Functional Studies On The Different Spiliceosomes Of BmYki Gene In Silkworm, Bombyx Mori

Hippo pathway is a kind of very important signaling pathway for the regulation of organ growth. There is a core in the Hippo pathway which is consisted of a series of phosphorylated components, including serine / threonine Ste20 kinase HPO, nuclear Dbf-2 related（NDR） family kinases Warts（WTS）, a scaffold protein sav and as a tumor suppression gene（mats） of the mob.When any component occurs to mutation, it will cause the abnormal growth. For example, during the development of the imaginal discs,the Wts mutant will cause cell proliferation in the development of multiple and tissues.Yorkie, as a transcriptional co-activator, has WW domain in the core of Hippo signaling pathway. It is the most important downstream element in a kinase cascade of the Hippo pathway. We also define Yorkie as the downstream element of Wts-Mats complex which is activated by the phosphorylated Hpo-Sav complex. The activation of the Wts-Mats complex resulted in the phosphorylation of Yorkie（Yki）, which is associated with the14-3-3 protein in the cytoplasm, and was inactivated and retained in the cytoplasm.When inhibiting the Hippo signaling pathway, the inhibition of Yki will be disappeared.Then Yki will enter the nucleus and be combined with the transcription factor Sd.Regulating the target genes of Hippo pathway including Diap1, cyclinE and Bantam can cause the process of the cell proliferation and the inhibition of apoptosis.1. The screening and identification of the different spiliceosomes and mutation of BmYki geneIn order to study the function of the different spilicesomes of the key transc ription co-factor BmYki in the Hippo signaling pathway of Silkworm, Bombyx m ori, we need to acquire the stable transgenic cell lines. First, we need to obtain the recombinant plasmids: pIZT-V5/His-BmYki2 and pIZT-V5/His-BmYki3. Secon d, we transfect the recombinant plasmids including pIZT-V5/His-BmYki1 and pIZT-V5/His-BmYki1S97 A which have been obtained by QianYing, pIZT-V5/His-BmYki2, pIZT-V5/His-BmYki3 and empty pIZT-V5/His vector into BmN cells. Cellular l ocalization results showed that BmYki1, BmYki2, BmYki3 and BmYki1S97 A protein can express in the BmN culture cells. BmYki1 mainly distributed in the cytoplas m. BmYki2 and BmYki3 distributed in the nucleus. We can also find BmYki1S97 A i n the nucleus which indicates that BmYki1 phosphorylation plays an important ro le in the location of the protein expression. The pIZT-V5/His-BmN indicated that BmYki distributed in the cytoplasm. Thus, Bm Yki2 and BmYki3 play a positive r ole in the growth of cells.2. The effects on the BmN cells and moth wing after over-expressing BmYki1,BmYki2, BmYki3, BmYki1 S97AIn order to study the function of the different spilicesomes of the key transcription co-factor BmYki in the Hippo signaling pathway of Silkworm, Bombyx mori, we perform an experiment on the two aspects : BmN cell and moth wing.When BmYki1, BmYki2, BmYki3 and BmYki1 S97 A have expressed, there are d ifferent changes on the cell migration, cell proliferation, cell size and cell cycle.The results show that pIZT-V5/His-BmYki1-BmN cell, pIZT-V5/His-BmYki2-BmN cell, pIZT-V5/His-BmYki3-BmN cell, pIZT-V5/His-BmYki1^S97A-BmN cell, pIZT-V5/His-BmN cell and wild type BmN cell transferred 1294.96μm, 1097.62μm, 1327.60μm, 1098.87μm, 633.59μm and 1027.00μm during 78 hours respectively. pIZT-V5/His-BmYki3-BmN cell has the longest migration distance. pIZT-V5/His-BmYki1-BmN cell and pIZT-V5/His-BmYki3-BmN cell have faster split speed than wild type B mN cell. pIZT-V5/His-BmYki2-BmN cell, pIZT-V5/His-BmYki1^S97A-BmN cell and p IZT-V5/His-BmN cell have slower split speed than wild type BmN cell. Thus, th e size of cells will gradually become large according to the time, and the sizes of pIZT-V5/His-BmYki1-BmN cell, pIZT-V5/His-BmYki2-BmN cell, pIZT-V5/His-B mYki3-BmN cell and pIZT-V5/His-BmYki1^S97A-BmN cell are bigger than the size of wild BmN Cell