The Structure Characteristics And Artificial Inducible Promoter Construction Of BmNPV 39K Promoter

Silkworm is an important economic spinning insect that plays a very important role in the agricultural production of our country. The nuclear polyhedrosis caused by the Bm NPV is the most deadly disease of silkworm. There is no effective method to treat the disease caused by Bm NPV; so culture the anti- Bm NPV transgenic silkworm has been attracting more and more attention. In transgenic engineering, as the transcript expression of constitutive promoters has no defect of space-time limitation, the inducible promoters get more and more favors for the advantage that it can only be expressed when special stimulations existed. The 39 K promoter is a kind of Bm NPV inducible promoter. The sequence of 39 K promoter is relatively long, which goes against for the construction of transgene vector and limites the utilization in transgenic engineering. Therefore, it is of great significance to analyze the structural characteristics of 39 K promoter and construct the artificial inducible promoter for the utilization of 39 K promoter.In this study, we identified the regulatory gene of the 39 K promoter through q RT-PCR and the dual luciferase reporter gene assays; analyzed the structure characteristics of the 39 K pro moter by 5’ truncated, 3’ truncated or deletion assays and detecting promoters’ transcriptional activity; constructed and screened the efficient artificial inducible promoters by the stratagem of the point mutations and the structure features of the 39 K promoter.The major results are as follows:1. Identification of the regulatory genes of 39 K promoterBased on the nucleotide sequences of the five immediate early genes of Bm NPV in NCBI, we analyzed and cloned the five genes, and constructed the corresponding over expression vectors of the 5 genes: p IZ-ie0-HA, p IZ- ie1-HA, p IZ- ie2-HA, p IZ-pe38-HA and p IZ-me53-HA. The results of the q RT-PCR analysis and the dual luciferase reporter gene test show that only the over expression vector of the ie-1 can activate the 39 K promoter to initiating the express of genes in the downstream of the 39 K promoter, meaning the regulatory gene of the 39 K promoter among the five Bm NPV immediate early genes is the ie-1.2. Structure characteristics of 39 K promoterTo analyze the structure characteristics of the 39 K promoter, the 5’ end truncated, 3’ end truncated and deletion for 39 K promoter assays were conducted, and the corresponding luciferase reporter gene detection vectors of 39 K promoter was built in this study. We detected and analyzed the relative luciferase activity of each vectors, and found that the fragments with function to enhance the inducible activity of the 39 K promoter are the +116 to +76, +62 to +1, +1 to-223 and-273 to-473 fragments; the fragments which are able to inhibit the inducible activity of the 39 K promoter are the +136 to +116, +76 to 62 and-223 to-273 fragments; the fragment that is correlative with the constitutive promoter activity is the +1 to-273 fragment.3. Construction of the artificial inducible promotersIn order to reform the 39 K promoter better, we cloned 6 recombine promoters of the 39 K promoter that have the CAAT point mutation and constructed six luciferase report gene detection vectors in this study. The results of the relative luciferase activity assay of the detection vectors show that the promoter activity is respectively increased by 164.37% and 64.09% if conducted mutation for CAAT at 329 and 399 point; however, there is no significant change on the promoter if C AAT at 93,248 and 560 point mutate to CGGT; meanwhile, the promoter activity of the 39 K promoter is decreased by 37.31% if conducted mutation at-306 th point.According to the structure characteristics of the 39 K promoter that the transcriptional activity of the 39 K promoter of +1 to the-273 fragment raises if the +136 to +62 fragment deleted and the transcriptional activity of the 39 K promoter decreases if the-573 to-773 fragment deleted, as well as the mutation of the-329 and-399 point CAAT raise transcriptional activity of the 39 K promoter, we chose the five elements as the material to reform the 39 K promoter. We have successfully constructed 11 artificial inducible promoters: from P-39K-1 to p-39k-11. The transcriptional activity testing result shows that: compared with the 39 K promoter, the transcriptional activity of the p-39k-9 has increased 13.31%; the transcriptional activity of the p-39k-1, p-39k-5 and p-39k-6 are 87.24%, 75.94% and 63.57% of that of the 39 K promoter. The transcriptional activity of the other 7 promoters is greatly decreased compared with the 39 K promoter. It’s worth noting that the length of the p-39k-1 is 362 bp, accounting for 40% of 39 K promoter and lowering operative difficulty index of genetic engineering. Therefore, the p-39k-1 can be used as the favourable candidate promoter in silkworm transgenosis anti-Bm NPV breeding engineering.