黄粉虫胰蛋白酶样酶基因TMTLSP全长cDNA的克隆和序列分析

Cloning and Analysis of Trypsin-like Serine Protease cDNA of Yellow Mealworm(Tenebrio molitor) Larvae

以黄粉虫(Tenebrio molitor)幼虫全RNA逆转录得到的cDNA为模板,参照地鳖(Eupolyphagasinensis)纤溶酶(fibrinolytic enzyme)简并引物,进行温度梯度PCR.以得到的扩增产物为基础,采用RACE得到基因全长cDNA,命名为黄粉虫胰蛋白酶样丝氨酸蛋白酶(Tenebrio molitor trypsin-likeserine protease,TMTLSP).TMTLSP全长869 bp(GenBank No.JN662461),开放阅读框为777 bp,编码258个氨基酸,并具有蛋白酶样特有的起始位点、活性中心预计底物结合位点.经过比对分析,该基因编码的氨基酸序列与赤拟谷盗、谷蠹、光亮扁角水虻、美洲大蠊等多种昆虫的胰蛋白酶或丝氨酸蛋白酶有较高的相似性.本研究将为胰蛋白酶样丝氨酸蛋白酶的提取及研究提供更为广泛的材料及研究依据.

In this study,the template cDNA which was reversely transcribed from the total RNA of Tenebrio molitor larvae,was subjected to temperature gradient polymerase chain reaction（PCR） with the degenerate primers of Eupolyphaga sinensis fibrinolytic enzyme.The full-length cDNA,achieved with Rapid Amplification of cDNA End method（RACE） from the amplification product,was named Tenebrio molitor Trypsin-like Serine Protease（TMTLSP）.TMTLSP includes 869 bp（GenBank No.JN6624614） with the open reading frame of 777 bp and 258 coding amino acids.It also has the unique trypsin-like initiation sites and expected binding sites of substrate active sites.The results of comparative analysis illustrated that the gene encoding of amino acid sequence was highly similar to those of a variety of insects.Based on the results,this study could be considered as a valid reference for the further investigation of trypsin-like serine protease extraction and examination.