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持续恶化的自然环境使得植被不断受到各种各样的非生物胁迫导致生物量不断减少。对基因工程研究的不断深入,使科研人员对植物在抗性方面的研究取得了显著成就。与植物抗逆相关的基因主要包括可以编码渗透压物质的基因、抗氧化基因、编码保护生物大分子及膜结构蛋白质的基因、编码转录因子的调节基因以及编码可在逆境中诱导的植物蛋白激酶基因[1]。
碱性亮氨酸拉链转录因子(bZIP)普遍存在于真核生物当中,参与多种生物学功能,如植物的生长[2]、损伤修复[3]、逆境胁迫的抵御[4-9]以及病菌的防御[4-5]。
bZIP转录因子结构域含有大约60到80个氨基酸残基,由一个16至20个保守氨基酸残基组成的碱性氨基酸区和一个亮氨酸拉链共同组成[10]。保守的碱性氨基酸区含有核定位的信号序列,紧随其后的是DNA的识别区域,该区域可以与特异的DNA序列相互作用。亮氨酸拉链区是一个具有两亲性的α螺旋[11],α螺旋中,每个重复的七肽都包含一个亮氨酸或者其他的疏水残基。该螺旋结构参与bZIP蛋白与DNA结合之前的二聚体化[12]。植物的bZIP转录因子主要存在于细胞核中[13]。
干旱和高盐等条件下,bZIP转录因子可以与ABFs和AREBs结合,从而调控下游的靶基因,ABFs是元件ABRE的结合因子,AREBs 是元件ABRE的结合蛋白,其核心序列为ACGT。拟南芥的抗逆相关基因ICK1、rd29B、rab18、KIN2/KAT2、ADH1、CHS、racS、SUS1的启动子区域都发现了ABRE顺式作用元件[14]。Choi等第一次克隆了拟南芥中的bZIP转录因子,主要包括参与低温应答反应的ABF1、参与高盐、干旱等逆境应答反应的ABF2/AREB1、ABF3以及ABF4/AREB2[15]。大豆中鉴定了10个亚家族共131个bZIP基因,其中,有近三成bZIP基因可以响应高盐干旱等逆境胁迫 [16]。Liao等选取其中的4个GmbZIP基因的过表达载体转入拟南芥中,发现其均能对逆境胁迫产生相应的应答[17]。Xiang等鉴定出了水稻中的bZIP基因OsbZIP23,该基因可以显著提高水稻中的耐旱、耐盐性[18]。Kranner等将bZIP家族中的ThbZIP1基因的过表达载体转入烟草中,可以提高烟草的部分保护酶(超氧化物歧化酶(SOD)和过氧化物酶(POD))的活性,以此减少活性氧的数量来有效的克服逆境环境[19]。
白桦( Betula platyphylla Suk)作为东北地区重要的绿化及用材树种,鉴定重要抗旱耐盐基因,研究分子调控机制,具有重要的理论及应用价值。本研究克隆白桦bZIP转录因子,验证抗旱耐盐功能,为研究白桦抗非生物胁迫性状形成的的分子基础研究及抗性分子育种提供理论数据和研究材料。
白桦BpbZIP1基因抗旱耐盐分析及ABRE元件结合鉴定
Drought and Salt Tolerance Analysis of BpbZIP1 Gene in Birch and ABRE Element Binding Identification
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摘要:
目的 在白桦(Betula platyphylla Suk)中鉴定抗非生物胁迫bZIP基因,为研究白桦抗逆性状形成的分子基础及抗性遗传改良提供数据和材料。 方法 从白桦苗克隆白桦bZIP基因,利用生物信息学对该基因进行系统进化分析。利用瞬时转化法进行快速基因功能分析,通过组织化学染色和生理指标测定鉴定抗旱耐盐功能。利用酵母单杂交初步鉴定转录因子与元件的结合。 结果 获得白桦bZIP基因,命名为BpbZIP1,其与已知抗性相关基因蛋白序列聚为一组。BpbZIP1基因受NaCl处理的诱导,在甘露醇处理下,表达变化不明显。在NaCl胁迫条件下,转基因株系清除活性氧的能力强于野生型,SOD和POD活性提高;在甘露醇胁迫条件下,转基因株系活性氧清除能力与野生型差别不明显,SOD活性升高,POD活性上升不显著。BpbZIP1能够与ABRE元件特异性结合。 结论 BpbZIP1基因能够通过清除活性氧来提高白桦瞬时转化株系的耐盐能力,抗旱能力不明显。鉴定出具有耐盐能力的白桦bZIP基因及其结合元件,为进一步分析白桦bZIP基因的耐盐分子调控机制提供基础数据。 Abstract:Objective To identify basic region/leucine zipper motif (bZIP), the gene resistant to abiotic stress, from Betula platyphylla Suk. Method The bZIP gene of B. platyphylla was cloned with genomic information, and the phylogenetic analysis of resistance related genes was carried out with bioinformatics analysis. The function of drought and salt tolerance was identified by histochemical staining and physiological index. The binding between transcription factors and elements was identified by yeast 1 hybrid. Result A bZIP gene was cloned from B. platyphylla, named as BpbZIP1, which was clustered into a group with the known resistance related gene protein sequence. The expression of BpbZIP1 gene was induced by NaCl treatment and did not change significantly under mannitol treatment. Under NaCl stress, the ability of scavenging ROS was stronger in transgenetic lines than that in wild-type control as the activities of SOD and POD increased. Under mannitol stress, the ability of scavenging ROS in transgenetic lines was not significantly different from that of the control, the POD activity increased, SOD activity increased less. BpbZIP1 can specifically bind to ABRE element. Conclusion BpbZIP1 gene can improve the salt tolerance in transient transformed birch by scavenging active oxygen, but the ability of drought resistance is not obvious. The bZIP gene of B. platyphylla with salt tolerance and its binding elements were identified. The results of this study provide data and materials for the molecular-based study of stress resistance and genetic improvement of in B. platyphylla. -
Key words:
- BpbZIP1 gene
- / drought resistance
- / salt tolerance
- / functional analysis
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