College of Life Sciences, Hubei University, Wuhan 430062, China.
Z Naturforsch C J Biosci. 2010 Jan-Feb;65(1-2):109-18. doi: 10.1515/znc-2010-1-218.
A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used to clone diverse trypsin-like protease gene transcripts from midguts of the brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae). Six individual trypsin-like protease transcripts were identified. On the basis of one nucleotide sequence of the six clones, a full-length cDNA sequence (1902 bp) was obtained by rapid amplification of cDNA ends (RACE). The cDNA contained an 1128-bp open reading frame encoding a putative protein of 375 amino acids with typical features of the trypsin-like protease. Heterogeneous expression of the coding sequence for the mature peptide in Escherichia coli cells showed that the expressed protease with a molecular weight of 27.0 is active, for its BApNAse activity assayed by using BApNA (N-benzoyl-D,L-arginine-p-nitroanilide) as substrate. The protease had its maximum activity at pH 8.0 and 35 degrees C. A much better stability was observed at pH values above 4.0 and temperatures below 40 degrees C. The enzyme was strongly inhibited by serine protease inhibitor. The trypsin-like protease is therefore likely one of the major digestive proteases responsible for protein hydrolysis in N. lugens gut, and multiple gene families encoding digestive proteases may help in adaptation of this sap-sucker to different rice varieties.
采用反转录-聚合酶链反应（RT-PCR）策略，从褐飞虱Nilaparvata lugens Stål（半翅目：飞虱科）的中肠克隆了多种胰蛋白酶样蛋白酶基因转录本。鉴定出 6 种个体胰蛋白酶样蛋白酶转录本。基于 6 个克隆之一的一个核苷酸序列，通过快速扩增 cDNA 末端（RACE）获得了全长 cDNA 序列（1902bp）。cDNA 包含一个 1128bp 的开放阅读框，编码一个具有胰蛋白酶样蛋白酶典型特征的 375 个氨基酸的假定蛋白。成熟肽编码序列在大肠杆菌细胞中的异源表达表明，表达的蛋白酶具有 27.0 的分子量是活性的，因为其 BApNAse 活性是通过使用 BApNA（N-苯甲酰-D，L-精氨酸-p-硝基苯胺）作为底物测定的。该蛋白酶在 pH8.0 和 35°C 时具有最大活性。在 pH 值高于 4.0 和温度低于 40°C 时观察到更好的稳定性。该酶被丝氨酸蛋白酶抑制剂强烈抑制。因此，胰蛋白酶样蛋白酶可能是负责褐飞虱肠道蛋白水解的主要消化蛋白酶之一，并且编码消化蛋白酶的多个基因家族可能有助于这种吸汁昆虫适应不同的水稻品种。
