转BpLTP4烟草耐盐性分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0498

摘要/Abstract

摘要：

从白桦（Betula platyphylla Suk）中克隆获得CDS为357 bp的BpLTP4。该BpLTP4蛋白为疏水性蛋白且具有一个潜在的跨膜区,蛋白三级结构含有一个疏水腔,该蛋白具有LTP家族基因特有的8个Cys残基结构。BpLTP4与其他物种LTP耐盐基因的亲缘关系较近。qRT-PCR分析表明,NaCl胁迫处理48 h高度诱导BpLTP4表达。通过叶盘法获得BpLTP4过表达烟草转基因株系,用150 mmol/L NaCl胁迫处理转基因和野生型株系后发现,转基因株系耐盐能力高于野生型,其平均发芽率是WT的5.6倍;根长是WT的1.099倍;DAB、NBT、Evans blue染色均浅于WT,POD活性比WT增高了0.28倍,SOD活性较WT增高了0.12倍。BpLTP4响应盐胁迫处理,通过提高保护酶活性、清除活性氧、维持细胞完整性来提高转基因草的耐盐能力。

关键词: 白桦, BpLTP4, 耐盐性分析, 烟草

Abstract:

The BpLTP4 gene with CDS of 357 bp was cloned from Betula platyphylla Suk. The BpLTP4 protein was a hydrophobic and had a potential transmembrane region. The tertiary structure of the protein contained a hydrophobic cavity. The protein had 8 Cys residue structures unique specifically to LTP family genes. BpLTP4 was closely related to LTP salt- tolerant genes of other species. qRT-PCR analysis showed that NaCl stress treatment for 48 h highly induced BpLTP4 expression. BpLTP4 overexpressed tobacco transgenic lines were obtained by leaf disc method. After 150 mmol/L NaCl stress treatment of transgenic and wild-type（WT）lines,it was found that the transgenic lines had higher salt tolerance than the WT,and the average germination rate was 5.6 times that of WT. The root length was 1.099 times that of WT. DAB,NBT,Evans blue staining were lighter than WT,POD activity was 0.28 times higher than WT,and SOD activity was 0.12 times higher than WT. In response to salt stress treatment,BpLTP4 improves the salt tolerance of transgenic grass by increasing the protective enzyme activity,removing reactive oxygen species,and maintaining cell integrity.

Key words: Betula platyphylla Suk, BpLTP4, salt tolerance analysis, tobacco

石晶静, 郭依萍, 于颖, 周美琪, 王超. 转BpLTP4烟草耐盐性分析[J]. 生物技术通报, 2020, 36(12): 34-41.

SHI Jing-jing, GUO Yi-ping, YU Ying, ZHOU Mei-qi, WANG Chao. Analysis of Salt Tolerance of Transgenic BpLTP4 Tobacco[J]. Biotechnology Bulletin, 2020, 36(12): 34-41.

图/表 10

图1 BpLTP4的PCR扩增 M：DNA Marker DL 2000; BpLTP4：BpLTP4基因PCR产物

图2 BpLTP4的生物信息学分析 A：亲水性预测分析;B：跨膜区域预测分析;C：蛋白二级结构预测分析;D：蛋白三级结构分析

图3 LTP4蛋白与其他LTP抗性基因蛋白序列比对分析

图4 BpLTP4蛋白与LTP其他耐盐蛋白系统进化分析为LTP4蛋白; 为耐盐蛋白

图5 NaCl胁迫处理后白桦BpLTP4表达量分析

图6 BpLTP4转基因烟草DNA分子检测 M：DNA Marker DL 2000; 1-3：转基因烟草;WT：野生型烟草

图7 NaCl胁迫处理转BpLTP4烟草种子发芽情况分析 A：0 mmol/L NaCl胁迫下烟草发芽情况;B：150 mmol/L Nacl胁迫下烟草发芽情况;C：株系分布模型;D：NaCl胁迫处理转BpLTP4烟草发芽率分析使用独立样本t检验进行显著性分析（* P <0.05）

图8 NaCl胁迫处理下转BpLTP4烟草表型分析 A：NaCl胁迫下转BpLTP4烟草幼苗生长形态分析;B：NaCl胁迫下转BpLTP4烟草根长测定,分析使用独立样本t检验进行显著性分析

图9 NaCl胁迫下转BpLTP4烟草组织化学染色分析

图10 NaCl胁迫下转BpLTP4烟草生理指标分析 A：NaCl胁迫下转BpLTP4烟草SOD活性分析;B：NaCl胁迫下转BpLTP4烟草POD活性分析。使用独立样本t检验进行显著性分析（* P <0.05）

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