaLEA4和TaLEA5蛋白中均包含1个典型的LEA_4（pfam:02987）保守域，属于LEA_4类蛋白。蛋白质高级结构分析显示，α-螺旋分别占TaLEA4和TaLEA5蛋白的96.7%和96.3%，并可形成弓形的空间结构；在TaLEA4中，检测到1个配体PEV（C₃₉H₇₈NO₈P）的结合位点，而在TaLEA5中存在2个这样的配体结合位点。表达特性分析显示，2个LEA基因均可被植物激素ABA诱导而上调表达，其中，TaLEA4的表达水平显著高于TaLEA5；TaLEA4在干旱、高盐和高温胁迫过程中均受胁迫诱导而迅速上调表达，但TaLEA5却只受干旱胁迫的诱导，且其表达水平显著低于TaLEA4；2个LEA基因对冷胁迫均无响应；干旱和高盐胁迫过程中，TaLEA4在根系中的诱导表达水平显著高于叶片，而热胁迫过程中该基因在叶片中的表达水平要显著高于根系，这可能与根系直接感受渗透胁迫而叶片直接感受热胁迫有关。【结论】小麦TaLEA4和TaLEA5均属于LEA基因家族的LEA_4亚类，具有较强的亲水性，它们属于依赖于ABA胁迫响应基因调控网络；TaLEA4可能在干旱、高盐和热胁迫过程中均发挥重要功能，TaLEA5仅参与小麦对干旱胁迫的响应，其作用要弱于TaLEA4。

关键词: 普通小麦, LEA基因, 非生物胁迫, 表达模式

Abstract: 【Objective】 Drought is one of the major factors significantly affecting the growth and yield of wheat crop. In this study, two LEA genes were characterized and their response to abiotic stresses, such as drought, salt, heat and cold, were investigated. The biological functions of these two LEA genes during the stress tolerance of wheat plant were also discussed, which would provide important information for the application of these two LEA genes in wheat molecular breeding for the improvement of stress tolerance. 【Method】RT-PCR technology was used in the cloning of LEA genes and proper bioinformatic methods were applied for characterizing the LEA genes and their deduced proteins. The gene expression patterns responding to various abiotic stresses were analyzed by using the real time qRT-PCR. 【Result】 Two wheat LEA genes TaLEA4 and TaLEA5, with complete coding sequences, were cloned and sequencing analysis showed that these two LEA genes encode 180 and 163 amino acids, with a predicted molecular weight of about 18.8 and 16.9, pI of 5.6 and 7.2, respectively. Genomic structuring showed that one intron with 100 bp in length was detected in both of the cloned genes. Amino acid sequence analysis indicated that both of the wheat LEA proteins were rich in polar amino acids, which accounted for about 71%, and so they showed high hydrophilic. Conserved domain analysis indicated that both of TaLEA4 and TaLEA5 included a typical LEA_4 (pfam: 02978) domain and belong to LEA_4 subfamily of LEA proteins. Protein structural analysis demonstrated that the region of helixes accounted for 96.7% and 96.3% of TaLEA4 and TaLEA5, respectively. Both of these LEA proteins can form a bow shape structure, and one binding site of ligand PEV (C₃₉H₇₈NO₈P) was found in TaLEA4, while two of that were detected in TaLEA5. The expression profiling revealed that both of the wheat LEA genes were induced by plant hormone ABA, and the expression level of TaLEA4 was significantly higher than that of TaLEA5. Under the abiotic stresses of drought, salt and heat, TaLEA4 was greatly up-regulated, while TaLEA5 was only induced by drought-stress and its expression level was significantly lower than that of TaLEA4. Both of these LEA genes did not response to cold-stress. Interestingly, the expression level of TaLEA4 was obviously higher in root tissues than that in leaf tissues under drought- and salt-stresses, while that was higher in leaves under heat-stress, which might be due to that the osmotic stresses were directly sensed by roots, but leaf tissues were more sensitive to heat-stress. 【Conclusion】 Both of the wheat LEA genes TaLEA4 and TaLEA5 belonged to the LEA_4 subclass LEA family, they showed high hydrophilic and were involved in the ABA dependent stress-responding gene network. It was deduced that TaLEA4 might play important roles during the processes of responding to the drought, salt and heat stresses, while the TaLEA5 was only involved in the drought-stress responding and its function might be weaker than that of TaLEA4.

Key words: common wheat, LEA gene, abiotic stresses, expression patterns

刘露露，陈雷，张春艳，石瑞杰，任江萍，孟凡荣，尹钧，李永春. 两个小麦LEA基因的特征及其对非生物胁迫的响应[J]. 中国农业科学, 2014, 47(19): 3736-3745.

LIU Lu-lu, CHEN Lei, ZHANG Chun-yan, SHI Rui-jie, REN Jiang-ping, MENG Fan-rong, YIN Jun, LI Yong-chun. Characterization of Two LEA Genes and Their Response to Abiotic Stresses in Wheat[J]. Scientia Agricultura Sinica, 2014, 47(19): 3736-3745.

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