The Cloning And Functional Analysis Of Bmtdh And Bmsps 1, Aboundingly Expressing In Silkgland Of Bombyx Mori

The silkgland of Bombyx mori, the most powerful organ of protein biosynthesis in the insect kingdom, can efficiently synthesize a large amount of silk proteins during a rather short period. And it has become an ideal model in researching the regulation mechanism of gene space and temporal specificity. The silk protein is made up of fibroin and sericin, and many genes and regulation factors are concerned with their biosynthesis process. There are three genes fib-L,fib-H and P25, encoding the fibroin protein, being deeply studied. And five genes, Ser1, Ser2, S3, S4 and S5, encoding the sericin protein, have been isolated and cloned. The other well-studied genes and regulation factors concerning with the biosynthesis process of silk protein are listed as follows: the specific transcription factors of silkgland such as SGF-B, BmFA, Ub_2a and Ub_2b compounds, TRIO compound and PSFG; several tRNA genes such as tRNA^Ala, tRNA^Lys, tRNA^Glu, tRNA^phe, tRNA^Gly and their transcription factor TFIIIR; the eukaryotic translation elongation factor I (EF-1) and some of the other special regulation factors. With the discover of these genes and regulation factors, many good attempts have been undertaken to elucidate the efficient biosynthesis mechanism of the silk protein, however, there are many things reserved unclearly because of the complexity of the mechanism, and further research should still be carried on.In order to put some new light on revealing the complex mechanism of silk protein biosynthesis, based on the analysis of the silkworm genome and the large scale EST datas, this paper choosed two genes, Bmtdh and Bmsps1, which would probably participate in the course of silk protein biosynthesis. Both of them are cloned and sequenced. The space and temporal expression characteristics of these two genes were analyzed by RT-PCR, and the Bmspsl gene was expressed in the prokaryotic expression system additionally. The results are as follows:1. The Sequence and Structure analysis of Bmtdh The Bmtdh enconds the L-threonine 3-dehydrogenase ( TDH ) of Bombyx mori, and the 1310bp length cDNA( accession No. ABA43639.1) was cloned and sequenced, containing a 227bp 5'-untranslated region( 5'-UTR ). The Bmtdh gene, a single copy locating on the genome, has three extons: the Extron 1 locates on Scaffold006584 with the length of 333bp, but Extrons 2 and Extron 3 both locate on Scaffold003119 with the length of 79bp and 186pb respectively. The second intron locates on Scaffold003119 with the length of 1365bp, but the first intron remains uncertain because of its spreading between the two scaffolds. The entire ORF with the length of 1026bp encodes a...