| Study of photosynthetic response in medicinal plants provides vital information for artificial cultivation in terms of light selection,which is an important environment factor affecting the plant growth and development as well as the production of secondary metabolites.One of the antenna proteins located on photosysem II,called Lhcb2,has recently been proved to play a unique role in state transition during light acclimation.Also,as concluded from recent studies of medicinal plants Colobanthus quitensis and Sedum alfredii,Lhcb2 was demonstrated to be the key gene that determines the different photosynthetic ability in different ecotypes.Peltate yam(Dioscorea zingiberensis)is a climbing plant that can produce diosgenin for medical use.It has been found to have three ecotypes: low light intensity,mediate light intensity and high light intensity.They tend to show different photosynthetic response and survival rate to different light intensities.For now,all the studies focused on the photosynthetic traits and light adaptability of low light intensity,lacking the comparison of three photo-types,Therefore,the aim of this study is to explore the structure and function of Lhcb2 in peltate yam to provide molecular information for the future crosswise comparison of different photo-types.The study could be divided into five parts:(1)Using homology-based cloning and RACE technology to obtain the full length of Lhcb2 gene in peltate yam,referred as DzLhcb2.(2)Connecting the DzLhcb2 coding sequence to the vector p ET-32 a for prokaryotic expression,then verified by western blot.(3)Protein reconstitution in vitro by adding pigments,and then purified through Ni-column.(4)Protein characterization by analysis of absorbing spectrum and circular dichroism spectrum.(5)Identification and analysis of Lhcb gene family in peltate yam using the newly obtained Dioscorea zingiberensis genome.From the experiments above and statistics analysis,several conclusions have been reached :(1)The length of the DzLhcb2 coding sequence is 795 bp,coding for 264 aa.(2)the classic approach for LHC protein in vitro reconstitution can be applied to peltate yam,and the reconstituted protein has very similar secondary structure to that in other plants.(3)In total,14 Lhcb genes were identified in peltate yam,located on chromosome 1,2 and 6.Their coding protein all contain the CAB domain.(4)By constructing phylogenetic tree with Arabidopsis thaliana,Brachypodium distachyon and Chlamydomonas reinhardtii,Lhcb proteins could be divided into three clades: major LHCII,minor antennas,and the Lhcbm protein in algae.(5)The 14 Lhcb genes are mainly expressed in leaf and aerial stem.The expression in rhizome is extremely low,but the expression of Lhcb7 is relatively high comparing with other genes.This study successfully makes the classic reconstitution approach applied to medicinal plant.It also identifies the other 13 Lhcb genes in peltate yam.The identification and structural analysis of Lhcb2 in Dioscorea zingiberensis could promote the further study on the comparison of different photo-types in peltate yam on the molecular and protein level,and the identification of other Lhcb genes could provide information for the investigation of their roles in the regulation of photosynthetic performance in different photo-types of peltate yam. |
