| Silkworm（Bombyx mori）has a long history of domestication,and economic traits such as body weight and silk production of silkworm have been positively selected after long-term artificial selection.Compared with wild silkworms（Bombyx mandarina）,silkworms therefore need to take in more food to meet the needs of growth,development and reproduction.Existing research reports show that the salivary glands secrete salivary fluid during the feeding process of insects.On the one hand,it lubricates the food,and on the other hand,the digestive enzymes in the salivary fluid also have the function of preliminary digestion of the food.Whether the salivary glands play other distinct functions in insect nutrient utilization is currently unclear.The research work of Master Bingchuan Bai in our group shows that a fatty acid desaturase Bmdesat5 is specifically and highly expressed in salivary glands of wild silkworm,but not expressed in silkworm;and silkworms eat significantly less after feeding its enzymatic product cis-vaccenic acid and growth slowed down.Based on this,we put forward the hypothesis:Bmdesat5 plays a role in regulating feeding and satiety in wild silkworm through its enzymatic product;domestication selection leads to the inhibition of Bmdesat5 expression in silkworm,and then the synthesis of enzymatic product is reduced,and satiety regulation is weakened,resulting in the increase in the amount of mulberries it eats provides a guarantee of nutrients for humans who grow larger and produce more silk.To test this hypothesis,we conducted related studies and obtained the following findings:1.Analysis of domestication characteristics of Bmdesat5 sequencesCollecting the silkworm germplasm resources preserved in the laboratory,we obtained 27 genome sequences corresponding to the Bmdesat5 gene through multi-fragment PCR amplification,sequencing and assembly,including 21 Bombyx mori and 6Bombyx mandarina relationship was analyzed.Firstly,the phylogenetic tree clustering analysis based on the nucleotide sequence of Bmdesat5 gene was carried out.The phylogenetic tree of Bmdesat5 gene showed that the Bombyx mori were all clustered in one branch,and the Bombyx mandarina were clustered in another branch,and the two groups could be well distinguished.Second,we compared the polymorphisms in different regions（5’UTR,CDS,intron and non-coding region）of Bmdesat5 gene.The polymorphism of Bmdesat5 sequence in Bombyx mori and Bombyx mandarina is mainly concentrated in intron and non-coding region.Especially in the intron region and 5’UTR,the differences of nucleotide polymorphisms in the CDS region is small;the sliding window analysis of nucleotide polymorphisms showed that the polymorphisms of the Bmdesat5 gene of Bombyx mori are the same in the whole gene region higher than Bombyx mandarina.The SNP,πtotal andθw in Bombyx mori were lower than those in Bombyx mandarina,indicating that the genetic diversity of this gene was decreasing during evolution.Neutral tests of upstream regulatory regions with significantly different polymorphisms,Tajima’s D test shows that Tajima’s D in Bombyx mori is-2.27542,Tajima’s D in Bombyx mandarina is 0.05569;while Fu and Li’s D*silkworm value is-0.83043 and Fu and Li’s F*silkworm value is-0.67024 are both negative values,which indicates that the observed heterozygous loci in the population is less than the expected value,and the frequency of rare alleles increases,which is also the result of population expansion;it also indicates that this gene is under selection pressure in Bombyx mori,and some regions of Bombyx mori may have been selectively eliminated.2.Exploration of the mechanism of differential expression of Bmdesat5 in Bombyx mori and Bombyx mandarinaBmdesat5 was significantly differentially expressed in the salivary glands of Bombyx mori and Bombyx mandarina.We firstly aligned the protein sequences of Bmdesat5 in different species of Bombyx mori and Bombyx mandarina in different regions.The alignment results showed that its protein sequence was completely conserved in many Bombyx mori and Bombyx mandarina.In the detection of the promoter activity of 2700bp upstream of the translation initiation site of Bmdesat5,it was found that the promoter of Bmdesat5 in Bombyx mori was also active,and the relative activity was even higher than that of Bombyx mandarina.In order to further explore the expression inhibition of Bmdesat5 