Establishment Of A Simple And Stable System For Plastid Transformation And Its Application In Transplastomic Woody Plants

Plastid engineering has become a promising approach for plant biotechnology applications.The foreign genes were inserted into plasmid DNA by homologous recombination via the flanking sequences at the insertion site,while DNA integration in the nucleus is a random process.Compared with conventional nuclear transformation,plastid transformation offers several highly attractive features,such as high protein expression levels,stacking of transgenes into operons,compartmentalization of foreign proteins in plastids,and the excellent biosafety resulting from maternal inheritance.Thus,plastid engineering could be utilized as plant bioreactor for production of recombinant proteins and metabolites of commercial interest.However,large-scale application of this technology has been hindered by some technical bottlenecks,including establishment of high efficiency for plant regeneration,tedious and time-consuming cloning steps for the construction of plastid transformation vector,and lack of suitable selection condition for transplatomic plants.At present,stable plastid transformation system was established for only 6 herb plants.Both poplar and kiwifruit are important economic woody plants cultivated widely in China.However,workable plastid transformation protocols were not available for these woody plants.In order to expand the species range of plastid engineering,we developed plastid transformation systems for poplar and kiwifruit in this study,and the results can be summarized as follows:1.We describe the adoption of an in vivo Escherichia coli cloning?i VEC?technology toquickly assemble a plastid transformation vector.Firstly,the i VEC method enables simple and seamless build-up of a complete tobacco?Nicotiana tabacum?plastid transformation vector pYY12 from five DNA fragments in a single step,which consists of homologous recombination,left homologous recombination region?LHRR,containing psa B,rps14 and trnf M?,and right homologous recombination region?RHRR,containing psb Z and trn G?of tobacco plastid,aad A and green fluorescent protein?GFP?expression cassettes,as well as the vector backbone fragment from p Bluescript II SK?+?without multiple cloning site.Vector pYY12 was introduced into tobacco plastid genome via particle bombardment,and successful transformation was verified by Southern blot analysis plus seed assays.GFP fluorescence signal in chloroplasts of Nt-pYY12 tobacco plants was visualized by confocal microscopy,and GFP accumulation was approximately 9%of the total soluble protein?TSP?quantified by SDS-PAGE.Given the high accumulation of GFP in above results,expression cassettes,including aad A and gfp cassettes,of pYY12 could be utilized in constructing plastid transformation vectors for other plants,so as to broaden application of plastid engineering.2.We constructed a poplar transformation plastid vector pYY20,in which the site of integration is similar to pYY12,while LHRR and RHRR were cloned from a poplar hybrid?Populus davidiana×P.bolleana?.Then,pYY20 was transformed into poplar plastid genome using our established high efficient poplar leaf-explant regeneration system.As a result,homoplasmic transplatomic poplar plants were obtained after 2?3 rounds of regeneration under antibiotic selction.GFP accumulated only 0.005%of TSP in homoplasmic Pa-YY20 poplar plants.Two other poplar plastid transformation vectors,pYY25 and pYY26 were derivatives of pYY20,which harbored Bacillus thuringiensis?Bt?-cry1C and Bt-cry3Bb,respectively,by replacement of gfp gene of pYY20.The Cry1C insecticidal protein in transplastomic poplar showed a differential expression pattern in different tissues with highest expression level in young leaves?up to 20.74?g/g fresh weight?and relatively low level in mature and old leaves,and hardly detectable levels in non-green tissues such as phloem,xylem and roots.For detached leaf bioassay,transplastomic poplar Pa-YY25 showed 100%mortality of 1^st-4^th instar larvae of Hyphantria cunea after 3 days feeding the leaves,while Pa-YY26 poplar plants exhibited100%mortality of 1^rd instar larva and adult insect of Plagiodera versicolora after 1 days and 6days feeding the leaves,respectively.The results reported here the first successful example of generating insect resistant poplar via expressing Bt gene from plastid genome,thereby open a new avenue for future genetic improvement of poplar plants.3.Further,a‘Red Sun'kiwifruit?Actinidia chinensis?plastid transformation vector pYY34was constructed based on pYY12.In the leaves of the single homoplasmic Ad-pYY34 kiwifruit plant,GFP accumulated approximately 0.02%of TSP.Although only one homoplasmic event was obtained via two biolistic bombardments,this is the first case of generating transplastomic plants in a fruit tree.This system paves the way to efficient production of edible vaccines,pharmaceuticals,and antibodies in kiwifruit.Overall,in this study,we constructed a plastid transformation vector using i VEC approach,and established plastid transformation system for both poplar and kiwifruit.Notably,generation of Bt-transplastomic poplar plants with strong resistance to H.cunea and P.versicolora confirmed the importance of plastid engineering,and provide a new way of genetic improvements for other forestry trees in the future.