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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。
In order to elucidate the function of Nt LTP1.1, a non-specific lipid transfer protein gene, in Nicotiana tabacum, the gene was cloned from Nicotiana tabacum by Rapid-amplification of c DNA ends (RACE) Length, and analyzed the DNA sequence structure, protein physical and chemical properties, conserved motif, tertiary structure and phylogenetic relationship by bioinformatics methods; using q RT-PCR to detect the expression pattern of the gene in different tissues as well as the stress Expression difference under the conditions. The results showed that the full length of Nt LTP1.1 gene was 615 bp and the encoded protein contained 129 amino acids, which was a kind of small molecule alkaline soluble protein. The N-terminal of the protein sequence contains a signal peptide that is predicted to localize in the secretory pathway. Blast comparison showed that tobacco nonspecific lipid transfer protein has the highest similarity with tomato ns LTP1 sequence and contains 8CM conserved motif (conserved motif consisting of 8 cysteine residues). Prediction of the tertiary structure of protein showed that Nt LTP1.1 has the typical tertiary structure of ns LTP, including four α-helices, four pairs of disulfide bonds and one hydrophobic cavity that can bind and accommodate lipid molecules. Multiple alignment results showed that the Nt LTP1.1 8CM domain pattern was C1-X9-C2-X13-C3C4-X19-C5XC6-X22-C7-X13-C8. Phylogenetic analysis showed that there are 5 types of ns LTP evolution and Type 1 Nt LTP1.1. Expression pattern analysis showed that Nt LTP1.1 gene was mainly expressed in tobacco leaves and had obvious tissue expression specificity. The expression was inhibited under the condition of low temperature stress and the expression was up-regulated under the condition of salicylic acid induction. Therefore, it is speculated that the Nt LTP1.1 gene may play a role in tobacco defense response.