Molecular Cloning And Expression Analysis Of DoHDS And DoIPI In Dendrobium Officinale Kimuraet Migo

Dendrobium officinale Kimura et Migo is a traditional Chinese orchid herb which has both ornamental value and broad rage of therapeutic effects.D.officinale stems are rich in active compounds,especially polysaccharides and alkaloids.At present,the researches showed that the alkaloids of D.officinale may be derived from the terpenoids metabolic pathway,which include MVA and MEP pathway.4-hydroxy-3-methyl-2-(E)-butenyl-4-phosphate synthase(HDS)and isopentenyl diphosphate isomerase(IPI)are key enzymes of the MEP pathway.In the present experiment,HDS and IPI genes were cloned from D.officinale.For further analysis the roles of both genes in the MEP pathway and their regulation function,transcript levels of HDS and IPI in different organs and subcellular localization were determined,respectively.The main results are as follows:(1)DoHDS and DoIPI cDNA full-length sequence(GenBank accession number,DoHDS:KJ161312;DoIPI:KJ161311)had been cloned by using RT-PCR and RACE techniques.The full-length DoHDS cDNA comprised 2666 bp with an ORF of 2238 bp,5-UTR 149 bp,3-UTR 279 bp,encoding 745 amino acids.And the full-length DoIPI cDNA comprised 1102 bp with an ORF of 726 bp,5-UTR 153 bp,3-UTR 223 bp,encoding 241 amino acids.(2)Tissue specific expression analysis showed that DoHDS and DoIPI could express in all of organs which were tested in this study.The highest expression of Do HDS in stem was 5.06 times of that in the protocorm.While,The highest expression of DoIPI was 1.93 times of that in the protocorm.(3)The prokaryotic expression vector pET-28a-DoHDS and pET-28a-DoIPI were constructed successfully.SDS-PAGE results showed that the expression of the two fusion proteins were successfully induced.(4)After the construction of eukaryotic expression vector,the subcellular localization and the complementation test were done.Subcellular localization analysis showed that DoHDS and DoIPI are located in the chloroplast.Using complementation of Arabidopsis thaliana ipi mutant by DoIPI,we found that ipi mutant is serious adverse growth,such as the small plant,the leaves turning yellow and the root shortened.While,DoIPI transgenic plants are growing slightly worse than the wild types.The Arabidopsis mutant complementation test,confirmed the key role of DoHDS and DoIPI in terpenoid biosynthesis.