| Biomineralization is common in nature,minerals in the organism play a role of protection,induction,calcium regulation,support and so on.At present,the researches on biomineralization mainly focus on mollusks,but the mineralization in insects is rarely reported.Most Lepidopteran insects secrete a large number of silk fibers to form a cocoon during pupation,so as to protect the fragile pupa and improve survival.The cocoon of oriental moth,Monema（Cnidocampa）flavescens Walker,a member of the Lepidoptera Limacodidae,is the hardest insect cocoon ever reported,due to its layered structure and composition.Previous studies have shown that the mineral in the cocoon is calcium oxalate and that proteins in the cocoon are rich in aspartic acid/asparagine,serine and glycine.However,until now,the specific types of calcium oxalate,protein components and proteins related to cocoon mineralization have not been reported,so this study focused on these problems.In this study,the mineral composition and content of the cocoon were determined by ICP-OES,FTIR and XRD,and the mechanical properties of the cocoon were characterized by universal testing machine.By means of LC-MS/MS,proteomic analysis was conducted on cocoon proteins of oriental moth,and it was found that in addition to silk fibroin and sericin,there were also a large number of proteins rich in asparagine（Asn,N）,so it was named Asn（N）-rich protein（NRP for short）.By quantitative PCR analysis,we found that NRP is a silk protein specifically expressed in the silk gland.NRP protein was obtained by prokaryotic expression and its related functions were investigated.The main results obtained are as follows:1.Analysis of mineral composition and determination of mechanical properties of oriental moth cocoonsOriental moth is an insect of the Lepidoptera Limacodidae,its development experience egg,larva,pupa and adult four periods,the cocoon of the oriental moth is an oval shape with distinct brown and white stripes on the surface,with a long axis of about11-15 mm,short axis 8-10 mm.The surface of the cocoon was observed by scanning electron microscope（SEM）,the white part was mineral crystal and the brown part was organic matter.In order to identify the minerals in the cocoon,we used Inductively Coupled Plasma Optical Emission Spectrometer（ICP-OES）to analyze the elemental composition of the cocoon.The results showed that the mass of calcium accounted for18.7%of the total cocoon weight,and the content of other elements was less than 1%,indicating that the minerals in the cocoon were calcite minerals.The white mineral powder was further characterized by Fourier Transform Infrared Spectrometer（FTIR）and X-ray diffraction（XRD）,the results showed that the mineral content of the cocoon was mainly calcium oxalate monohydrate（COM,99.03%）,Also contains a small amount of calcium formate（CF,0.97%）.Through anatomical observation of oriental moth,we found that the Malpighian tubules of the larvae of the first day of wandering were full of white contents.Further detection by FTIR and XRD revealed that it was composed of COM,indicating that the minerals in the cocoon of the oriental moth were discharged from the Malpighian tubules.Through the observation of the stereomicroscope and electron microscope,it was found that the cocoon was obviously divided into the inner layer of silk fiber and the outer layer of mineral,the outer layer of mineral accounted for94%of the total cocoon weight,and the inner layer of silk fiber accounted for 6%.The mineral content was then determined by combustion and Na OH solution methods.The results showed that the mineral crystal calcium oxalate monohydrate in the cocoon was41%.After conversion,insoluble calcium accounted for 11%of the whole cocoon.By means of hydrochloric acid-ammonia water method and ICP-OES,the total calcium content of cocoon was 17%,suggesting that 6%of calcium existed in soluble form.Finally,the stress-strain tests were conducted on the silk slice and cocoons of oriental moth.The results showed that there was no significant difference in stress between the oriental moth and the Bombyx mori silk slices,the toughness of the silk slices was significantly higher than that of Bombyx mori,but the strain and Young’s modulus of the silk were significantly lower than that of Bombyx mori.The compressive stress on the horizontal axis of the cocoon was 45 N,and that on the vertical axis was 70 N,about 1.5times that of the horizontal axis.There was no obvious difference in the strain between the horizontal axis and the vertical axis.2.Proteomic and metabolomic analysis of oriental moth cocoonsBased on