Studies On Producing The Transgenic Silkworm Harboring Bh-EGase â…¡ Gene And Its Related Features

Feed efficiency has always been the economic trait which gets the attention of silkworm breeders. Many scholars’ research suggests that although the cocoon silk scores has been a significant increase, the digestibility and feed efficiency of commercial variety in the1980s compared with the early, were no change. At present, the feed efficiency of commercial variety still could not been improved in China’s production.Mulberry leaf is the prime sources of feed for silkworm, silkworm absorbs nutrient content from it in order to maintaining bodily functions and promoting growth. The dry matter of mulberry leaf is composed of protein, carbohydrate, lipid, etc, most of proteins and lipids can be absorbed by silkworm. The cellulose is the main ingredients of mulberry leaf cell walls, its absorption rate is only0.04%because of the lack of enzymes of cellulose degradation in midgut, that is the major factor affecting digestion and absorption of mulberry leaf.Exogenous gene can integrate and express in host cells using transgenic technology. Make use of transgenic technique to break germ line isolation under natural conditions, can help gene to flow between the distant organisms of the germ line. In this study, we first cloned endogenous cellulose Ⅱ (β-1,4-endoglucanase Ⅱ from the beetle, Batocera horsfieldi, Bh-EGase Ⅱ) gene, expressed recombinant Bh-EGase Ⅱ enzyme in BmN cells and larvae of silkworm, analyzed activity of Bh-EGase Ⅱ enzyme, constructed the transgenic vector of secreting Bh-EGase Ⅱ enzyme, preliminarily discussed the effect of Bh-Gase Ⅱ gene on the glucose metabolism of larvae and the quality of cocoon. Main results for this study are as follows:1. cDNA cloning, expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldiIn the present study, we cloned a novel cellulase gene, named Bh-EGase Ⅱ, from the midgut tissues of beetle Batocera horsfieldi using RACE method. The Bh-EGase Ⅱ gene contains only one exon and without an intron. The Bh-EGaseⅡ enzyme belongs to glucoside hydrolase family45(GHF45) has a very conservative catalytic site of GHF45at amino acid residues36-48and three N-glycosylation sites.The Bh-EGaseⅡ enzyme consists of a single catalytic domain and lacks both a cellulose-binding domain as well as a spacer sequence that is similar to the amino acid sequences from Ag-EGase Ⅰ, Ag-EGase Ⅱ, and R. speratus hindgut protein. The amino acid sequence of Bh-EGase II cDNA has93.72%homology with Ag-EGase Ⅱ,73.64%similarity with Ag-EGase land67.78%homology with β,4-endoglucanase Ⅱ of O.albomarginata chamela.The Bh-EGase Ⅱ cDNA was expressed in baculovirus-infected insect BmN cells and fifth instar larvae of silkworm. The results of SDS-PAGE and Western blot show that there are bands of about28kDa from cell lysates and33kDa from cell supernatant as well as larva hemolymph. The different mobility on SDS-PAGE can be explained by a possible post-translational modification at three potential N-glycosylation sites,56-58(N-K-S),99-101(N-S-T) and237-239(N-Y-S) respectively, and14conservative cysteine (C) residues.The molecular weight of proteins that were secreted out of the cell increased because of protein folding after translation and galactosylated modification.We discovered that Ag-EGase Ⅱ shared93.72%identity with Bh-EGase Ⅱ. Its N-linked glycosylation site at amino acid residues99-101(NST) was essential to its enzymatic activity. The result of Congo red staining test indicates that the enzymatic activity of recombinant Bh-EGase Ⅱ is closely related to the recombinant protein modification, which is conserved in known beetle GHF45. The unglycosylated recombinant Bh-EGase Ⅱ from cell lysates lost its enzymatic activity, in contrast, the glycosylated recombinant Bh-EGase Ⅱ from cell supernatant as well as larva hemolymph has enzyme activity.the enzymatic activity of recombinant Bh-EGase Ⅱ from BmN cell supernatant as well as larva hemolymph is about28U/mg, the Optimum pH and temperature of this enzyme are pH6.0and50℃. The enzymatic activity could maintain at about60%, when the pH and temperature are pH8.0-9.0and20-30℃, accord with the acid and alkali environment of silkworm midgut and the optimum temperature of Silkworm breeding. Our results strengthen the understanding of the gene structure and enzyme activity of insect cellulases. Bh-EGase Ⅱ can be used as a candidate gene for transgenic silkworm research to improve food efficiency.2. The construction of Trans-Bh-EGase Ⅱ gene vector and the test of in BmN cellsConstruction transgenic expression vector based on piggyBacs Bh-EGase Ⅱ, and introduction recombinant plasmid into the BmN cells by transfection methods, then proved eGFP reporter gene can express in the recombinant plasmid by fluorescence detection method; The molecular biology functions of BmN cells may represent the basic functions of the silkworm larvae, our results lay the foundation for promoting the Bh-EGas Ⅱ gene transfer vector successfully expressing in silkworm larvae.3. A look at taking advantage of exogenous Bh-EGase Ⅱ gene to raise the cellulose’s use efficiency of silkworm.According to the standard methods, we microinjected the early embryos of Dazao, gain transgenic positive individuals expressing green fluorescent protein, established a single transgenic strain. At the same time, we also detected the insertion sites of transgenic individuals at molecular level using genomic PCR, reverse PCR technology. The results show that foreign genes are inserted into Dazao’s genome, and the insertion sites located on chromosome4.By means of DNS colorimetry the OD540nm value of reducing sugar from food in the lumen of transgenic silkworm midgut was determined, the results showed that the mean OD540value of reducing sugar in transgenic silkworm was0.429, mean square error was0.0024; the mean OD540value of reducing sugar in control was0.381, mean square error was0.0021, showing that the exogenous Bh-EGase II gene being integrated into silkworm genome has been expressed and secreted into the lumen of transgenic individuals; the Bh-EGase Ⅱ enzyme in the lumen degraded the cellulose of food, causing the raise of the content of reducing sugar in food.Bombyx mori6-phosphofructokinase-1(Bombyx mori phosphofructokinase, BmPFK) is the key enzymes in glycolysis. Real time RT-PCR was used to detect the expression quantity of BmPFK mRNA in transgenic silkworm. The results showed that the expression quantity of BmPFK mRNA in midgut of the transgenic silkworm was al.58-fold compared with the control group, and the difference has reached the significant level(P<0.05). the results indicates using Bh-Gase Ⅱ gene can not only increase reducing sugar content in the intestine, but also accelerate glycolytic cycle in the midgut cells.Glycolysis is close tied to protein metabolism. the middle product of glycolysis can be used to synthetize the carbonaceous framework of amino acids, after transamination, they can generate amino acids; the energy produced in glycolysis can be used for the synthesis of amino acids and proteins. A survey of the cocoon quality score of silkworm with Bh-Gase Ⅱ gene found that the average amount of cocoon and cocoon layer of male and female transgenic silkworms were1.073g and0.134g, increased by3.5%and3.1%, respectively, suggesting cellulose is a principal factor to promote the peristaltic movement of food. The degradation of cellulose can reduce the peristalsis speed, delay the remains time of food in the midgut, make midgut cells fully absorb the nutrient composition in food, and accelerate glycolysis, they are the main factors improving the cocoons quality of transgenic silkworm. The mechanisms of interaction between sugar metabolism and protein metabolism has yet to be further research.This study will lay a foundation for breeding commercial silkworm varieties.