Cloning And Transcriptional Profile Of Serine Protease CDNAs From Midgut Of Diamondback Moth, Plutella Xylostella, And Related Proteinase Activity

Serine proteases including trypsins and chymotrypsins have been reported to be involved in immune response, food protein digestion and molting process in insects. Because food digestion is important for insect growth and development, digestive enzymes in the midgut of polyphagous lepidopteran insects have been the subject of many studies due to their importance as potential targets for insect control.1) The full cDNA sequences of four serine proteases were obtained using Rapid Amplification of cDNA Ends (RACE). They were designated as Pxsp-A, Pxsp-B, Pxsp-C and Pxsp-D, which were914,924,933and1038bp long respectively The full cDNA sequences consisted of open reading frames of795,768,816and879bp long encoding264,255,271and292amino acid residues respectively. The four predicted mature serine protease enzymes had molecular masses of28.8,27.6.29.7and30.9kDa for Pxsp-A, Pxsp-B, Pxsp-C and Pxsp-D, respectively. The results of of sequence analyses predicted that the four genes all contained signal peptides. The multiple sequence alignments revealed that four Pxsps were clearly divided into three different groups:Pxsp-A, Pxsp-B and Pxsp-C, and Pxsp-D. Within Pxsps, the phylogenctic relationship was ((Pxsp-B+Pxsp-C)+Pxsp-A)+Pxsp-D.2) The mRNA expressions of Pxsp-A, Pxsp-B, Pxsp-C and Pxsp-D genes in different developmental stages and larval instars of Plutella xylostella are different. Midguts of Plutella xylostella larvae were collected at middle2nd instar, early and later3rd instar, early, middle, and later4th instar, and then the transcript levels were detected by rt-qPCR. The results indicated that the transcription levels of Pxsp-A, Pxsp-B, Pxsp-C and Pxsp-D at each detection time-point increased slowly and reached their peak levels at middle4th instar, and then went down at later4th instar. The transcript abundances of Pxsps in midgut indicated Pxsp-B and Pxsp-C were predominately higher than those of Pxsp-A and Pxsp-D.3) We analyzed the impact of parasitization of C. vestalis on the mRNA expressions of Pxsp-A, Pxsp-B, Pxsp-C and Pxsp-D. The time-specific analysis by rt-qPCR were conducted to show the transcripts of these genes in the parasitized larvae by C. vestalis from0h to96h after parasitization and in the nonparasitized larvae from the same detected time points. The results indicated that all four serine protease genes were down-regulated in the midgut of parasitized P. xylostella larvae by C. vestalis at all time points, and the effects of parasitization of C. vestalis on the transcripts of Pxsp-C was significantly greater than those of Pxsp-B and Pxsp-D, then followed by Pxsp-A.4) We tested the serine protease activities in response to the parasitization by C. vestalis. The results revealed that the casein activity of the nonparasitized larvae was slightly higher than that of larvae parasitized by C. vestalis (not significantly) during the stage from second instar to later fourth instar. Moreover, the protease composition in the midgut was significantly affected by parasitization, and the balance among the proteinases in the midguts was disrupted. Activities of casein/BApNAase/SAAPFpNA in the parasitized larvae were also drastically changed. The effect of preincubation of enzymes prepared from P. xylostella midguts with selected proteinase inhibitors on activity against casein in our assays is presented. When two inhibitors,4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF) and (S)-l-chloro-4-phenyl-3-tosylamido-2-butanone (TPCK), were used with casein as a substrate, both equally inhibited proteolysis caused by P. xylostella gut homogenate. However, with BApNA as a specific substrate for trypsin, APMSF showed significant inhibition of the trypsin activity whereas TPCK only moderately inhibited the activity. However, with SAAPFpNA as a substrate for chymotrypsin, the presence of the inhibitor TPCK led to significant inhibition of the chymotrypsin activity while APMSF has no inhibitor effect. Coupled with down-regulated transcript abundance, the trypsin and chymotrypsin activity were also decreased, therefore, the decreasing trypsin and chymotrypsin activity after parasitization of C. vestalis is most probably caused by parasitization induced down-regulating serine protease gene transcripts. In addition, the effect of the parasitization of C. vestalis on trypsin activity in midgut is consistent after parasitization, while on chymotrypsin activity strengthenes over time.