in Bombyx mori,we crossed Bombyx mori and Bombyx mandarina,and successfully obtained the first generation of the hybrid line,and then detected the expression level of Bmdesat5.The results showed that the expression level of Bmdesat5in salivary glands of Bombyx mori and Bombyx mandarina was significantly higher than that of Bombyx mori and hybrid lines,which indicated that the individual had in the case of Bombyx mori genetic background,the expression of Bmdesat5 was inhibited to a certain extent.This indicated that the lack of expression of Bmdesat5 in silkworm was a dominant trait relative to its expression.Therefore,we speculate that there may be a strong transcriptional repressor targeting Bmdesat5 in Bombyx mori to suppress the transcription of Bmdesat5;The specific reasons remain to be further explored.3.The molecular mechanism of Bmdesat5 regulating silkworm feeding and the biological significance of expression inhibition in Bombyx moriThrough the data survey of Bombyx mori and Bombyx mandarina,the single time of eating mulberry is significantly longer than that of Bombyx mandarina.Bombyx mori quickly entered the mulberry-eating state after feeding on mulberry leaves,and the average single-time mulberry-eating time was 39 min.The average single feeding time of Bombyx mandarina was 26 min;and the expression of Bmdesat5 also changed with the different satiety state of Bombyx mandarina.The product cis-vaccenic acid can up-regulate the expression of Bombyx mori satiety regulator gene Bm SK.This preliminarily suggested that the high abundance of Bmdesat5 in Bombyx mori plays a role in regulating food intake and satiety through its products.Whether the endogenous expression of Bmdesat5 in Bombyx mori produces a phenotype similar to that of product feeding will be a direct evidence to prove whether Bmdesat5 is involved in the regulation of satiety in Bombyx mori.Through genetic engineering,we successfully constructed a Bmdesat5 overexpression vector.Through microinjection technology and fluorescence screening,a stable transgenic line A4-desat5that can express Bmdesat5 protein and synthesize cis-vaccenic acid was obtained.In the statistics of the body weight and food intake of the fifth instar larvae,it was found that the male and female individuals of the A4-desat5 transgenic silkworm were lower than those of the wild-type silkworm.The pupa weight of the male silkworm was 13.01%lower than that of the wild type,but there was no difference in the cocoon shell rate.All the above results indicated that Bmdesat5 was involved in regulating satiety and food intake of silkworm,did not affect silkworm development.4.Exploration of functional application of Bmdesat5 enzymatic productsGiven that the Bmdesat5 product cis-vaccenic acid plays a role in regulating feeding and satiety in the silkworm,does this role also apply to mammals?Related research has been carried out on this topic.We explored the possible application value and prospect of this gene and its products through mammalian system.We explored the effect of cis-vaccenic acid on food intake and satiety in mice by adding cis-vaccenic acid to mice;the results showed that the weight of mice in the cis-vaccenic acid group decreased by 16.1%compared with the control group,the food intake was lower than 34.1%of the control group;at the same time,we also investigated the effect of cis-vaccenic acid on the expression of satiety-related genes in mice.The results showed that,compared with the control group,the cis-vaccenic acid group increased CCK by about 11.4 times and PYY by about 1.8 times.This also shows that cis-vaccenic acid can increase the satiety of mice.At the same time,we found that cis-vaccenic acid can also reduce the accumulation of fat in the liver of mice and accelerate the metabolism of fat;it has a significant effect on relieving liver steatosis caused by high-fat diet.At the same time,the observation of the mice and the statistics of the organ index during the experiment showed that cis-vaccenic acid had no adverse effect on the growth and health of the mice.The above research results show that Bmdesat5 and its product cis-vaccenic acid also have certain application prospects in the fields of human body weight control,improvement of sub-health-related symptoms caused by obesity,and non-alcoholic fatty liver disease. |
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