Cloning,Eukaryotic Expression And Analysis Of HDS Gene In Huperzia Serrata

Huperzia serrata（Thunb.ex Murray）Trev.),a kind of perennial rare medicinal plant,was featured with slow growth speed and a long breeding cycle,which made it fail to meet the market demand.Thus,the genetic engineering was significant in increasing the output of active ingredients.4-hydroxy-3-methylbut-2-en-l-yl diphosphate synthase might play a key role in the process of synthesizing Terpene,but what specific effects it caused still need further study.In order to ensure a better understanding of the synthetic routes of terpene of Huperzia serrata,this test cloned the HDS gene of Huperzia serrate and analyzed its function.RACE technology was used to separate and clone the Huperzia serrata’s HDS gene order with the length of 3050bp and 755 amino which contained 2267 bp open reading frames and 288 bp 5’-UTR and 495 bp 3’-UTR.The cloned HDS gene was nominated as HsHDS.Besides,bioinformatics method was also adopted to predict and analyze the chloroplast transit peptide and trans-membrane domain of the HsHDS encoded sequences of proteins and to compare and analyze the HDS protein similarity with other species.The results showed that the HsHDS proteins’molecular weight was 8.3 kDa,pI is 5.98 and its molecular formula was C₃₆₇₂H₅₉₁₀N₁₀₂₀O₁₁₁₇S_37.It contained 102 negative charge residue（Asp+Glu）and 92 positive charge residue（Arg+Lys）.Its instability index was 40.13,so it was unstable protein.It was hydrophobic protein since its aliphatic index was 90.53 and grand average of hydropathicity was-0.217.The HsHDS proteins had aβ-strand of 15.50%,α-helix of 35.63%and loop of 48.87%.Chloroplast transit peptide was found in the HsHDS proteins,a trans-membrane domain and a GcpE conserved domain were contained in it.So it was speculated that the subcellular localization was located in the chloroplast.Besides,HsHDS protein had a closest connection with ginkgo.The above indicated that the HsHDS protein of Huperzia serrate was successfully gained and this provided a bioinformatics basis for the investigation the synthesizing of the effective components in it.In order to investigate the function of Huperzia serrate HsHDS protein,eukaryotic expression vector pINT121::HsHDS was built and transfered to Agrobacterium LBA4404.q-PCR（Quantitative Real-time PCR）method was applied to detect the demonstration of the HsHDS proteins in root,leaf and stem.The results showed that the relative expression of HsHDS was the highest in leaf,and the least was in root.It was indicated that the bioinformatics of this experiment successfully predicted the function of Huperzia serrate HsHDS proteins,and agreed well with the theory of MEP pathway positioning in plastid.This revealed that HsHDS was synthesized in the cytoplasmic matrix and exerted its function after being transported to chloroplast through post-translational translocation.