Cloning And Functional Analysis Of Chloroplast Transit Peptide Of The Small Subunits Of Ribulose 1,5-bisphosphate Carboxylase/oxygenase In Maize

Many chloroplast proteins are encoded by nuclear genes,which are translated into protein precursors or further processed into mature functional proteins in the cytoplasm,and then are imported into chloroplasts,therefore playing their respective functions.Transportation of chloroplast proteins from the cytoplasm to the chloroplast is usually achieved by mediation of transit peptide(TP).The TP is generally located in the N-terminus of the transported proteins.The primary structure of TP can be divided into three domains:the N-terminal domain,central domain and C-terminal domain.It has been found that primary sequenes of cTP display high variabilities and lack consensus sequence or functional motifs either within the same domain between different species,or between different domains within the same speices.Each motif plays a crucial role in a specific process of protein transportation into the chloroplast.Chloroplast transit peptide(cTP)of the small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase(RbcS)is one of widely studied cTPs.Currently,the structural domain of RbcS cTP and parts of its functional motifs have been well characterizaed in Arabidopsis,but how the structural domains and motifs of RbcS cTP in monocots function in transportation of chloroplast proteins remain less studied,especially it is unclear whether there exists species specific function of cTP between monocot and dicot.In this study,we first used the bioinformatic approach to predict the size of cTP and accordingly cloned the gene of maize RbcS cTP.We used a similar approach to clone the RbcS homolog in rice and Arabidopsis.We performed a more detailed analysis of the sequence characteristics of structural domains and potential motifs between maize and Arabidopsis RbcS cTP,and functionally validated them.This study was designed to clarify the sequence basis of monocot and dicot RbcS cTP functional specific.The main results are as below:1.Cloning and sequence characterization of RbcS cTP gene in maizeThe cTP sequence was initially identified from the promoter region of the RbcS gene in maize,and the size was then predicted as 47AA using ChloroP.By constructing transient expression vector using the GFP gene as a reporter,which contains mutated vectors with a series of point mutations at individual amino acid or deletion at partial amino acids eithet at N-or C-terminus,we conducted PEG mediated protoplast transient transformation.The result showed that the complete cTP can successfully target the GFP signal into chloroplast prepared from both greening or etiolated maize leaf tissue,and the first or second ATG mutation did not affect its chloroplast targeting,but its targeting efficiency apparently decreased;in contrast the third ATG mutation of cTP resulted in the loss of the chloroplast localization.Similarly,we observed that deletion of one or a few more amino acids starting from the C-terminus of the cTP caused the loss of function cTP chloroplast targeting.Therefore,these analyses show that chloroplast targeting of maize RbcS cTP is not light-dependent,which requires at least 47AA of cTP to excert the full function of chloroplast targeting.2.Functional divergence in the chloroplast targeting of RbcS cTP between maize and ArabidopsisTo compare the function of RbcS cTP in chloroplast targeting between monocot and dicot,we further obtained RbcS cTP sequence of Arabidopsis and rice through homologue of maize RbcS cTP blasted in NCBI.Their functions of chloroplast targeting were confirmed using PEG mediated protoplast transient transformation method.The results of subcellular localization showed that GFP signal deried from maize and rice RbcS cTP were observed in monocot chloroplasts,such as maize,rice or wheat,but did not seen in Arabidopsis chloroplasts;In contrast,the GFP signal from the RbcS cTP was only detected in Arabidopsis chloroplast,but not in maize or rice chloroplast.In summary,these analyses demonstrated that the RbcS cTP between monocot and dicot displays a species-specific chloroplast targeting.3.The N-terminal domain of RbcS cTP plays an important role in chloroplast targetingIn order to clarify the role of the N-terminal domain of RbcS cTP from in the role of monocot and dicot in the corresponding chloroplast targeting function,we intentionally switched exchanged the N-terminal domain of RbcS cTP between maize and Arabidopsis RbcS cTP to form a series of artificial chimeric cTPs.Through PEG mediated protoplast transient transformation,we expectedly observed that GFP signal derived from AhTP:GFP(contains the N-terminal domain of maize RbcS cTP plus central and C-terminal regions of Arabidopsis RbcS cTP)is localization was specifically localized in the maize chloroplast compared with the GFP signal,but not in Arabidopsis chloroplast.However,unexpectedly MhTP:GFP(contains the N-terminal domain of Arabidopsis RbcS cTP plus central and C-terminal regions of maize RbcS cTP)can target mediate GFP signal targeted to both Arabidopsis and maize chloroplast.Hybrid DNAMTP of N-terminal domain of Arabidopsis RbcS cTP inserted between the N-terminal domain and middle central domain of maize RbcS cTP,the GFP signal was targeted into Arabidopsis and maize chloroplast.But hybrid DNAMTP of N-terminal domain of maize RbcS cTP inserted between the N-terminal domain and middle central domain of Arabidopsis RbcS cTP,the GFP signal was targeted into the maize chloroplast,not in Arabidopsis chloroplast.Taken together,we concluded that the N-terminal domain of Arabidopsis RbcS cTP is the key domain for chloroplast targeting,while the N-terminal domain and the other two domains of maize RbcS cTP may functionally coordinate in chloroplast targeting in maize.