烟草非特异性脂质转移蛋白基因的克隆与表达分析

Cloning and Expression Analysis of Non-Specific Lipid Transfer Protein Gene from Nicotiana tabacum

为了阐明非特异性脂质转移蛋白基因Nt LTP1.1在烟草抗病抗逆反应中的功能,用c DNA末端快速扩增技术(Rapid-amplification of c DNA ends,RACE)从普通烟草中克隆了该基因全长,并通过生物信息学方法分析了c DNA序列结构、蛋白理化性质、保守基序、三级结构以及系统进化关系;利用q RT-PCR检测了该基因在不同组织中的表达模式以及胁迫条件下的表达差异。结果表明:Nt LTP1.1基因全长615 bp,编码蛋白含有129个氨基酸,为一种小分子碱性可溶性蛋白。蛋白序列N端含有信号肽,预测其可能定位于分泌途径中。Blast比对结果显示,烟草非特异性脂质转移蛋白与番茄ns LTP1序列相似性最高,含有8CM保守基序(8个半胱氨酸残基组成的保守基序)。蛋白三级结构预测表明,Nt LTP1.1具有ns LTP典型的三级结构,包括4个α-螺旋,4对二硫键,1个可结合和容纳脂质分子的疏水腔。多重比对结果显示,Nt LTP1.1 8CM结构域模式为C1-X9-C2-X13-C3C4-X19-C5XC6-X22-C7-X13-C8。系统进化分析结果表明,ns LTP进化形成5类,Nt LTP1.1属于Type I。表达模式分析显示,Nt LTP1.1基因主要在烟草叶片中表达,具有明显的组织表达特异性。在低温胁迫条件下表达受到抑制,在水杨酸诱导条件下表达上调。因此,推测Nt LTP1.1基因可能在烟草防御反应中发挥作用。

To clarify the function of non-specific lipid transfer protein gene NtLTP1.1 in resistance against disease and stress, full-length NtLTP1.1 was cloned from Nicotiana tabacum by RACE technology, and cDNA sequence structure, protein physicochemical properties, conserved amino acid motifs, tertiary structure and evolutionary relationships were analyzed with bioinformatics methods. The expression patterns in different tissues and expression difference under stress of NtLTP1.1 were determined by qRT-PCR. The results showed that NtLTP1.1 was 615 bp in length and encoded a protein of 129 amino acids, the protein was a micromolecular soluble basic protein, and its N-terminal contained a signal peptide that might target the protein in secretory pathway. Blast analysis revealed that the homology of sequence was the highest between tobacco NtLTP1.1 and tomato nsLTP1, NtLTP1.1 contained a conserved 8CM motif composed of eight-cysteine-motif. The prediction analysis of protein tertiary structure indicated that NtLTP1.1 possessed the typical tertiary structure of nsLTP, which was composed of 4α-helices, 4 pairs of disulphide bonds, an internal hydrophobic cavity capable of binding and accommodating lipid molecule. The multiple alignment results showed the structural domain pattern of NtLTP1.1 8CM was C1-X9-C2-X13-C3C4-X19-C5XC6-X22-C7-X13-C8. Phylogenetic analysis revealed that NtLTP1.1 belonged to the Type I among the 5 clustered types of nsLTP. The results of qRT-PCR analysis showed that NtLTP1.1 was a tissue-specific gene and mainly expressed in tobacco leaves, its expression was suppressed under low temperature stress, while promoted by salicylic acid induction; it suggested that NtLTP1.1 gene might play a role in tobacco defense reaction.