| Cyclin is one of the most important regulatory factors to promote cell cycle progression, and also plays roles in eukaryotes from yeast to humans. Cyclin regulates cell cycle in the multi-level by activating corresponding cyclin-dependent kinase (CDK). Cyclin also decides phosphorylation processes of CDK substrates, and which drives the cells to span the phases of the cell cycle checkpoint, ahd promote cell cycle progression. Cyclin also plays a role in individual development and the formation of tumors.There are 5 type cyclins in silkworm: CyclinA、CyclinB、CyclinB3、CyclinL1 and CyclinE. We investigated the expression characteristics of cyclin genes and function of CyclinE in Bombyx mori. The main results are as follows:1、Cloning, structural characteristics and expression analysis of cyclin genes in silkwormIn order to explore the expression of Cyclin genes, we designed primers and cloned the 5 cyclin genes according to silkworm genome database information. The result of sequence analysis showed that CyclinA、CyclinB、CyclinB3 contained 7 exons, respectively,CyclinE contained 8 exons,CyclinL1 only had 1 exon. CyclinA and cyclinE have more alternative splicing, Cyclin A mainly contains 2 141bp and 2 173 bp transcripts with its variation at 7 exon; Cyclin E contains 1 422 bp、1 290 bp、1194 bp and other many ones,the variation differentiated at 5-7 exon; the transcripts of CyclinB、CyclinB3 and CyclinL1 are relatively regular. The expression analysis of silkworm Cyclin family genes shows that cyclinA gene only expressed in testis, cyclinB3 expressed in testis and ovarian apparently, cyclinB and cyclinE genes express in silk gland, brain, fat body, midgut, Malpighian tubule, testis, ovarian and blood, CyclinL1 gene expresses in 7 tissues except for the silk gland and blood. The result of this study provides references for further study of functions in insect molting, metamorphosis and other physiological processes.2、Splicing variants and expression characteristics of CyclinE in different tissues in 5th instar silkworm larvaeTo study the expression of cyclin E gene in different tissues of silkworm (Bombyx mori), we designed primers by using information deposited in the silkworm genome database and cloned cyclin E cDNA sequences expressed in different tissues of the 5th instar silkworm larvae. As a result, 6 different splicing variants of this gene were obtained. The first 4 exons and the last exon of the 6 splicing variants were the same. Splicing variation mainly existed in the 5th to 7th exons. These results suggest that the change of splicing variants in different tissues may be related to specific types or specific cell cycle stage in cell cycle, and diverse and unique CyclinE-CDK2 complexes may regulate the development of cell cycle in different tissues. Homology comparison revealed that the cyclin box regions of cyclin E proteins from different species were highly conserved, among which 35 amino acids were identical. This region might also be the functional domain of silkworm cyclin E that involves in specific recognition and binding with CDK. Real-time quantitative PCR analyses showed that cyclinE was expressed in various tissues in all tested tissues of day 3 silkworm larvae of the 5th instar, and the expression level was relatively high in the fat body. It suggests that silkworm cyclinE gene plays an important role in controlling the cell division in fat body.3、The expression of cyclin genes in the 3rd days of 5th larvae in different tissues of B. moriWe used a real-time fluorescent quantitative PCR method to detect the transcription levels of cyclins in different tissues of the 5th instar larvae. The results indicated that the transcription level of CyclinE was high in silk gland,CyclinA and Cyclin L1 have low transcription level,and there were no transcription level detected of CyclinB、CyclinB3. The silk gland cells belong to endoreduplication, they alternate between G1 phase to S phase without a transition period of G2 phase to M phase. This result shows that Cyclin E is a key factor in the regulation of cell cycle from G1 to S phase and CyclinB、CyclinB3 play an important effect from G2 phase to M phase. We also detected the transcription levels of CyclinA、CyclinB、CyclinB3、CyclinL1 in other tissues of the 5th instar larvae. The result shows that CyclinA、CyclinB、CyclinB3、CyclinL1 have high expression level in spermary, we suppose that these genes play an important role in the development in spermatocytes divided into sperm in silkworm testis.4、CyclinE-interference RNA affects cell multiplicationTo study the BmN cell proliferation and cell cycle after inhibition of siRNA against CyclinE. We designed siRNA gene fragments of CyclinE, then transfected them into BmN silkworm cells using liposomes Metafectene, and detected CyclinE mRNA level after siRNA interference by a real-time fluorescent quantitative PCR method, and detected cell cycle by flow cytometry. The results indicated that siRNA-1252 (target the sequence from 1252 nt of the cyclin E ORF and siRNA-568 decreased the CyclinE mRNA level by 68%、90% respectly after transfection 48h; the result of flow cytometer showed that the ratio of cells in G0/G1 increased while S and G2/M decreased. These results indicate that the import of siRNA to CyclinE in BmN cells can decrease the expression of CyclinE, and slow down the cell proliferation. It means that CyclinE plays an important part in G1/S. Meanwhile, we found that the activity of the siRNA designed in the different coding position was different. The site of 1252 nt ia more active than that of 568 nt. We suppose that gene silencing effect may be also related with the selection of the target gene encoding region.5、The effect of exogenous substance on the transcription levels of cyclin genes in BmN cell lineTo study the change of expression levels of cyclin genes affected by exogenous substance in BmN cell, this research added BmNPV and extracts of cordyceps as stimulus into medium, and detected the changes of transcription levels of cyclin genes in BmN cell with florescence quantitative PCR.. The results shows that transcription levels of cyclin genes decreased rapidly for the first 12 hours after infected, and the expressions of cyclinB3 and cyclinA reached the minimum. At the 12~24 h, the expressions of cyclinB and cyclinL1 decreased significantly, and the cyclinB reached the minimum. The expressions of cyclinE and cyclinL1 reached the minimum, and after 36 h, there were no significant changes in mRNA levels. The results indicated that the expression levels of cyclin genes in BmN cell were affected by the duplication of BmNPV DNA, and the inhibitions of different genes were not same in several times after infection. In this research, the extracts of cordyceps prepared through frozen in vacuum and radiation inhibited the BmN cell, and the inhibition was more significant with the higher concentration and longer time. On this basis, this study wanted to clarify further the change of expression levels of cyclin genes affected by extracts of cordyceps. The expression levels of cyclinA, cyclinB, cyclinB3 and cyclinL decreased at 24 h after addition of extracts of cordyceps with the concentration 1mg/mL, and the change of expression levels of cyclinB3 was significant, it was 35.14% of normal. After 48 h, the expression levels of cyclin genes declined to 10% of normal, and recoverd 72 later. In addition, the expression levels of cyclinE began to rise at 24 h after induction, it rearched 2.43 times of control, and we speculated that between 0-24 h, the extracts of cordyceps promoted mitosis in BmN cell, so the expression levels of it rised and the others decreased. |
PDF Full Text Request