Improving The Silk Synthesis Efficiency Of Silkworm Using Amino Acid Transporters And MTORC1 Signaling Pathway

Silkworm,Bombyx mori,has become an important economic insect due to artificial domestication for thousands of years.Silk is mainly economic products for Bombyx mori.Therefore the economic value of the silkworm depends on the silk yield.Cocoon shell ratio（cocoon shell weight/cocoon weight）is used for measuring silk yield in the industry.With the improvement of the cocoon shell ratio,silk yield increased and economic value is further enhanced.Hence,the cocoon shell ratio becomes a trait that breeders always focus on it.Through long-term artificial breeding,the cocoon shell ratio of the silkworm has increased from 10%to 25%.However,there are some restrictions in the traditional breeding technology,leading to a dilemma for the improvement of the cocoon shell ratio.In order to increase the cocoon shell ratio and economic value,it is necessary to break traditional breeding methods and find a sally port using new technology.The essence of increasing the cocoon shell ratio is to improve silk protein yield.Therefore,improvement can be viewed from two aspects:the efficiency and time of silk protein synthesis.In fact,increasing time of silk protein synthesis extends developmental period of the larvae,which adding consumption of manpower and mulberry leaf.And the actual income has little increase in the perspective of the industry.Therefore we focus on the improvement of silk synthesis efficiency.Firstly,amino acids are raw materials in the process of protein synthesis.By providing more raw material,protein production increased.Therefore,increased raw material of silk protein synthesis is regarded as a potential method to improve the silk protein synthesis efficiency.Preliminary analysis is showed that the major amino acids（glycine,alanine and serine）of silk protein are a sally port,and the three amino acids account for 85%in fibroin protein.Interestingly,glycine is the main amino acid which accounts for 45.9%in fibroin protein,and it can be converted into alanine and serine by transamination and deamination.In order to improve the efficiency of silk protein synthesis and cultivate a strain of increasing cocoon shell ratio,we plan to increase the glycine content through transporting glycine from the larval haemolymph to the silk gland.Secondly,m TORC1 signaling pathway plays important roles in protein translation,which responds sensitively to the nutritional signal and regulates the formation of translation initiation complex to modulate protein synthesis.A larva eats 20 g mulberry leaves,then producing 0.5 g silk proteins,which indicate vigorous protein synthesis in the silk gland.Therefore,silk gland is also regarded as a protein production factory.During the fifth instar,consumption of mulberry leaf,silk protein synthesis and silk gland size are rapidly increased.We speculate that nutritional signal may be involved in the regulation of the silk protein synthesis via m TORC1signaling pathway at the fifth instar.Therefore,we want to explore the relation between nutritional signal and silk protein synthesis and m TORC1 signaling pathway.In addition,we plan to increase the m TORC1 activity and improve the capacity of the silk protein synthesis using amino acid transporters.Using a series of techniques of bioinformatics and molecular biology,the influences of the major amino acid（glycine）of silk protein and m TORC1 signaling pathway on the silk synthesis efficiency were studied in the protein translation process.To increase the silk synthesis efficiency,the level of amino acids and the activation of m TORC1 signaling was genetically modified using the transgenic methods.Then,we cultivated three transgenic silkworm strains that exhibited increased silk yield and cocoon shell ratio.Finally,the results showed that Bm SLC7A5 modulated leucine content to affect the m TORC1 activity and control the synthesis of silk proteins and transcription factors.The major conclusions and achievements are as following:1.Identification of the silkworm solute carrier 6/7 family gene and analysis of evolutionary relationship and expression patternsWe selected the solute carrier 6/7 gene family which is involved in transporting glycine or activation of the m TORC1 signaling base on the major amino acids of silk protein and m TORC1 signaling pathway.Firstly,we identified the solute