MEMBERS OF A TRYPSIN GENE FAMILY IN ANOPHELES-GAMBIAE ARE INDUCED IN THE GUT BY BLOOD MEAL
MEMBERS OF A TRYPSIN GENE FAMILY IN ANOPHELES-GAMBIAE ARE INDUCED IN THE GUT BY BLOOD MEAL
复制标题
DOI：
10.1002/j.1460-2075.1993.tb05951.x
复制
发表时间：
1993-07-01
期刊：
影响因子：
11.4
通讯作者：
CRISANTI, A
中科院分区：
文献类型：
--
作者：
MULLER, HM;CRAMPTON, JM;CRISANTI, A
丝氨酸蛋白酶是在蚊子肠道消化血粉过程中起关键作用的酶之一。这些基因的鉴定对于蚊虫的控制和蚊媒疾病的防治具有重要意义。这些基因的基因组组织的分析可能会导致肠道特异性，诱导型启动子的分离，用于在转基因蚊子中表达抗寄生虫剂。此外，可以根据酶的结构特性设计特异性抑制剂。我们在这里报告的胰蛋白酶基因家族的冈比亚按蚊，在非洲疟疾的蚊子载体的鉴定。以吸血蚊RNA为模板，通过PCR扩增出蚊胰蛋白酶相关序列。PCR产物的克隆揭示了两个不同的序列。获得相应的全长cDNA克隆并测序。Antryp1和Antryp2分别编码274和277个氨基酸的蛋白质，在氨基酸水平上显示75%的同源性。推导的氨基酸序列清楚地将它们鉴定为胰蛋白酶。在重叠的基因组克隆中发现了五个额外的胰蛋白酶序列。所鉴定的基因在11 kb内紧密聚集，测序表明不存在内含子。北方和PCR分析表明，Antryp1和Antryp2的转录均受吸血诱导。此外，使用抗重组Antryp1的抗血清，在吸血蚊子的中肠裂解物的蛋白质中检测到Antryp1蛋白。此外，来自在大肠杆菌中表达的Antryp1和Antryp2的重组多肽对不同的血液蛋白表现出很强的蛋白水解活性。我们的结论是，Antrp1和Antryp2的产品在蚊子肠道消化血粉过程中的蛋白质分解中发挥了重要作用。
Serine proteases are among the enzymes that play a crucial role during the digestion of the blood meal in the gut of mosquitoes. The identification of the corresponding genes would have important implications for the control of mosquitoes and mosquito-borne diseases. Analysis of the genomic organization of these genes may lead to the isolation of a gut-specific, inducible promoter tor the expression of anti-parasitic agents in transgenic mosquitoes. Moreover, specific inhibitors could be designed on the basis of the structural properties of the enzymes. We report here on the identification of a trypsin gene family in Anopheles gambiae, the mosquito vector of malaria in Africa. Mosquito trypsin-related sequences were amplified by PCR using as template cDNA derived from RNA of blood fed mosquitoes. Cloning of the PCR product revealed two distinct sequences. Corresponding full-length cDNA clones were obtained and sequenced. Antryp1 and Antryp2 code for proteins of 274 and 277 amino acids respectively, showing 75% homology at the amino acid level. The deduced amino acid sequences clearly identify them as trypsins. Five additional trypsin sequences were found in overlapping genomic clones. The genes identified are tightly clustered within 11 kb and sequencing indicates that no introns are present. Northern and PCR analysis indicated that the transcription of both Antryp1 and Antryp2 is induced by blood feeding. Moreover, the Antryp1 protein was detected among the proteins of a midgut lysate of blood fed mosquitoes using antisera against recombinant Antryp1. In addition, the recombinant polypeptides derived from Antryp1 and Antryp2 expressed in Escherichia coli showed a strong proteolytic activity against different sets of blood proteins. We conclude that the products of Antrp1 and Antryp2 play an important role in the breakdown of the proteins during the digestion of the blood meal in the mosquito gut.