| The visual organs of insects,compound eyes and ocellus,are exquisite special structures that appear in the process of insect evolution in the face of specific visual challenges and ecological needs,and are the structural basis of vision.Among them,compound eyes are the main visual organs of insects,and ocelli is an auxiliary organ of vision,and their structural changes play an important role in visual formation.In the visual formation mechanism of insects,opsin is one of the determinants,it binds to the chromophore(11-cis-retinal)and is activated by allosteric and chromophore detached under the action of light,and then under the mediation of G protein,the activation of kinase C initiates subsequent pathways,and finally transmits the signal to the central brain center to send a visual response signal.Studies have shown that Nina E in Drosophila melanogaster(Dm Nina E)is a family of opsins,and the deletion and mutation of Nina E will lead to retinal degeneration,and mutations in this gene can also affect the development and photosensitivity of R1-R6 photoreceptor cells.In this study,the external morphology and internal morphology of the monocular and compound eyes of Lepidoptera insect silkworms were first observed by scanning electron microscopy,HE sectioning,transmission electron microscopy and other techniques.Then,the opsin in the silkworm genome was identified by bioinformatics analysis,and their spatiotemporal expression patterns were further detected,and finally the homologous genes Bmcop and Bmopsin1 of Dm Nina E in silkworms were selected for prokaryotic expression,subcellular localization and gene knockout,and their functions were explored.The main results obtained are as follows:1.The Structure of ocellus in silkworm larvaeThe Ocellus of the silkworm larvae is distributed on both sides of the lateral skull plate of the head of the silkworm,with 6 on each side.Each monocular external bulge is hemispherical and approximately 80 μm in diameter,and the main structures include the cornea,corneal cells,vitreous,crystals,omental cells,pupils,and optic nerve.2.Compound eye development in the pupal stage of silkworms.At pupal 1 d,the eye disc of the compound eye can be clearly seen,sandwiched between the mouthparts and antennae.The eye disc is superficially divided into three parts: anterior,differentiation center and posterior parts.From pupal 1 d to pupal 8 d,the pigmentation of the compound eye gradually deepened,and the boundary between the anterior part of the eye disc,the center of differentiation and the posterior part became more obvious.At the moth stage,the compound eye is completely pigmented,and no obvious difference can be observed in the area of the compound eye.The development process of compound eye from 1 d-8 d to silkworm moth stage was observed by HE section,and the results showed that the cornea and crystal cone were the first to develop in the compound eye,followed by pigment cells,basement membrane,optic lobe and rod.The basement membrane and optic lobe develop before the optic rod,but the formation of all structures lags behind the rhabdom,and after the rhabdome is formed,the basement membrane and optic lobe are separated,and the optic nerve develops last.3.Structure of compound eye of silkworm moth.Firstly,the compound eye of silkworm moth was supervisibly observed by scanning electron microscopy,which was hemispherical in shape,with a radius of about 580 μm,distributed on both sides of the head,and a compound eye probably contained more than 3000 small eyes.Then,transmission electron microscopy(TEM)was used to perform cross-cutting and centering longitudinal sections at depths of 40 μm,200 μm and 400 μm.The results showed that the basic structure of the small eye in the compound eye of silkworm moth was,from the outside to the inside:cornea,crystal cone,pigment cell,transparent area,rhabdome,basement membrane,and optic lobe.The cornea is located in the outermost layer,is a structure of about 50 layers of chitin thin layer,below the cornea is the crystal cone bundle,composed of 4-5 crystal cone cells,surrounded by two large primary pigment cells,the pigment granules at the lower end of the crystal cone are composed of secondary pigment cells,basically spread throughout the entire small eye structure,mainly concentrated at the end of the crystal cone and the tip of the rhabdom.The main structure is 10 retinal cells,arranged in a "9+1" style,the retinal cells form a sensory rod,which fuses into a large sensory rhabdom in the nucleus area at the top of rhabdom,and the basement membrane and optic lobe are below the sensory rod bundle.4.Bioinformatics