Evolutionary Analysis Of Cocoonase Gene And Its Application As A Target Gene In Silkworm Breeding

Insects are the most prosperous animal groups on the earth,due to their adaptabilities to the environment during the long-term evolutionary process,and the related astute life-cycle strategies,such as diapause,aposematic signals and mimicry,long distance migration,etc.,which are conducive to the survival and population expansion.Spinning and cocooning is one of the effective strategies for holometabolous insects to protect the immobile pupa metamorphosing into adult safely.A considerable number of insects from Coleoptera,Lepidoptera,Hymenoptera and Neuroptera are able to spinning and cocooning.The mature larva usually uses silk or silk embedded with surrounding materials to construct a cocoon.Cocoon not only provides a shelter for the pupation of the insect,but also protects the pupa from mechanical damage,predators,parasites and other adverse factors before adult emergence.For the insects spining sealed cocoons,after the pupa metamorphosing into adult,the adult must break out of the cocoon and complete the mission of succession.There are various ways for insects to spin cocoons,and the decocooning ways are also different.Previous researches reported that,when the silkworm and tussah insects escaped from the sealed cocoons,their adults could secrete alkaline spit to infiltrate the cocoon layer,and then the cocoonase,dissolved in the moth-vomiting fluid,degraded the sericin of the cocoon layer.So that,the cocoon became soft,allowing the moth to pull aside the loose silk and emerge.However,besides cocoonase,the vomiting fluid also contains 28 proteins,some of which are enzyme proteins.It is not clear whether other proteins are involved in the process of cocoon dissolution.And,many insects can construct a cocoon,it is also unclear whether cocoonase plays a role in the emergence of other insects from the sealed cocoon.In this study,we analyzed the origin,evolution and distribution range of cocoonase gene,and detected the selection effect on cocoonase gene during the evolution.Meanwhile,the silkworm,Bombyx mori was used as the model of insects spining sealed cocoons to explore the role of cocoonase via genome editing technology.Finally,we evaluated the application value of using cocoonase gene as the target to create a"cocoon-invulnerable"novel silkworm breeding material.The main results of this research are as follows:1.The cocoonase is specific to LepidopteraIn order to reveal whether cocoonase is widespread and pervasive among insects and understand its origin,we conducted evolutionary analysis.A total of 295homologous proteins with more than 40%identity were retrieved from NR database,Insectbase and Lepbase.Phylogenetic analysis showed that 70 proteins among these homologous sequences were clustered together with the cocoonase enzyme of silkworm,Bombyx mori.Further domain analysis confirmed 72 homologous proteins as the orthologs of cocoonase,and all of them contained the cleavage site of the precursor,the catalytic triad?His-Asp-Ser?and the key cysteine residues forming three disulfide bonds.Further investigation showed that all the cocoonase enzymes were only from moths and butterflies,which indicated that cocoonase was specific to Lepidoptera.At the same time,the synteny analysis of the cocoonase loci showed a conserved gene organization and confirmed the identification of cocoonase.2.Cocoonase is under purifying selection in the evolution of LepidopteraIn order to understand the molecular evolutionary basis of these cocoonase genes,we used the branch model of Easycode ML software to detect the selection pressure on cocoonase gene in the evolution process.Assuming that the?values of all branches in the phylogenetic tree are equal,the result of one-ratio model showed that cocoonase gene in each branch was mainly affected by purifying selection during evolution??=0.16308?;when assuming that cocoonase gene is subject to different selection pressures between lepidopteran insects spining unsealed cocoons??₁?and sealed cocoons??₂?,the detection results of two-ratio model showed no significant difference with the result of one-ratio model??₀=0.16603,P=0.06688?,but the purifying selection was more intense in the moths spinning sealed cocoons??₁=0.13840,?₂=0.04881?.The results of selection pressure analysis suggest that cocoonase genes are more functionally conservative in insects spinning sealed cocoons,consisting with the result of multiple sequence alignments.Hyposmocoma kahamanoa,a kind of amphibious insects,belongs to the genus Hyposmocoma?Cosmopterigidae?and is located in the base of the Lepidoptera lineage in the evolutionary tree of species.Although it does not construct a cocoon,this species also has the cocoonase gene.Combined with the differentiation time of species and the cocooning behaviors of lepidopterans,it seems that cocoonase gene exists before the emergence of Lepidoptera insects spinning cocoons,ensuring the escape of adults from their cocoons.In addition,insects spinning cocoons are distributed in multiple families that are not closely related,indicating that the cocooning behavior may evolved parallelly.3.Knockout of cocoonase gene in the silkworm Bombyx mori based on the CRISPR/Cas9 systemTo uncover the internal relationships between cocoonase