Characterization Of The Promoter Of The CIb1 Encoding Gene-Ict-H From The Hemolymph Of Bombyx Mori

The achievement of genomic sketch map of Bombyx mori is the start symbol of post genome era of silkworm biological research. Silkworm, Bombyx mori is one of the most important model insect of Lepidoptera, study on its physiology, pathology and innate immunity will not only be helpful to understand its mechanism of disease-resistance and further to breed disease-resistant varieties, but also provides the theoretical base to the study of new methods for control of pest in agriculture and forestry.There are 69 genes relating to the immunity and recovery of silkworm according to analysis of Bombyx mori genome. Proteinase inhibitors are important and necessitated in the immunity system of insects. The Kunitz-type chymotrypsin inhibitor b1 (CIb1) is involved in the control of melanization induced by infection of bacteria, implying that it might play an important role in the innate immunity of silkworm.In this study, we aim to elucidate the structure, and upstream regulation elements of the promoter of Clbl encoding gene Ict-H by means of characterization of promoter, and then to confirm the main viral factor of Bombyx mori nucleopolyhedrovirus (BmNPV). In addition, we also investigate the effects of challenge of heat shock, foreign insect hormone etc. This study is not only the important step to study the disease-resistant and immunity mechanism of silkworm, B. mori, but also provides feedback information to study other related insects.1. The structure and upstream elements of Ict-H promoter was preliminary elucidatedAccording to the sequence of Ict-H promoter published in BGI-Sikworm Genome Database (accession No. AADK01017234.1), five forward primers and one reverse primer for PCR amplification of Ict-H promoter fragments were designed and synthesized. They are PF1, PF2, PF3, PF4, PF5 and PR, corresponding to the following sites upstream the transcriptional initiation site Ict -H: -824 to -816 nt, -672 to -649 nt, -506 to -482 nt, -302 to -282 nt, -72 to -51 nt, and -16 to +10 nt of the complementary strand, respectively. Ict -H promoter fragments were cloned from the genome of p50 silkworm strain. Report plasmids were constructed by using firefly luciferase gene (luc) as reporter driven by Ict-H promoter. The luciferase activity of the report plasmids were assayed in vitro in the Bombyx mori cultured cell line BmN. And deletion assays were carried out for study of Ict-H promoter structure and its upstream elements.These results indicate that within the cloned 844 bp of Ict-H promoter there are some DNA motifs including a Bml (highly repetitive retroposon-like DNA element), a TATA box, a CCAAT motif (on the complementary strand), a lipopolysaccharide-response element CATTW, a transcription factor NF-κB binding motif GGGAACTCCT and a GATA box. The heterogeneous promoters of Ict-H showed significantly different activities. The 82 bp promoter fragment containing the basic elements of eukaryotic promoter showed a certain transcription level. The 312 bp promoter revealed the highest transcriptional activity, significantly higher than that of the 82 bp fragment, suggesting that there is a positive cis-acting element from -302 to -73 nt upstream the transcriptional initiation site. When the promoter fragment increased to 516 bp in length, its transcriptional activity was dropped significantly to 1.88% of that of 312 bp, implying that there is a strong negative cis-acting element from -506 to -303 nt. However, when the promoter increased to 682 bp and 844 bp containing a partial and a complete Bml element, respectively, the transcriptional activities raised again, perhaps Bml element functioned as a positive cis-acting element.2. Ict-H promoter was found to be polytypic among silkworm strainsBy using p50 genomic DNA as the template for amplification of Ict-H promoter with primers PF2 and PR, only one fragment was obtained. However, using Suju×Minghu mass genomic DNA instead of p50's as the template, four different fragments of 682 bp,696 bp,550 bp and 564 bp in length were obtained. These four fragments showed high similarity in DNA sequence, and therefore they are called Ict-H promoter-like fragments. What are these fragments? Whether they are different promoters of different genes or different promoters of the same Ict-H gene? In order to understand these questions, we used genomic DNA of single parent of Suju X Minghu (Su, Ju, Ming and Hu) and C108 as the template to amplify Ict-H promoter with primers PF2 and PR, respectively.The sequence analysis revealed that Ict-H promoter like fragments amplified from p50, Su and Ju three silkworm strains are identical each other, and the similarity is 99.17% to the Ict-H promoter published in BGI-Sikworm Database, denominated as the basic type Ict-H promoter. While the Ict-H promoter like fragments amplified from C108, Ming and Hu three strains are identical each other too, but different from those of above three strains. They lack a 132 bp fragment from -517 nt to -386 nt upstream