analysis and identification of silkworm opsin genesIt is known that the opsins of insects is divided into five categories: LW(Rh6),UV(Rh5),blue(Rh4),Rh7,and c-opsin,and 6 opsin genes were identified in the silkworm genome through bioinformatics analysis,of which 2 genes belong to Rh6 opsin;The remaining four genes belonged to four taxa of Rh5,Rh4,Rh7 and c-opsin,and the six opsin genes were constructed with other insect opsins,and the results were consistent with the previous analysis,XP＿037871704.1,XP＿037871515.1 and Drosophila melanogaster Nina E were clustered with Sympetrum frequens LW opsin;KWMTBOMO05053 and Rh7 opsin in Drosophila melanogaster were clustered in one group;XP＿028030904.1 and Rh5(UV)opsinin of Papilio glaucus were clustered in one group;XP＿004932925.1 and Helicoverpa armigera blue opsin were clustered in one group;XP＿021205037.1 and Anopheles gambiae str.PEST c-opsin were clustered in one group.According to their annotations and taxa in NCBI,the two opsin genes of the Rh6 group in silkworms were named Bmcop,Bmopsin 1,the opsin gene of the Rh5 group was named Bmrh5,the opsin gene of the Rh4 group was named Bmrh4,the opsin gene of the Rh7 group was named Bmrh7,and the opsin gene of the c-opsin group was named Bmc-opsin.Among them,two Rh6 group opsin genes of silkworms are homologous genes of Dm Nina E,both located on silkworm chromosome 15,which are transmembrane proteins,containing 7 transmembrane structures,and neither protein predicted obvious signal peptide sequence,and both proteins contained 7tm＿1 domains,and the two genes were further clonely identified and functional studies continued.5.Expression patterns of silkworm opsins and localization analysis of Bmopsin1 and Bmcop.RT-PCR was used to detect the expression patterns of the six opsin genes identified in the tissues of the large silkworm cultivars at different periods and five instar and three days,and the results showed that Bmcop,Bmopsin 1 and Bmrh5 were expressed in small amounts during the 2 d period of silkworm-upper cluster.In the pupal stage 1-8 days and moths,regardless of male or female,Bmcop had a small amount of expression in the head at each stage,but basically no expression in the pupal body and moth body.The expression of Bmopsin 1 gradually increased in the head at all times,especially in the pupal 7 and 8 days and the high amount of the moth head,but it was basically not expressed in the pupal body and moth body.Bmrh4 is expressed in trace amounts in female pupae for 7-8 days and in moth heads,but not in male pupae;Bmrh5 was microexpressed in both male and female pupal heads and moth heads in the late pupal stage.In the embryonic stage,except for Bmcop expressed in small amounts at 2 h-5 days in embryos,and high amounts in 6 d-8days,the expression gradually increased with the development time.The other three genes were not expressed at the embryonic stage.In the tissues of the fifth instar of silkworms,except for Bmopsin 1 in the seminal nest,the other three genes were basically not significantly expressed in each tissue.In addition,cloning sequencing analysis confirmed that Bmrh7 was expressed in higher amounts in moth heads.Bmc-opsin expression was not detected in various developmental stages and tissues of silkworms.Detection at the cellular level showed that Bm N-SWU1 cells were not significantly expressed in the nucleus and expressed in the cytoplasm.Bmopsin 1 is expressed in high amounts in both the cytoplasm and the nucleus.6.Prokaryotic expression and gene knockout of Bmcop and Bmopsin1 genes in silkwormsp ET-28a＿Bmcop and p ET-28a＿Bmopsin1 expression plasmids were constructed,and two recombinant plasmids were introduced into the expression strain BL21 for prokaryotic induced expression.After changing the induction conditions several times,the results of SDS-PAGE showed that no bands consistent with the size of the band of interest were found in the supernatant and precipitate,which showed that the prokaryotic expression was unsuccessful,which was speculated to be related to the opsin containing 7 transmembrane domains.Furthermore,the two genes were designed with knockout targets,and the three targets of each gene design were successfully constructed onto the Piggy Bic knockout vector,and the mixed plasmid of the three targets of each gene was mixed and injected with the volume of auxiliary plasmid A3 after the plasmid was used for purity detection.Two microinjections were performed,but neither of them screened positive individuals and failed to conduct more in-depth studies of Bmcop and Bmopsin1 function. |
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