respectively. pIZT-V5/His-BmYki1-BmN cell, pIZT-V5/His-BmY ki2-BmN cell and pIZT-V5/His-BmYki3-BmN cell mainly distributed in the prolife rating phase which were compared with pIZT-V5/His-BmN cell in the cell cycle.When over-expressing BmYki1, target gene in the Hippo pathway has changed.The expression levels of kibra, iap, fj and ex were significantly increased compared with pIZT-V5/His-BmN cell. When over-expressing BmYki2, the expression levels of wnt,kibra, iap, fj, ex and bmpr have increased. When over-expressing BmYki2 and BmYki3,the expression levels of wnt, kibra, iap, fj, ex and bmpr have increased. When over-expressingand BmYki1S97 A, the expression levels of wnt, kibra, crb, fj, ex and bmpr have increased. Besides, the expression levels of cat, dpp, serr and stat have decreased in the transgenic cells.When over-expressing BmYki1, BmYki2 and BmYki3 in the wings of silkworm moth, we can conclude that the right wing of silkworm moth has grown up abnormally,and the abnormal ratio were 30%, 40% and 30%. Thus the enlargement ratio of front wing is generally higher than the expand ratio of posterior wing. When over-expressing BmYki1 in the wings of silkworm moth, the target gene of Hippo pathway such as myc,ex, kibra, wts2, iap, fj, bmpr, wnt and dpp have decreased, and cat. cycE and stat have increased. When over-expressing BmYki2 in the wings of silkworm moth, the target gene of Hippo pathway such as myc, ex, kibra, wts2, iap, fj, bmpr, wnt and dpp have decreased. cat and cycE have increased. When over-expressing BmYki3 in the wings of silkworm moth, the target gene of Hippo pathway such as myc, ex, kibra, fj and dpp have decreased. cat, cycE, crb and wts2 have increased.3. Comparative study of transcription groups on the over-expressing BmYki3In order to get a more comprehensive understanding of the changes in the over-expressing BmYki3 BmN cells, we use the RNA-sequence technology so that we can obtain the information of transcription groups. We can conclude that there are10291 up-regulated genes and 4444 down-regulated genes. From the GO enrichment analysis of differential gene expression, we find that there are 49 GO classifications,included cellular compent, molecular function and biological process. From the KEGG enrichment analysis of differential gene expression, we have a conclusion that these genes can exist in MAPK, Wnt, Hippo, JAK-STAT and mTOR signaling pathway which contact with cell proliferation, cell differentiation, apoptosis and cell size. Thus,sometimes just one gene can exist in multiple signalingpathways, and make several signaling pathways linked together to have regulation effects.In Hippo signaling pathway, spiliceosomes include BmYki1, BmYki2 and BmYki3 have different function. BmYki1 has the second important positive function, and BmYki2 has a tiny and balance function in Hippo Signaling Pathway. Besides, BmYki3 has the most important effect on Hippo Signaling Pathway. When overexpress BmYki1, it can increase the expression level of BmYki3. The protein locates in the cytoplasm and increases the multiplication rate. BmYki1 can also increase the cell numbers and the migration rate. It can enlarge the cell size. When overexpress BmYki2, it can decrease the expression level of BmYki3. Because of the deletion of a WW domain, the protein locates in the nucleus and cytoplasm. This can make the speed of multiplication become a little quickly and increase the cell numbers. Besides, it can increase the migration rate and enlarge the cell size. But all of these effects aren’t very extrusive. When overexpree BmYki3, it will increase the expression level of BmYki1. But the expression level of spiliceosome BmYki3 increase a little. It can increase the cell numbers and the migration rate. It can enlarge the cell size. The protein locates in the nucleus and cytoplasm and decreases the multiplication most quickly. Because at the 3’end of the 5th exon has 15 bases and at the 5’end of 6th add 5 bases. BmYki3 has the most important positive effecs.