荞麦BTI全长基因的克隆及表达分析

【研究目的】扩增荞麦胰蛋白酶抑制剂(Buckwheat trypsin inhibitor,BTI)基因,并对其表达特性和氨基酸同源性进行分析。【方法】根据荞麦胰蛋白酶抑制剂氨基酸的保守序列设计简并引物,采用RT-PCR和RACE技术,以荞麦cDNA为模板,扩增BTI基因并进行序列及其表达谱分析。【结果】克隆得到了BTI基因。它的cDNA全长为459bp,含有一个216bp的完整阅读框,编码72个氨基酸。同源性分析表明,其蛋白质序列与已报道的荞麦种子提取的BWI-1的氨基酸序列的同源性达96%,与笕属植物ATI、亚麻属植物Luti、马铃薯蛋白酶抑制剂PPI-I的氨基酸序列的同源性分别为65%、59%、48%。RT-PCR分析表明,BTI基因可在不同的组织中进行表达,但经伤害处理后的叶片中其表达量略高于生长各阶段。这可能与逆境情况(如外界损伤)下的应答等生理过程有关。【结论】荞麦BTI基因的获得,可为荞麦胰蛋白酶抑制剂的基础及应用研究奠定基础。

Molecular Cloning and Expression Analysis of a Full-Length Gene (BTI) in Buckwheat

[OBJECTIVE]To obtain the nucleotide sequence of buckwheat trypsin inhibitor (BTI) and to further analyze its expression and regulation mechanism.[METHOD]Based on the amino acid information of trypsin inhibitor of buckwheat, degenerated primers were designed and a full-length cDNA sequence of BTI was amplified from cDNA by using RT-PCR and rapid amplification of cDNA ends (RACE).[RESULTS]Sequence analysis shows that the 459 bp cDNA contained an open reading frame (ORF) of 216 bp, encoding 72 amino acids residues. The deduced amino acid sequence exhibits 96%homology with BWI-1, which trypsin inhibitors from buckwheat seeds. It shared 65%, 59%, and 48% homology with ATI from Amaranthus hypochondriacus, Luti from Linum Usitatissinum, potato inhibitor I-type family protein, respectively. By using RT-PCR, the expression pattern of BTI gene was detected. It was demonstrated that BTI gene was expressed in every period, and high levels of expression were accumulated during in leaves and by wounding treatment leaves, suggesting that BTI gene was correlated with the growth environment, as wound-induced defense response of plants against herbivores and pathogens.[CONCLUSION]This work will be useful for further studying on the molecular mechanism and the potential applications of buckwheat trypsin inhibitor.