Functional Study Of BmJHBPd2 In The Formation Of Silk Protein

The life of silkworm is mainly regulated by juvenile hormone(JH)and20-hydroxyecdysone(20E).Among them,JH is synthesized and secreted into hemolymph by corpus allatum,and is bound to free juvenile hormone binding protein(JHBP)in hemolymph,and transported to corresponding target organs or target cells to regulate the growth,development and metamorphosis of silkworm.Previous studies have shown that there is a specific expression of juvenile hormone binding protein gene(BmJHBPd2)in the posterior silk gland of Bombyx mori.In order to study the function of BmJHBPd2 in silk protein synthesis,we conducted research through transgenic overexpression technology and CRISPR/Cas9 knockout technology.The main findings are as follows:1、 The effect of knocking out BmJHBPd2 on silk protein synthesisIn order to explore the function of BmJHBPd2,we designed and synthesized three sg RNAs according to the gene sequence of BmJHBPd2,and injected them with Cas9 protein at the early embryo stage of silkworm.First,we performed genomic deletion detection on G0 generation individuals injected,and we found that there were individuals whose BmJHBPd2 was successfully knocked out.Then,we selected knockout individuals and bred them for subsequent generations,and extracted the genomes of some of the offspring for testing.We found that the knockout individuals had three types of knockout forms,namely,base deletion,insertion and mutation.We cloned and sequenced the open reading frame(ORF)of BmJHBPd2 that was knocked out,and found that the knocked out individuals were in heterozygous form with different knockout forms,which was consistent with the genomic results.At the same time,the protein sequence prediction results showed that the gene appeared the termination codon in advance.We used q RT-PCR to detect the expression level of BmJHBPd2 in the silk gland,and found that the expression level of this gene was significantly reduced.We investigated the expression of individual silk gene Bm Fib-H,Bm Fib-L and Bm P25 by q RT-PCR,and found that compared with the control group,the expression of silk protein genes Bm Fib-H,Bm Fib-L and Bm P25 were significantly down-regulated.Then,we investigate the cocoon quality,and the results of the survey showed BmJHBPd2 gene was knock out individual whole cocoon weight and cocoon shell weight,pupa weight were significantly lower,silk protein synthesis is reduced,and 25% of the naked pupa.These results indicate that BmJHBPd2 is involved not only in the development of individual silkworm,but also in the synthesis of silk protein.2、The effect of overexpression of BmJHBPd2 on silk protein synthesisBy comparing the expression levels of BmJHBPd2 in the silk gland tissues of different silk producing varieties,it was found that the expression level of BmJHBPd2 in the high silk producing varieties was significantly higher than that in the low silk producing varieties.In order to explore whether the overexpression of BmJHBPd2 could increase the synthesis of silk protein,we cloned the ORF of BmJHBPd2 and constructed a transgenic overexpression vector with silk fibroin light chain(Fib-L)as the promoter,piggy Bac vector as the connecting skeleton and Myc exogenous expression tag.The BmJHBPd2 overexpression strain was successfully obtained by microinjection and positive individual screening.Firstly,the BmJHBPd2 expression level of overexpressed transgenic strain was detected,and the results showed that the expression level of BmJHBPd2 in the posterior silk gland of the positive individuals at the third day of the fifth age was significantly higher than that of the wild type.At the protein level,the overexpressed individuals detected the protein bands expressed by the foreign Myc tag through Western blot detection,which was consistent with the detection results of the RNA level.We observed the positive individual silk glands at the same developmental stage and found no significant difference.The results of cocoon quality investigation showed that the total cocoon weight,cocoon layer weight,pupa weight and cocoon layer rate of BmJHBPd2 positive individuals were significantly reduced.At the later stage,we applied Juvenile hormone analogue(JHA)coating on the positive individuals at the 5th age and the 1st day to investigate its effect on silk synthesis,and found that the cocoon silk quantity and cocoon layer rate of overexpressed BmJHBPd2 were still lower than those of the wild-type control.The results showed that the overexpression of BmJHBPd2 in the posterior silk gland did not increase the synthesis of silk protein,but produced inhibitory effect.3、Determination of juvenile hormone III in posterior silk gland of silkworm by HPLCBmJHBPd2 is a juvenile hormone binding protein of the silk gland.According to the three-dimensional structure prediction,it has a similar structure to the reported JHBP and should be able to bind to JH.In the first part,we successfully knocked out this gene through knockout technology,which seriously affected the silk protein synthesis,indicating that this gene played a role in the silk protein synthesis process,but whether there was JH in the silk gland was still unclear.To explore this problem,HPLC was used to determine the content of silk gland JH.We randomly selected ten silkworms of 932 strains at the 5th age and the 5th day,dissected the posterior silk gland,ground the liquid nitrogen,and finally obtained 2.69 g of posterior silk gland powder.We used methanol and n-hexane extraction method for JH extraction,and then concentrated the extraction liquid by means of spin evaporation method,and melted it back into the methanol flow phase,finally obtaining 1m L of the posterior silk gland hormone extract.Before the sample was tested,we first detected the peak time of JH I and JH III standard and blank control dimethylsulfoxide(DMSO).It was found that JH I and JH III had their own characteristic peaks at 53.078 min and 26.945 min respectively.Then,we performed mechanical detection on the posterior silk gland hormone extract,and a peak was detected at 26.887 min,which was basically consistent with the peak time of JH III,but no peak was detected at 53 min.In order to further confirm that this characteristic peak is the peak value of JH III,we also used the internal standard method for determination,that is,adding JH III to the sample.We found that the peak area of the sample increased significantly at 26.887 min,but the peak did not shift.This result indicated that there was JH III in the posterior silk gland tissue of silkworm strain 932 on the 5th day of the 5th age,but the existence of JH I still needs further verification.According to the standard curve of the JH III standard product,the formula for calculating the concentration of JH III is y=30452x-1E+06,and the concentration of JH III in the posterior silk gland of the silkworm is 12.39 μg/g.Based on the results,we speculated that BmJHBPd2 of the posterior silk gland and JH III of the silk gland of silkworm might combine with JH III and play corresponding functions.In summary,BmJHBPd2 is involved in the synthesis of silk protein and plays a very important role in the synthesis of silk protein.Too high or too low expression of BmJHBPd2 will affect the synthesis of silk protein and have a certain influence on the development of silkworm.It was speculated that BmJHBPd2 balanced the titer of JH in the silk gland by binding to the free JH in the silk gland in this process,so as to coordinate the synthesis of silk protein.