the analysis of the mineral components in the cocoon of oriental moth,we analyzed the organic components in the cocoon.A total of 467 cocoon proteins were identified by LC-MS/MS mass spectrometry.These cocoon proteins were divided into 12types according to their functions.The highest abundance of cocoon proteins was uncharacterized protein,followed by silk protein.A total of five silk proteins were identified in the oriental moth cocoons,including Fibroin heavy chain（Fib H）,Fibroin light chain（Fib L）,Fibroin p25（Fib p25）,sericin 1 and seroin 1.Among the uncharacterized proteins,we found 25 Asn-rich protein（NRP）with very high abundance,indicating that they may be related to the mineralization of oriental moth cocoons.Bioinformatics analysis showed that NRP proteins contain signal peptides,are secreted proteins.Besides asparagine N,they also contain a high proportion of glycine G and Serine S.The molecular weight of NRP mature protein ranges from 7.7 to 16.8 k Da,and the isoelectric point ranges from 5.2 to 11.3.In order to detect the expression characteristics of NRP,silk glands were collected and divided into 5 segments according to their morphology.Firstly,the silk fibroin protein was detected by q PCR,and the results were found that Fib H and Fib L were only expressed in the v region of the posterior silk gland,while Fib p25 was mainly expressed in the v region of the silk gland,with a small amount of expression in the iv region.Then,multiple NRP genes were detected,and it was found that NRP 1,NRP 3 and NRP 4 were only expressed in silk glands but not in other tissues and organs,indicating that they belonged to silk proteins.Further studies were conducted to find the expression amount of NRP 1,NRP 3 and NRP 4 gradually increased from anterior silk gland i to posterior silk gland v.We are using the metabonomics method analyzes the oriental moth cocoons of small molecular components,from the identification to more than 200 kinds of small molecule metabolites,found that methyl dopamine and 4-ethyl catechol content is higher.They are presumed to be metabolites of dopa and dopamine.On the one hand,dopa and dopamine react to produce melanin,which causes the oriental moth cocoon to blackening and appear brown color.On the other hand,they are tanning agents,which cause the cross-linking of silk fibroin proteins,which causes the hardening of the oriental moth cocoon.3.Functional study of asparagine-rich protein NRP 3 in oriental moth cocoonsIn order to explore the function of NRP protein in oriental moth cocoons,we cloned NRP 3 coding genes and connected it to the prokaryotic expression vector PGEX-4T-2,the pure NRP 3 protein was obtained by nickel column affinity chromatography.The interaction between NRP 3 and calcium ion was determined by fluorescence spectrometry.It was found that NRP 3 could combine with calcium ion,which decreases its fluorescence value,the Kd value was 1.754±0.358 m M.NRP 3 and NRP 3-Ca were analyzed by Fourier Transform Infrared Spectrometer.The absorption peak of 3338 cm-1in the spectra shifted to a lower frequency(3314 cm-1),indicating that NRP 3 protein binds to Ca via imino（-NH）.The interaction between NRP 3 and calcium ion does not change the structure of NRP 3 which is dominated by random curling by circular dichroism.The calcium-binding content of NRP 3 was then determined using the calcium ion content chromogenic kit.The results showed that 1 mg of NRP 3 could bind 19μg Ca.The binding capacity of NRP 3 to calcium oxalate was then determined,compared with the control BSA,NRP 3 had stronger binding capacity with calcium oxalate and was not easy to be eluted by high concentration salt solution.NRP 3 was incubated with calcium chloride and sodium oxalate in vitro.SEM results showed that NRP 3 inhibited the growth of calcium oxalate crystals at the early stage of mineral formation,and was beneficial to the formation of dumbbell type calcium oxalate monohydrate（COM）.Subsequently,we tested the antibacterial activity of NRP 3 protein.The results showed that NRP 3 could significantly inhibit the growth of fungi Candida albicans and Saccharomyces cerevisiae,could not inhibit the growth of gram-positive Staphylococcus aureus,but could strongly and significantly inhibit the growth of gram-negative Escherichia coli,suggesting that NRP proteins in oriental moth cocoons may play multiple roles.It can not only regulate the mineralization process of calcium oxalate to strengthen the physical defense of cocoon,but also improve the biochemical defense of cocoon to pupa through bacteriostasis. |
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