The Polymorphism Of Bombyx Mori Ubiquitin Gene, Cloning And Expression Of Bombyx Mandarina Ubiquitin Gene

Ubiquitin is the highly conservative multi-peptides chain which iscomposed by 76 amino acids. The name comes from its widely distribution toeach kind of cell. Its molecular weight is about 8.6 KDa. Ubiquitin's structure isalmost identical from different species of the eukaryotic. It is known thatubiquitin efficiently and highly selects to break down the proteins in thecytoplasm and nucleus particularly short-lived protein mainly throughubiquitin-proteasome pathway. In the process of Lepidoptera's metamorphosis,the insects not only need new protein's synthesis, but also break down theabnormal and old proteins, by which they can maintain the life and body'sbalance. Bombyx mandarina and other Lepidopteras have similar living habits.Protein's metabolism in the body is extremely complex. Therefore the researchof Bombyx mandarina's ubiquitin gene and its expression has certain guidingsignificance to understanding insect's protein metabolism pathway.Many insects' ubiquitin are coded by ubiquitin gene family including UBI1,UBI2, UBI3 and UBI4. This study discovered an encoding 15 ubiquitin DNAsequences from Bombyx mori's gene (No.AADK01019318). These 15 ubiquitingenes are conservative in the nucleotide level, and also conservative in theamino acid level. We can use Bombyx mandarina's genomic DNA as a templateto amplify ubiquitin gene which showed no introns in the ubiquitin gene. Andwe used Bombyx mandarina's skin cell's cDNA as a template which is amplifiedsingle ubiquitin gene coding region, GenBank Accession No.DQ839401. Thelength of this opening reading frame is 228 bp, encoding a protein (76 aminoacids) with molecular weight of 8.6 KDa and isoelectric point of 5.73. From thehomology analysis we can know there is 83% homology at the nucleotide levelbetween Bombyx mandarina's ubiquitin gene and other eukaryotic ubiquitin genes, and 94% similarity at the amino acid level. We linked Bombyxmandarina's ubiquitin gene fragment into pGEX-4T-2 to set up the prokaryoticexpression vector pGEX-4T-2/ubi. After recombinant vector Transformed intoE.coli BL₂₁, the ubiquitin is highly expressed at 37℃and 30℃conditions.