Identification Of Key Metabolites In Pupal Nutrient Allocation From Silkworm, Bombyx Mori

Bombyx mori is a lepidopteran silk-spinning insect and was domesticated from the wild silkworm. After a long-period artificial selection, silkworm has a well-developed silk gland. Compared with the wild silkworm, the domestic silkworm have better secretory ability and better silk quality. During the thousands of years domestication, the cocoon shell’s weight and size increased, at the same time, the pupal weight and size also become greater. Through deeply study of the correction of major economic traits in silkworm, it is found that the cocoon layer weight and pupal weight is highly correlated (genetic correlation coefficient is 0.856), suggesting that the cocoon layer weight and pupal weight is increased or decreased simultaneously.For thousands of years, the main purpose of raising silkworm is for the silk reeling to obtain the silk products that needed in life. Now people are more concerned about their health and comfort, thus silk products, as an excellent natural fibers, will undoubtedly become the darling of people. However the main obstacle for the sericulture is the low silk production, and the traditional methods for improving the silk production have their limitations. Thus it is very important for us to look for other ways to increase the silk production.In the fifth instar silkworm will eat more leaves to get nutrients,during the early fifth instar of silkworm, the nutrients intake from mulberry is mainly used to build the body. At the last stage of the fifth instar, some of which is used for the synthesis of silk, and the other nutrients is translated to energy for the life activities of the pupa and moth. During the actual feeding prosess, neither the strain that with big cocoon and small pupa nor the strain with light cocoon and great pupa was found,it suggest that there is a dynamic balance the allocation of material and energy between the pupa and silk. This laboratory applied Zinc finger nuclease (ZNF) technology to knockout the Fib-H gene ((?)oin heavy chain gene, which encodes a silk protein) and won the silk synthesis is blocked in the knockout strains (Bombyx mori fib-h gene knock-out, Bmfib-H KO), but the pupal size and weight is increased significantly. The silk protein is synthetic by silk gland, so we design the artificial removal of the silk gland surgery. After completely block the synthesis of fibroin, we got the same phenotype knockout strains.In the thesis,we successfully prevented the synthesis of the silk protein and changed the distribution of nuritents through the above two methods.In order to find out the key metabolites that may regulate the distribution of nutrients,we further analysis the pupal hemolymph from two experimental materials by non-target metabolomics technology.The main results are as follows:1、Hemolymph metabonomics based on GC-MC of silkworm at the stage of pupa in the group of Bmfib-H KO and silk glands extirpation group:Hemolymph metabonomics data analysis method using GC-MC was established. According the results, we identified 38 differential metabolites in the group of Bmfib-H KO and 42 differential metabolites in the group of silkworm silk glands extirpation. Differential metabolites mainly contained tricarboxylic acid cycle intermediates, sugar and sugar alcohols, amino acids, fatty acids, sterols. The major metabolic pathways involved tricarboxylic acid cycle, glucose metabolism, amino acid metabolism, fatty acid biosynthesis and sterol biosynthesis.20 differential metabolites were identified by two sets of data at the same time,6 differential metabolites expression level increased after biocking the silk synthesis, including proline, valine, isoleucine, tryptophan, threonine and trehalose.8 differential metabolites expression level decreased after biocking the silk synthesis, including sitosterol, cholesterol, phosphoric acid, mannitol cellose and propionic acid and so on.2、Hemolymph metabonomics based LC-MC of silkworm at the stage of pupa in the group of Bmfib-H KO and silk glands extirpation group:Hemolymph metabonomics data analysis method using LC-MC was established. According the results, we identified 177 differential metabolites in the group of Bmfib-H KO and 236 differential metabolites in the group of silkworm silk glands extirpation. Differential metabolites mainly contained tricarboxylic acid cycle intermediates, amino acids, fatty acids,ester amide, carbohydrates, heterocyclic and so on. The major metabolic pathways involved tricarboxylic acid cycle, glucose metabolism, amino acid metabolism, fatty acid biosynthesis, purine metabolism and biotin metabolism and all.52 differential metabolites were identified by two sets of data at the same time,25 differential metabolites expression level increased after biocking the silk synthesis, including amino acids differential metabolites such as Leucine, lysine, tyrosine, lysine arginine, serine, heterocyclic differential metabolites such as nicotinamide, quinoline, morpholine, benzimidazole, sulfone, pyrimidinones, and tricarboxylic acid cycle intermediates such as succinic acid, butyric acid, octanoic acid. 6 differential metabolites expression level decreased after biocking the silk synthesis, including palmitic acid, stearic acid, phosphoric acid, spermidine, quinuclidine and biotin.3、The integration analysis of differential metabolites by GC/LC-MS::Integration analyzes of data from the third chapter and the fourth chapter. GC-MC data analysis showed that there were 20 common differential metabolites were identified both in the group of Bmfib-H KO and the group with the silkworm silk glands extirpation were abstracted, as for the LC-MS data analysis method, we got 52 common differential metabolites. Further data analysis of 72 different metabolites from two methods, network topology diagram analysis results show that leucine, tyrosine, lysine, proline plays a role of critical information nodes in the map of the entire network.Differences in metabolic pathway analysis showed that the amino acid involved in major differences in metabolic pathways. The analysis results from the abundance of amino acid showed that they are increased significantly after biocking the silk synthesis The above analysis results showed that leucine, tyrosine, arginine, proline is the key metabolites and may play an important role in the regulation of the distribution process of the nutrients.