gene?Bm Coc?expression and development,and predict the phenotype after Bm Coc knockout,we investigated the temporal expression profile of Bm Coc through the entire life cycle.The results showed that Bm Coc was specifically expressed in the middle and late pupal stages,being initiated on the 5th day and gradually increasing to a peak on the 8th day and then rapidly declined.The expression could not be detected in the newly emerged silkworm moths.The specific expression pattern of Bm Coc suggests that it may play an important role during or after the emergence of moths.In order to explore whether cocoonase is the unique factor of the moth-vomiting fluid to assist the adult escaping from the sealed cocoon,we used the silkworm,Bombyx mori,as the model and knocked out Bm Coc using the CRISPR/Cas9 system.We designed a binary transgenic CRISPR/Cas9 system,in which Cas9 and g RNA targeting Bm Coc were expressed in two independent transgenic lines.After a series of hybridization,the homozygous individuals(Bm Coc^-/-)were identified by fluorescence screening and mutation sequence analysis.The expression level of Bm Coc^-/-was investigated at the m RNA and protein levels in Bm Coc^-/-homozygotes.The m RNA level of Bm Coc was extremely reduced compared to that in wild type individuals.Meanwhile,the silkmoth-vomiting fluid from wild type and Bm Coc^-/-moths were collected and used to implement SDS-PAGE experiment.Consistent with the results of q RT-PCR,there was no detectable mature cocoonase?predicted to be 23.7 k Da?in the samples of Bm Coc^-/-silkworms,confirming the complete disruption of Bm Coc.4.Cocoonase is the determining factor allowing silk moths to escape from the cocoonDuring the rearing process,we observed that the Bm Coc^-/-homozygotes were able to grow,spin and cocoon normally.Eleven days after pupation,all the wild type moths emerged,leaving pierced cocoon shells.Meanwhile,the chimeras and heterozygotes of Bm Coc edited were also able to escape from the cocoon.However,all the Bm Coc^-/-homozygotes could not escape from the cocoon,no matter whether the cocoon layer was thick or thin.After splitting the cocoons,successfully metamorphosed adult moths could be observed,indicating that there was no adverse impact on moth eclosion after knocking out Bm Coc.Moreover,there was no escapers in the offspring of the homozygous mutants.Although there were detectable extra proteins in the silkmoth-vomiting fluid,the moth stayed trapped in the cocoon due to the loss of cocoonase,which confirmed the determining role of Bm Coc in facilitating moth escape from cocoon.5.Evaluation of the practicability of the novel silkworm strain free from cocoon dryingBm Coc^-/-homozygous moths died naturally in the cocoons because they could not escape from the cocoon after eclosion.After being stored under natural conditions for up to 6 months,the cocoons were still bright and intact and there was no softening or mildewing of the moths.These phenotypes were in line with our assumption of the silkworm strain free from cocoon drying.In order to evaluate the practicality of the novel"cocoon-invulnerable"strain,we introduced the mutation site of Bm Coc gene from the basal strain-Dazao into the current commercial strain-872 through crossing and6 generations backcrossing.And the homozygous mutants——"cocoon-invulnerable"transgenic line 872,were obtained from the inbred individuals of BC6 generation.Then,we conducted comprehensive and detailed tests on Bm Coc^-/-homozygous mutants of Dazao and 872.First of all,we investigated the oviposition amount and hatching rate.The results showed that the two traits were not different between Bm Coc^-/-homozygous mutants and wild-type individuals,indicating that knocking out Bm Coc did not affect the reproduction.Secondly,we investigated the health status from five aspects:the duration of the 5th instar,the duration of the whole instar,the rate of diseased silkworms?the fourth and fifth instars?,the cocooning ratio,and the ratio of survival pupae.It showed that there was no significant changes in the homozygous mutant individuals compared with wild-type individuals,indicating that the normal growth and development of silkworm would not be affected after knocking out Bm Coc.Finally,we investigated the cocoon quality traits from three aspects:whole cocoon weight,cocoon shell weight,and cocoon shell rate.The results showed that these three key cocoon quality traits were not significantly different between Bm Coc^-/-homozygous and wild-type male and female individuals,which indicated that knocking out cocoonase gene did not affect the cocoon quality of the silkworm.In conclusion,cocoonase gene is Lepidoptera-specific,and it is under purifying selection in the evolutionary process.This gene is a necessary factor for adult emergence of Lepidoptera insects spinning sealed cocoons.This study provides a new strategy and target for lepidopterous pests control that either inhibition or inactivation of the enzymatic action of the cocoonase or gene drive strategies in lepidopteran pests spinning sealed cocoons could control the pest population.In addition,the homozygous mutant line of Bm Coc knockout,as a novel breeding material of cocoon drying-free silkworm strain,should bring significant innovation to the silk industry upon further evaluation and utilization.