carrier 6 gene family in the silkworm genome,then we performed a simple analysis that contains the amino acid sequence alignment,evolutionary relationship,expression patterns and potential physiological functions.As a result,a total of 16 transporters were identified as SLC6 family gene in the silkworm genome.And the SLC6 gene family in insects was commonly separated into five subfamilies,including nutrient amino acid transporters（NATs）,amino acid transporters（AATs）,INE（inebriated genes）,neurotransmitter transporters（NTTs）,Orphan transporters.Certainly,silkworm SLC6 family genes were found to be broadly distributed across all subfamilies.Subsequently,expression patterns revealed that Bm NAT1,Bm NAT2,Bm GT1-L,Bm SLC6-15,Bm INE and Bm GT1 were highly or specifically expressed in the midgut.Midgut-enriched genes showed persistent expression at the fifth instar,which may be involved in absorbing nutrient amino acids.In addition,the brain-enriched genes,Bm GAT1,Bm GT2,Bm DAT,Bm SERT,Bm B（0）AT3-1 and Bm B（0）AT3-2,may be involved in mating and wandering behavior.A total of 12 transporters were identified as SLC7 family gene in the silkworm genome using other known SLC7 sequences.We performed a simple analysis that contains the amino acid sequence alignment,evolutionary relationship and expression patterns.Consequently,the silkworm SLC7 family gene was divided into two subfamily,including CATs and HATs,which were similar to other insects.Moreover,SLC7 members showed extensive tissue distribution,and most members were expressed in the malpighian tubules.However,Bm SLC7A5 was highly expressed in the silk gland.Therefore we speculated that the physiological function was different between Bm SLC7A5 and other members.2.Functional study and utilization of Bm GT1-L geneIn the following studies,glycine transporter was selected to further analyse,because we mainly focused on the major amino acids（glycine）of silk protein.There were three glycine transporters in the silkworm genome.However,Bm GT1-L gene was specifically expressed in the midgut,which was selected for the follow-up studies.The expression patterns of Bm GT1-L in the transcripts and proteins were detected by real-time PCR,quantitative real-time PCR and western blotting,respectively.Furthermore,immunofluorescence localization of Bm GT1-L was carried out.The results were exhibited that Bm GT1-L was specifically expressed in the midgut and highly expressed in the microvillus.Therefore,we speculated that Bm GT1-L may be involved in nutrient absorption of the microvilli in the silkworm.The expression level of Bm GT1-L displayed significant difference between molting larvae and newly molted larvae,and it implied that Bm GT1-L may be affected by JHA or diet.In order to examine the differences,the treatment of JHA and starvation was performed in ex-vivo midgut and larvae,respectively.As a result,the expression level of Bm GT1-L was affected by starvation and JHA.Furthermore,the capacity of transporting glycine was indirectly proved by ¹⁵N-glycine and inhibitor.The results inferred that Bm GT1-L may be responsible for transferring and absorbing glycine in the midgut.In conclusion,Bm GT1-L can be considered as a molecular target to increase glycine content in the silk gland.In order to determine whether the silk synthesis efficiency is enhanced by providing more raw material,the function of Bm GT1-L was directly used to improve the silk yield.The Bm Fib HP,which is a posterior silk gland-spicific promoter,was cloned from the silkworm genome.We used Bm Fib HP and Bm GT1-L gene to construct the piggyback[3×P3-Red,Bm Fibh P-Bm GT1-L]vectors.Subsequently,we obtained a transgenic silkworm strain that Bm GT1-L was ectopic expression in the posterior silk gland（PSG）.The silk yield and economic value were markedly improvement.Moreover,cocoon shell weight increased 11%and cocoon layer ratio increased 1%.Ectopic expression of Bm GT1-L in the posterior silk gland promoted glycine biosynthesis,and enhanced silk yield via increasing fibroin synthesis.Together,our results will further enhance functional studies of Bm GT1-L and provide a novel insight to increase silk yield on the perspective of raw materials of silk protein synthesis.3.m TORC1 signaling pathway regulates silk protein synthesisIn this chapter,we focus on the silk synthesis efficiency.Therefore factors influencing protein translation were assessed.In the translation