Studies On The Function And Mechanisms Of BmYki In Regulating The Development Of Silkworm Wings

Wings are essential organs for insects to survive,and the insect world is colorful because of wings.But for pests,wings are an weapon to expand their expansion,such as the Spodoptera exigua that invaded many countries in South Asia via Africa in 2016 and attacked our country last year,and the desert locust that started in East Africa in 2019and recently ravaged the Middle East and South Asia,have amazing flight capabilities and devastating crops.how do insect wings occur and how are they regulated?This is an urgent content that the field of entomology is committed to solving.Bombyx mori is an insect of the subclass Lepidoptera domesticated from the wild silkworm.It has been reared and used by humans for more than 5000 years due to its ability to produce a large amount of silk.Compared with the wild silkworm,one of the biggest changes of silkworm is that it has lost its ability to fly,and its wing color and markings are also seriously degenerated.However,the basic morphology and development characteristics of insect wings are still there,so it is considered to be a unique model for studying the regulation mechanism of insect wing development and pest control strategy.Bombyx mori wing development is a complex process regulated by many genes.As early as the 1960s,researches on the regulation mechanism of silkworm wing development have been carried out at home and abroad,and much progress has been made,but its regulation mechanism has not been proved yet.In recent years,researchers have discovered an important signaling pathway called Hippo signaling pathway that controls organ size in drosophila.Its downstream transcriptional coactivator Yki is a"switch"of signal transduction,which turns on or off the expression of downstream target genes by entering or exiting the nucleus,thereby regulating organ size and development.Yki has been proved to play an important role in regulating the development of drosophila wing,but there are few reports on whether it has a similar function in silkworm and how to regulate it.In our previous study,we cloned BmYki,a transcriptional coactivator of Hippo signaling pathway,from silkworm.In order to further explore the function and mechanism of BmYki regulating the wing development of silkworm,this paper first constructed a Gal4/UAS transgenic line that could express target genes in high quantity in the wing primordium of silkworm.On this basis,genetic materials of overexpression,knockdown and knockout of BmYki were prepared,and wing mutant phenotypes and target genes of BmYki were analyzed.The main results are as follows:1.Preparation of GAL4/UAS transgenic silkworm with high expression of target gene in wing discWith the proximal promoter of the Actin4（A4）gene as the regulatory element and EGFP as the reporter gene,Our group use silkworm Nistari produced the Gal4transgenic line A4-GAL4（AG4）and UAS-EGFP（UEGFP）,respectively.However,the spatiotemporal characteristics of EGFP expression driven by A4 promoter have not been analyzed.In this study,AG4 was first hybridized with UEGFP,and positive individuals AG4/UEGFP,abbreviated as EGFP^OE,were screened from their progeny.The fluorescence results showed that EGFP expression started from the third day of embryo and continued until the adult stage,and EGFP expression was high in the wing primordia and inner epidermis of the pupal stage,which are closely related to the wing development of silkworm,as well as in the wing and scale hairs of the adult worm.By observing the main tissues of silkworm on the fifth day of the fifth instar,it was found that EGFP was highly expressed in the forewing primordia and inner epidermis,only a small amount was expressed in adipose body and trachea,and no expression of EGFP was observed in other tissues.The results of qPCR and Western blot were consistent with those of fluorescence observation.These results indicate that the Gal4/UAS system with A4 as the promoter is an effective tool to regulate the high expression of target gene in the wing disc of silkworm.2.Production of BmYki overexpression,knockdown and knockout systemsOur research group has previously produced and obtained UAS-based transgenic lines for overexpression,knockdown and knockout of BmYki,including UAS-BmYki,UAS-dsBmYki and UAS-BmYki^KO.In this study,three UAS transgenic lines were interbred with AG4 transgenic lines,and the corresponding Gal4/UAS positive individuals were screened from their hybrid progeny,which were successively named AG4/UAS-BmYki(Yki^OE,overexpressed BmYki)and AG4/UAS-dsBmYki(Yki^DS,Knockdown BmYki)and AG4/UAS-BmYki^KO(Yki^KO,knockout BmYki).Genome PCR and sequencing results showed that the above BmYki overexpression,knockdown and knockout systems were all successful,and could be used for further research.3.Phenotype analysis of overexpression,knockdown and knockout of BmYki silkworm wing disc（wing）With wild-type silkworm as a control,the wing primordia（wing）phenotypes of Yki^OE,Yki^DS and Yki^KO silkworms were investigated,and the results showed that:（1）Yki^OE larvae became smaller and the area of wing disc became smaller;adult wing shrank severely,The handsare dry and hard;the front and rear wings are sticky,almost without scale coverage;the body surface has fewer scales,and the antennae and feet are covered with only a few scales;the silkworm moth has weak mobility,and some silkworm moths cannot crawl out of the cocoon shell.