the transcriptional initiation site while inserted a 14 bp tandem motif AACGATATCATGCA between -122 nt and -121 nt, named deletion-insertion type promoter. Besides these two types as their parents, two new types of promoters were amplified from Suju X Minghu. One is named as deletion type which lacks the 132 bp fragment compared with the basic type, and the other as insertion type in which a 14 bp tandem motif was inserted compared with the basic type.A new PCR primer locating at the first extron of Ict-H gene was used for amplification of the extron-jointed promoter fragments with PF2 by using the genomic DNA of silkworm strains Su, Hu and Suju X Minghu hybrid as the template, respectively. The result indicated that only one type of extron-jointed DNA fragment was generated from Su and Hu strain, respectively, the basic-and deletion-insertion type. Those from Suju X Minghu were four types as described above. This result suggests that all four types of Ict-H promoter like fragments joint with Ict-H gene, namely, the Ict-H promoter is polytypic.The polytypic promoter fragments provide a natural deletion model for study of the Ict-H promoter. The results of in vitro assay in BmN cells showed that the transcriptional activities of different Ict-H promoters were significantly different. The 682 bp basic type promoter revealed a lower level transcription activity. In contrast, the transcriptional activity of the 550 bp deletion type promoter was the highest which was about 5000 times of that of the basic one, suggesting that there is a strong negative cis-acting element suppressing the transcription activity of the Ict-H promoter in the 132 bp fragment. The insertion type promoter showed the lowest activity and the activity of deletion-insertion type promoter was significantly lower than that of the deletion type, implying that the insertion of the 14 bp tandem strongly suppresses the transcriptional activity of the Ict-H promoter.3. BmNPV infection increases the transcriptional activity of Ict-H promoterThe effect of BmNPV infection on the transcriptional activity of Ict-H promoter in BmN cells was studied. The results showed that infection of BmNPV increased the transcriptional activity of the Ict-H promoter, suggesting that the Ict-H promoter response to the challenge of BmNPV. However, the increases varied with the lengths of the promoter, the 682 bp and 516 bp promoters increased much more than those of 844 bp and 312 bp. Time-course expression in vitro indicated the function of BmNPV occurred at about 3 hours post infection. The immediate-early protein IE-1 of BmNPV also increased the activity of Ict-H promoter, implying IE-1 is perhaps one of the most important viral factors increasing the activity of Ict-H promoter. As an enhancer of transcription, BmNPV hr3 increased the activity of Ict-H promoter significantly. While as a trans-acting viral factor, the function of hr3 required the stimulating of BmNPV or IE-1.4. BmNPV infection changes CIb1 content in the hemolymph of silkwormSilkworm larvae of Jingsong×Haoyue hybrid were infected with wild type BmNPV and larvae treated with distilled water served as the control. By using native-polyacrylamide gel electrophoresis followed by activity staining, the changes of activity of CIb1 in the hemolymph of silkworm were investigated. The results revealed that within 24 hours post infection of BmNPV either by oral or by injection, the CIb1 content reduced. But from 24 hours post infection, CIb1 content recovered to the same level as the control, suggesting farther that CIb1 plays an important role in the innate immunity of silkworm.5. The deletion type of Ict-H promoter shows both constitutive and inducible promoter characteristicsReport plasmid with a luc gene as reporter driven by the deletion type of Ict-H promoter,550 bp in length, expressed not only in ovary-delivered BmN cultured cells and in the silkworm larvae, but in non-silkworm-delivered Sf (Spodoptera frugiperda) cultured cells, indicating that it is a constitutive promoter. However, the expression levels in silkworm-delivered cells were higher than in non-silkworm-delivered cells, implying it is species specific to some extent.In addition, the effects of foreign stimulation on the activities of deletion type Ict-H promoter have been investigated in vitro (BmN cells) including BmNPV infection, co-transfection of IE-1 expression plasmid pT-ie-1, administration of lipopolysaccharide (LPS) or foreign insect hormone ecdysone (MH), juvenile hormone analogues (JHA), diapause hormone (DH), and 37℃challenge. The results showed that BmNPV infection, co-transfection of pT-ie-1 and heat challenge increased the activity of 550 bp deletion type Ict-H promoter. This result suggests that the deletion type promoter also possesses the characteristics of inducible promoter. When viruses invaded into the body, the transcriptional level of Ict-H increased so as to enhance the content of CIb1 to resist the infection of viruses. While LPS, MH and JHA showed suppressing effects and DH showed no detectible effect on the promoter.