TGF-β1、Smad4 Gene Eukaryon And RNA Interference Expression Vector Construction And Their Effectiveness Verification In Ctenopharyngodon Idellus

Grass carp(Ctenopharyngodon idella)is currently the largest produced aquaculture species globally,with an annual production of more than 4 million tons in China alone.It is not only an important source of cheap protein in developing or undeveloped regions,but also it makes great contribution to the supply of high-quality animal protein in the world[1].In fact,the research of crisp grass carp nutrition has already started.Consider the situation of current study,we have also done some work to explore the regulated mechanism of grass carp nutrition.Scholar have found the muscle hardness of grass carp fed with bean increased significantly[2] and it was liked by comer.There have been study show that the increase of muscle hardness is significantly relative with the expression of Ⅰtype collagen[3],the expression of Ⅰtype collagen is regulated by the TGF-β1/Smad4 signaling pathway[4].For exploring the regulated mechanism of grass carp nutrition,the study mainly constructed TGF-β1、Smad4 gene eukaryotic overexpression vector pcDNA3.1(+)-TGF-β1 and pcDNA3.1(+)-Smad4 and RNA interference expression vector pRNA-6.1/Neo-TGF-β1(I)、pRNA-6.1/Neo-TGF-β1(Ⅱ)、pRNA-6.1/Neo-TGF-β1(Ⅲ)、pRNA-6.1/Neo-Smad4(Ⅰ)、pRNA-6.1/Neo-Smad4(Ⅱ)和 pRNA-6.1/ Neo-Smad4(Ⅲ)and then detected the liveness of these vector in cell and fish level.We hope to lay the foundation for the last study.The main content of this paper has been summarized as follows:1.The ORF(open reading frame)sequences of TGF-β1,Smad4 gene were first cloned.The full-length ORF of TGF-β1 was 1134 bp and the full-length ORF of Smad4 was 1644 bp.Secondly,the specific restriction sites were added to the both ends of ORF sequence and then ORF sequences with enzyme sites were inserted into the eukaryotic expression vector pcDNA3.1(+)which was double digested by restriction enzyme.Twoeukaryotic expression vector pcDNA3.1(+)-TGF-β1,pcDNA3.1(+)-Smad4 were constructed.On the other hand,according to TGF-β1、Smad4 gene sequence,three pairs siRNA sequences which were consisted of 48 bp have been designed.Then the completed shRNA was inserted into the carrier pRNA-U6.1/Neo.We successfully constructed the RNA interference expression vectors pRNA-6.1/Neo-TGF-β1(I)、pRNA-6.1/Neo-TGF-β1(Ⅱ)、pRNA-6.1/Neo-TGF-β1(Ⅲ)、pRNA-6.1/Neo-Smad4(Ⅰ)、pRNA-6.1/Neo-Smad4(Ⅱ)和 pRNA-6.1/ Neo-Smad4(Ⅲ).The construction of RNA interference expression vectors and eukaryotic expression vector based on TGF-β1,Smad4 gene will lay the foundation for the next study of TGF-β1/Smad4 signaling pathways in the muscle tissue of grass carp.2.For further detecting the effectiveness of the expression vectors constructed at cellular level,we separated the fibroblasts of grass carp and then transfected the overexpression and interference vectors to zebrafish ZF4 cell line because the fibroblasts can not form stable cell line.Because the transfective effection is not good,we made relative bioinformation analysis for different species(including protein structure domain and amino acid sequence homology comparison).The result show 90% homology for human and grass carp,and then transfer to 293 T cell line for further verification experiment.After 36h(the transfective efficiency reach to 80%),the relative expression of TGF-β1、Smad4 and Ⅰtype collagen(COL1-A1、COL1-A2)gene have been detected.The result showed that the expression of TGF-β1、Smad4 and Ⅰtype collagen(COL1-A1、COL1-A2)gene in overexpression group is significantly higher than the control group(P<0.05)and there is no significantly difference for human TGF-β1 、 Smad4 and Ⅰtype collagen(COL1-A1 、 COL1-A2)gene.The expression of TGF-β1、Smad4 and Ⅰtype collagen(COL1-A1、COL1-A2)gene in interference group is significantly decreased than the control group(P<0.05).We have found that the liveness of pRNA-6.1/Neo-TGF-β1(Ⅲ)and pRNA-6.1/Neo-Smad4(Ⅱ)were the best.The experimental results have been good enough to verify the validity of the expression vector at the cellular level.3.In this study,we have also detected the validation of the TGF-β1、Smad4 overexpression and RNA interference vector in fish level.The results show that the expression in overexpression group is significantly higher than the control group(P<0.05)in 36 h and 48 h.RNA interference expression vector pRNA-6.1/Neo-TGF-β1(Ⅲ)and pRNA-6.1/Neo-Smad4(Ⅱ)have some effect but not significantly.The expression rapidly increase in 48 h.The result in fish level also suggest that the liveness of overexpression is high and the RNA interference expression vector have liveness but not high.The experimental results have been good enough to verify the validity of the expression vector at the fish level.The TGF-β1、Smad4 gene eukaryon and RNA inteference expression vector construction and their active verfication in this study not only lay the foundation for exploring the mechanism of grass carp muscle development but also offer the reference value.