对多种已知植物巯基蛋白酶抑制剂（cystatin）基因的氨基酸序列进行比对分析,根据其高度保守的氨基酸序列设计一对简并引物,并从茶树品种龙井43鲜叶中提取总RNA,用RT-PCR法扩增出一204βbp的cDNA特异片段,然后通过3’/5’RACE的方法,分别扩增出3’端和5’端的序列,从而获得茶树巯基蛋白酶抑制剂基因的cDNA全长序列,所得序列全长627βbp,编码101个氨基酸,分子量约11.062βKDa。该基因在推测的氨基酸序列中含有巯基蛋白酶抑制剂家族中高度保守的、与其活性有关的QXVXG结构,且经Blast分析表明,该基因序列与其他植物巯基蛋白酶抑制剂基因的氨基酸序列同源性为54% ~ 77%。
Two degenerate primers were designed according to the conserved region among the known plant cystatins. A cDNA fragment of 204βbp was amplified by RT-PCR (reverse transcription polymerase chain reaction) of total RNA extracted from fresh leaves of Tea plant (Camellia sinensis cv Longjing43). A full-length cDNA of the cystatin gene was obtained by 3’/5’RACE (rapid amplification of cDNA ends). The cDNA sequence of this 627βbp clone contained an open reading frame encoding a polypeptide of 101 amino acid residues with a predicable molecular mass of 11.026βKDa. The deduced amino acid sequence contained the motif QXVXG conserved among most members of the cystatin superfamily. By using the program of Blast on GenBank database, the sequence presented a high match with the cystatin genes from other plants, such as European chestnut, Cassava, Cowpea, Tomato, Soybean et al. All researched out sequences were all cystatins, so we can conclude that the cloned sequence is a member of cystatin gene from Tea plant.
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