Overexpression Of Castor RcWRI1 Increases Oil Accumulation In Tobacco Leaves

Pant transcription factor WRINKLED1(WRI1)is a member of the AP2/EREBP transcription factor family,which regulate the expression of genes associated with glycolysis and plastid fatty acid synthesis.WRI1 plays a key role in the accumulation of oils in plants.Transcription factor WRI1 gene has been cloned in many crops such as Arabidopsis thaliana,Zea mays,Camelina sativa,and Brassica napus Castor bean(Ricinus connunis L.)is an important industrial oil crop,which is rich in ricinoleic acid(90%of total oil).Particularly,fatty acids and oil are mainly storaged in endosperm,different from other oil seeds where oil largely accumulated in cotyledons.However,regulatory networks underlying oil biosynthesis and accumulation in castor have not yet been well understood.The information of function and regulatory activity of the transcription factor WRI1 in castor has not been fully reported.In order to analyze the mechanism of oil accumulation and to identify WRI1 with highly regulation activity.The present study was conducted to clone RcWRI1 genes from castor development seeds and to analysis its gene structures and temporal-spatial expression patterns in castor beans.The overexpression vectors of RcWRI1s were constructed for its seed-specific expression in Arbidopsis wril-1 seeds and overexpression in tobacco plants.The phenotypic identification(contents of oil,starch and protein,and so on)on the transgenic plants were perfomed for fanctional characterization of castor RcWRI1 and its value in oil enginerring in plant vegetable organs.The main findings obtained in this study are described as the followings:1.Using the CDs of AtWRI1 as a probe,a BLAST analysis of the castor genome database revealed that the RcWRI1 gene(LOC8283400)has two alternative splices,named RcWRI1-A(1341 bp)and RcWRI1-B(1332 bp).High-fidelity RT-PCR was used to clone the RcWRI1 splice variants with different ORF lengths from the castor development seeds.Comparison with protein sequence of Arabidopsis AtWRI1 revealed that the RcWRI1-B protein sequence(443 AA)lacked the three amino acid "VYL"which presented in RcWRI 1-A(446 AA)and AtWRI 1 encoded by the third exon(9 bp)in the gene2.The expression patterns of RcWRI1-A and RcWRI1-B in different tissues and organs of castor were analyzed by qRT-PCR with specific primers.The results showed that RcWRI1-A was expressed in flowers,leaves,pericarp,and developing seeds,and its expression level in each organ was significantly different.RcWRI1-B was detected to express only in castor developing seeds.3.The expression vectors pJC-Gly-DsRED-RcWRI1A/B were constructed and then introduced into the wril-1 mutant through Agrobacterium tumefaciens-mediated transformation by floral dipping.When the two isoforms were individually introduced into an Arabidopsis wril-1 loss-of-function mutant,total fatty acid content was almost restored to the wild-type level,and the percentage of the wrinkled seeds was largely reduced in the transgenic lines of wril-1 mutant,indication that RcWRIls have the function of WRI1 transcrition factor.4.We constructed plant overexpression vector(pCAMBIA1303-RcWRI1-A/B)and transformed them into tobacco plants by Agrobacterium tumefaciens-mediated method to obtain transgenic tobacco lines.The controls were empty-vector transformed tobacco plants.The PCR and RT-PCR were used to detect RcWRI1s integration in ttobacco genome and their expression levels in the tissues/organs.5.Real-time PCR was used to detect the relative expression of WRI1 downstream target genes in transgenic tobacco.The result showed that the expression of these target genes was significantly increased 2-3 folds in leaves either expressing RcWRI1-A or RcWRI1-B relative to the WT and empty-vector controls.RcWRI1-B appeared to show a stronger activity in promoting the target gene expression than RcWRI1-A did.6.Total lipid content in the leaves expressing each of RcWRI1s was remarkably higher(4.3-4.9 folds)than that in the control leaves infiltrated with the WT and empty vector,with leaves expressing RcWRI1-B showing a higher level of oil content.However,the fatty acid profiles have no significant difference.The starch content reduced about 2%compared with control.And the total starch content is 21.6%in RcWRIl-A transgenic tobacco and 21.3%in RcWRI1-B transgenic tobacco.The content of protein in the leaves of was 10.6%in RcWRI1-A transgenic tobacco and 10.3%in RcWRI1-B transgenic tobacco.Respectively,in tobacco leaves expressed RcWRI1-A or RcWRI1-B,there was no significant different in protein content between the samples.In conclusion,both RcWRI1 isoforms have functional actively in induction of fatty acid/oil accumulation in heterogenous leaves when expressed in tobacco.The transgenic plants showed no significant negative phenotypes.These two RcWRI1 isoforms can be used as main transcriptional regulators to assemble metabolic pathways for the accumulation of oils in stem and leaf vegetative organs in order to produce edible or industrial vegetable oils from such high biomass vegetative organs.