| Philosamia cynthiacynthiais also known as silkworm or small sets of silkworm.It belongs to Samia,lepidoptera,one of the species of eating ecastor-oil plant.Philosamia cynthia lives on ecastor-oil plant,excoecaria sebifera,and heaven tree.It eats large amount of food,and is omnivorous with strong reproductive capacity,hence it is one of the important pests of garden plants.If prevention and control is not timely,the growth of trees will be affected;Philosamia cynthia cynthia is also one of the source insects in silk spinning.Its cocoon can be used for making silk,and silkworm pupa can be used as food or feed,thus it is an important economic insect.Hatching enzyme is protease secreted by embryonic hatching gland cells.Larvae is hatched and later growth is promoted through the degradation of the egg membrane / shell.The study of biochemical characteristics and gene structure of hatching enzymes is very significant for understanding embryonic development and embryo pharmacology.The spatial and temporal expression patterns analysis,tissue expression pattern analysis and prokaryotic expression analysis of Philosamia Cynthia hatching enzyme were carried out in the paper according to the reported gene sequence of lepidopteran insect hatching enzyme.The full-length sequence of Philosamia Cynthia hatching enzyme was also obtained by RACE-PCR technology.The main results are as follows:The full-length cDNA of hatching enzyme of Philosamia Cynthia was obtained by using RACE-PCR and RT-PCR.Philosamia Cynthia hatching enzyme genes were composed of 5 'untranslated region,3' untranslated region and 885 bp open reading frame.Its full length was 964 bp and 3 'UTR contained a polyadenylation signal AATAAA.Homologous sequence alignment results showed that Philosamia Cynthia and eri silkworm,wild silkworm,tussah,bombyx,mulberry white caterpillar,diamond back moth,fruit bat and mosquitoes and other insects belong to the same group,among which Philosamia Cynthia hatching enzyme and ERI silkworm hatching enzyme had the closest genetic relationship.The three stage structure of PccHE protein was predicted by software,using 3lq0.1.A as the template to model three stage structure diagram,the homology between the two was 30.88% with GMQE value 0.53,and QMEAN-3.15.Thus,the modeling results of this model was trustworthy.The temporal and spatial expression pattern analysis of Philosamia Cynthia embryonic development was did.The expression Pcc HE transcripts in early embryos remained at a low level,and there was a small increase on the third day,while it began to increase remarkably on the ninth day.The expression level reached the maximum before incubation.The transcription level of PccHE increased with hatching,which showed that the enzyme was closely related to embryonic development and hatching.In addition,the expression pattern of PccHE showed that the enzyme had expression in billiary vessels,and mid-intestine.Therefore,it is estimated that Pcc HE is related to functions of protein metabolism,and digestion.SDS-PAGE and Western bloting were performed to identify and analyze the protein of mature enzyme sequence,signal peptide sequence and ORF sequence of PccHE.It is beneficial to the expression of this enzyme in the future and identification of enzyme activity in vitro.The results of this study expounded the molecular mechanism of the Cynthia egg hatch,provide a theoretical basis for controlling Philosamia Cynthia hatching time and process,regulating Philosamia Cynthia as silk spinning economic insect and breeding materials of castor silkworm,and incubation conditions as forest pests.The study of incubation mechanism of Philosamia Cynthia provides a reference for studying hatching of other lepidoptera insects.Meanwhile,the paper offers a new idea to other studies to target hatching related genes as pesticide,inhibit the development of egg stage embryos to larvae,and kill lepidoptera pests in egg stage. |
PDF Full Text Request