process,m TORC1signaling pathway responds to the nutritional signal and regulates the protein synthesis.Therefore,we wanted to explore the relation between m TORC1 signaling pathway and silk protein synthesis.Firstly,the results of rapamycin treatment in vivo and in vitro showed that the silk proteins（Bm Fib H,Bm Fib L and Bm P25）were down-regulated,as well as transcription factors（Bmdimm and Bm SGF1）.In addition,4E-binding protein（4EBP）and p70 S6 kinase（S6K）,the effectors of TORC1,showed that phosphorylation levels were much lower than the control.Compared with the control,the cocoon size was significantly decreased,and the cocoon shell weight and ratio were obviously reduced about 45%and 7.3%respectively.Moreover,silk gland size was markedly smaller than that of control,however,the pupa weight was increased 21%.Whereafter,the Bm Rheb gene,which directly activates m TOR kinase,was overexpression in the PSG by transgenic technology.Finally,the transgenic silkworms showed that the phosphorylation levels of 4EBP and S6K were increased in the silk gland,leading to obvious activation of expression of silk protein and transcription factors.Furthermore,the size of the cocoon and silk gland was significantly enlarged,and cocoon shell weight and ratio increased 16%and 1%,respectively.In general,the m TORC1 signaling pathway can regulate the silk protein synthesis.4.Bm SLC7A5 mediates leu-m TORC1 signal regulating silk protein synthesisIn order to reveal the regulation mechanism of m TORC1 signaling pathway in silk protein synthesis at the fifth instar,we performed further analysis.In other species,SLC7A5 is involved in activating the m TORC1 signaling pathway.Interestingly,Bm SLC7A5 was highly expressed in the silk gland,which implied this gene may modulate the silk protein synthesis by the m TORC1 signaling pathway.Hence,we selected silk gland-enriched Bm SLC7A5 gene to perform further study.Firstly,the tissue expression profile of Bm SLC7A5 protein was detected,and immunofluorescence localization was performed.The results indicated that Bm SLC7A5 was mainly expressed in the silk gland,midgut and fat body,and the protein was located in the plasma membrane and endomembrane of the silk gland.In the PSG,Bm SLC7A5 was highly expressed from the first day to the third day of the fifth instar,and it was subsequently reduced.However,change of leucine content in the PSG was similar to the expression patterns of Bm SLC7A5,which implied that there was a relation between the two factors.Through the leucine injection and leucine supplementation in the diet,we found that leucine activated the m TORC1 signaling and promoted the expression of silk protein and transcription factors.Hence,we speculated Bm SLC7A5 was involved in modulating the leucine content in the silk gland,and further affected the m TORC1 signaling and silk protein synthesis.Then,we performed Bm SLC7A5 knockout using CRISPR/Cas9 system.The results showed that the growth and development of heterozygote were inhibited,and the body size became smaller,and larval periods of heterozygote were extended.Moreover,the m TORC1 signaling was decreased in the silk gland of heterozygote,leading to obvious inhibition of expression of silk protein and transcription factors.Finally,the size of the silk gland and cocoon became smaller,and the cocoon shell weight of mutants was reduced dramatically with 2.8 times.Moreover,the embryonic period of homozygote was extended,and homozygote died at the second instar.Similarly,the results of Bm SLC7A5knockout in PSG exhibited that the cocoon size was significantly decreased,and the cocoon shell weight and ratio were reduced about 31%and 2%respectively.Furthermore,we performed Bm SLC7A5 overexpression in the PSG.The results indicated the m TORC1signaling was obviously increased in the silk gland,leading to obvious promotion of expression of silk protein and transcription factors.Finally,the size of the silk gland and cocoon was increased,and silk yield and cocoon shell ratio increased～25%and～2%,respectively.Overall,we proved that the major amino acids of silk protein and m TORC1 signaling pathway were involved in regulating of silk synthesis efficiency in the translation process.In addition,we preliminarily revealed that the molecular mechanism of silk protein synthesis at the fifth instar on the perspective of nutritional signal,and provided a novel insight to increase silk yield.