（2）The mutant phenotype of Yki^DS and Yki^KO is opposite to that of Yki^OE,which is mainly manifested as the larger area of larval wing disc and adult wing,and the larger adult individual.（3）The traits of Yki^OE,Yki^DS and Yki^KO larvae such as body weight,pupa cocoon weight,mulberry consumption,growth cycle,lethality and mating rate were all affected to varying degrees.These results indicate that BmYki has an important regulatory effect on silkworm wing development.The phenotypes of wing disc（wing）of Yki^OE,Yki^DS and Yki^KO were investigated with wild type silkworm as control.The results showed that:（1）larvae of Yki^OE were smaller,and wing disc area was smaller;Adult wing wrinkled seriously,hand touch dry hard;Anterior and posterior wings conglutinate,almost without scaly hair cover;The body surface has few scale hairs,antennae and feet only a few scale hairs cover;The moth’s activity ability is weak,part of the moth cannot climb out of the cocoon shell.（2）The mutant phenotypes of Yki^DS and Yki^KO were opposite to those of Yki^OE.The main phenotypes were larval wing primordia,adult wing area enlarged,and adult individual enlarged.（3）Larval weight,cocoon weight,mulberry feeding,growth cycle,mortality and mating rate of Yki^OE,Yki^DS and Yki^KO were all affected to different degrees.These results indicate that BmYki plays an important role in regulating the wing development of Bombyx mori.4.A preliminary study on the molecular mechanism of BmYki regulating the wing development of silkwormIn order to explore the molecular mechanism of BmYki regulating silkworm wing development,transcriptome sequencing analysis was performed on the wing tissues of Yki^OE and Yki^DS at the fifth day of pupal stage.The results showed that:（1）516differentially expressed genes were found in the wing tissues of Yki^OE,among which302 were up-regulated and 214 were down-regulated.There were 165 differentially expressed genes in the wing tissues of Yki^DS among which 53 genes were up-regulated and 112 genes were down-regulated.A total of 57 differentially expressed genes were found in wing tissues of Yki^OE and Yki^DS.（2）Four types of differentially expressed genes were identified to be closely related to the wing development of silkworm,respectively:3 wing pattern determining genes（Dpp,ASH,Vg）,2 wing development related genes（Osiris,Notch）,and 5 hormone signaling related genes（HR3,E74,Jheh-2,Bm3DE-3α-reductase and Bm3DE-3β-reductase）.And 33 wing primordial epidermal protein genes（CPR3,CPH9,CPFL,CPG9,CPT3 and BmWCP10）.The results of fluorescence quantitative detection were basically consistent with the above results.These results suggest that wing pattern determining genes,hormone signal transduction genes and epidermal protein genes are downstream target genes of BmYki,and BmYki regulates the wing development of silkworm by regulating the expression of these genes.（3）Using hormone signal response factors HR3 and E74A as targets,transgenic lines AG4/UAS-HR3(HR3^OE)and AG4/UAS-E74A(E74A^OE)were prepared.It was found that both overexpression of HR3 and E74A could cause wings of adult silkworm to become smaller and curl,but the degree of mutation was slightly less than that of overexpression of BmYki.These results indicate that the regulation of wing development by hormone signaling response factors is one of the important functional ways of BmYki.In conclusion,on the basis of confirming that the Gal4/UAS system with A4 as the promoter can express a high amount of target genes in the wing disc（wing）of silkworm,the overexpression,knockdown and knockout lines of the Gal4/UAS system with BmYki as the target were made in this study,and then the mutant phenotype analysis confirmed that BmYki is an important factor regulating the wing development of silkworm.Through transcriptome sequencing,important candidate targets such as wing pattern determining genes,hormone signal transduction genes and Insect cuticular proteins genes regulated by BmYki were identified,and the overexpression of transgenic representative candidate targets was verified.These results lay a solid foundation for elucidating the molecular mechanism of BmYki regulating the wing development of silkworm,and provide useful clues for the study on the wing development of silkworm.