The Clone And Expression Analysis Of Genes Associated With Evolution In Onchidiidae

Evolutionary theory holds the opinion that the life originates from the ocean,and that organisms evolve from the sea to the land.Amphibians are the transitional species in evolution procession.Onchidiidae is Mollusca,Gastropoda,Pulmonata,Systellommatophora.Their larvaes live in the seawater,and become amphibious juvenile after metamorphosis.Onchidium struma,Platevindex mortoni,Paraoncidium reevesii and Peronia verruculata distributes in the east of China from the shallow sea to the wetland,in this migration process,they produce many phenotypic differences representing their regional features.We screen many genes related to evolution of phenotype in the main Onchidiid,which are Os-NMHC,Os-RYR,Os-IP3 R,Os-MEK and Os-IP3 K.Moreover,we use RT-PCR technology to explore the trend of relative expression in Onchidiids to reveal the evolution node of NMHC,and the evolution endency of two kinds similar calcium regulatory protein RYR and IP3 R.We cloned a full-length cDNA of nonmuscle myosin heavy chainII(NMHCII)isoform in Onchidium struma,based on the transcriptome sequencing of Onchidiids,and named it as Os-NMHC.The sequence was 6 057 bp,encoded 1 910 amino acids,including a 5 733 bp open reading frame,a 236 bp 5'-noncoding region and an 88 bp3'-noncoding region.Its protein isoelectric point was 5.26,molecular weight was 220.46KD;The protein tertiary structures was mainly composed of ?-helix and ?-sheet predicted by the Phyre2 program and the Pymol software.The comparison of the amino acid sequence induced by Os-NMHC showed the identity of NMHCIIA and NMHCIIB of Xenopus leavisis was 60.37% and 61.47%,respectively.The phylogenetic tree constructed by Bayes software showed that Aplysia california have the closest relationship with O.struma.The results of QRT-PCR showed that the four Onchidiids have different expression of Os-NMHC in its back,abdom,pleopod,pulmonary,cardic,ganglion.And the characteristic of relative expression in the four different Onchidiids is consistent with the evolutionary trend,in descending order is,O.struma?Platevindex mortoni>Paraoncidium reevesii>Peronia verruculata.A further study for Os-NMHC will help to explore the time nodes of NMHC subtypes evolution,and provide a new perspective to reveal the mechanism underlying the evolution of onchidiids from shallow sea to wetland.Os-RYR and Os-IP3 R are two kinds of calcium regulatory protein,and they are similar to the structure and function.We cloned their 3 'terminal conserved region.The nucleic acid sequence of Os-RYR are 1253 bp,and translate 376 amino acid residues,including 83 bp 3 'non encoding region.The identity of the 3'conserved sequence of RYR in Aplysia california and of Os-RYR in Onchidium struma is 94%.The nucleic acid sequence of Os-IP3 R are 4574 bp,and encode 936 amino acid residues,including1561 bp 3 'non encoding region.The identity of 3'conserved sequences of IP3 R in Aplysia and of Os-IP3 R in O.struma is 85%.The calcium channels of Os-RYR and Os-IP3 R are highly conserved in GXRXGGGXGD,compared by Clustal Omega,which proves that this sequence plays an important role in the formation of ion channel.The IP3 R ion channel in Aplysia and O.struma are very conserved,so their protein tertiary structure are very coincidence.Os-RYR and Os-IP3 R have a poor expression level in Peronia verruculata,and the expression of Os-IP3 R and the percentage expression of Os-IP3R/Os-RYR present a gradient increase in O.struma,Platevindex mortoni,and P.verruculata,but Os-RYR's expression is not,based on the analysis of QRT-PCR.This result prove that this two kinds of structurally and functionally similar calcium regulatory protein expression have an tropism for Os-IP3 R during the evolution of the three species of Onchidiids.We prove the opinion that the IP3K/AKT and mTOR signaling pathway are not exist in Onchidiidae,based on four kinds transcriptome databases of Onchidiids compared with the molecules of VEGF(Vascular endothelial growth factor signaling pathway)in Mammals.We screen Os-IP3 K associated with Os-IP3 R and downstream signaling molecule Os-MEK affected with epidermal growth.We clone 120 1bp nucleicacid sequence of Os-MEK,including 405 bp 5' non encoding region,83 bp 3' non encoding region,translating 368 amino acid residues.The consistent of MEK duced amino acid sequence in Aplysia california and in Onchidium struma are 82%.The protein tertiary structure of Os-MEK consists of alpha-helix,beta-sheet,and beta-loop,and some random coils.We cloned 3 661 bp partial length of Os-IP3 K cDNA nucleic acid,3 'and 5 ' non coding regions are not included,which translates 721 amino acid residues.The results of RT-PCR shows that the relative expression of Os-IP3 K and Os-IP3 R are consistent in O.struma.However,the ganglion of Platevindex mortoniin express a high level of Os-IP3 K,but a low level of Os-IP3 R.This result prompts that same function associated signaling pathway genes also have different function in different species,which is important to the evolution of signal transduction pathway.