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类甜蛋白是一种能在低温胁迫下诱导表达,增强植物抗逆性的关键蛋白质。本研究运用双向电泳结合质谱技术,筛选低温胁迫下陇油7号叶片差异蛋白点,从中分离出与抗寒密切相关的类甜蛋白。根据已发表植物类甜蛋白基因TLP的保守序列设计引物,采用RT-PCR扩增陇油7号的DNA,获得TLP基因开放阅读框,长度为732 bp,编码243个氨基酸的蛋白质。生物信息学分析显示,与甘蓝型油菜(Brassica napus)的蛋白质氨基酸序列同源性高达99.18%,该基因在进化上高度保守,其保守序列属于植物的GH69-TLP-SF超家族,TLP相对分子质量和理论等电点分别为26.02 k D和9.15。TLP含有一个信号肽,为亲水性蛋白,亚细胞定位预测其是在内质网中合成的蛋白。二级结构预测表明陇油7号的TLP是由不规则卷曲和延伸链组成的不稳定蛋白。实时荧光定量和半定量分析显示,在适当阈值的低温胁迫下TLP基因表达量上调,表明该基因在白菜型冬油菜陇油7号适应低温胁迫过程中发挥重要作用。
Quorcetin is a key protein that can induce expression under low temperature stress and enhance plant stress resistance. In this study, two-dimensional electrophoresis with mass spectrometry was used to screen the differential protein spots in leaves of Longyou No.7 under cold stress, and the quasi-sweet proteins closely related to cold resistance were isolated. According to the conserved sequence of the published TLR gene, primers were designed and the DNA of Longyou 7 was amplified by RT-PCR. The open reading frame of TLP gene was obtained, which contained a 732 bp open reading frame and encoded a 243 amino acid protein. Bioinformatics analysis showed that the amino acid sequence homology with Brassica napus was as high as 99.18%. The gene was highly conserved in evolution. Its conserved sequence belonged to the plant GH69-TLP-SF superfamily. TLP-related molecules The mass and theoretical isoelectric points were 26.02 kD and 9.15, respectively. TLP contains a signal peptide that is a hydrophilic protein that is predicted to be synthesized in the endoplasmic reticulum by subcellular localization. Secondary structure prediction shows that TLR of Longyou 7 is an unstable protein consisting of irregularly curled and extended chains. Real-time fluorescence quantitative and semi-quantitative analysis showed that TLP gene expression was upregulated under the appropriate low temperature stress, indicating that the gene plays an important role in adaptation to low temperature stress in Brassica campestris var.