Cloning And Functional Study Of CoFLAs Gene In Camellia Oleifera

Camellia oleifera is a unique woody edible oil trees in China,which belongs to the late-acting self-incompatibility(LSI)plant.Its molecular mechanism is unclear,which is not conducive to the optimization of variety allocation,the formulation of cultivation technical measures and the increase in yield per unit area.In order to analyze the molecular mechanism of self-incompatibility of C.oleifera,the laboratory’s research group constructed the transcriptome database of pistil and pollen of C.oleifera.It was found that FLAs were probably involved in the process of self-incompatibility of C.oleifera.So,in the subject,the gene cloning and biological function research of CoFLAs were carried out,with the main cultivars ’Hua shuo’ and ’Hua jin’ as materials.The main results are as follows:1.The cloning of CoFLAs.Based on transcriptome data,special primers were designed,and the full-length CDS sequences and gDNA sequences of the CoFLA genes were cloned.It was found that none of these seven genes had introns,which were sequentially named CoFLAl,CoFLA2,CoFLA3,CoFLA4,CoFLA5,CoFLA6 and CoFLA 7,with the length of 849 bp,1233 bp,1176 bp,1263 bp,1239 bp,771 bp,and 1401 bp.Homologous sequence alignment showed that they sequentially had the highest similarity to CsFLA3,NnFLA1,CsFLA2,CsFLA8,CsFLA4,CsFLA7 and CsFLA16.Phylogenetic tree analysis showed that they were sequentially closest to AtFLA14,AtFLAl,AtFLA1,AtFLA8 and AtFLA10,AtFLA4,AtFLA7,and AtFLA15～AtFLA18.CoFLAl,CoFLA2,CoFLA3,CoFLA4,CoFLA6 and CoFLA7 contained signal peptides,and CoFLAl,CoFLA2,CoFLA3 and CoFLA5 contained GPI anchor sites.They all had the main structural characteristics of FLA proteins with 1-2 FAS domains and three conserved domains of H1,[Y/F]-H and H2.2.The expression patterns of CoFLAs.The expression patterns of CoFLAs in different tissues,styles and ovaries at different time after self-/cross-pollination was explored by qPCR.In different tissues,CoFLA1 was specifically expressed in pollens,and CoFLA2 and CoFLA5 were mainly expressed in leaves,and CoFLA3 was mainly expressed in roots and leaves,and CoFLA4 was expressed in stems,and CoFLA6 was expressed in leaves,stems and pollens,and CoFLA7 was abundantly expressed in leaves and stems.In the styles and ovaries,the expression trend both was divided into four categories.Among the four types of styles,CoFLAl,CoFLA3 and CoFLA7 belonged to the first category,and CoFLA4 and CoFLA6 belonged to the second category,and CoFLA5 belonged to the third category,and CoFLA2 belonged to the fourth category.The large difference of these four categories was all in 2 to 48 h between self-and cross-pollination.Among the four types of ovaries,CoFLA2 and CoFLA7 belonged to the first category,and CoFLA3 and CoFLA5 belonged to the second category,and CoFLA4 and CoFLA6 belonged to the third category,and CoFLAl belonged to the fourth category.3.The subcellular localization of CoFLAs.By homologous recombination,the seven sequences of CoFLA1,CoFLA2,CoFLA3,CoFLA4,CoFLA5,CoFLA6 and CoFLA7 without signal peptides and four sequences of CoFLA1,CoFLA2,CoFLA3 and CoFLA5 without signal peptides and GPI anchor sequences were successfully ligated to the pCAMBIA1300 vector with GFP fluorescent protein,for subcellular localization.The results showed that the seven CoFLA proteins without signal peptides were all located on the plasma membrane,while the CoFLAl,CoFLA2,CoFLA3 and CoFLA5 lacking GPI anchor sequences were mainly located outside the plasma membrane.This showed that the CoFLAs was localized on the plasma membrane,and the GPI anchor sequence had a very important role in the localization of CoFLAs on the plasma membrane.4.Prokaryotic expression of CoFLAs.The CoFLAs were successfully ligated into pCold-TF vector with His-tagged by T4 ligase,and these seven recombinant vectors were transformed into E.coli Rosetta(DE3)for prokaryotic expression respectively.These seven recombinant proteins all could be induced efficiently by 0.5 mM IPTG,which were soluble proteins through Ni2+ purification experiments.The recombinant proteins were added to water or pollen medium to treat the pollens respectively,of which the final concentration was 5μg/mL.It was found that CoFLA3 could promote the pollen activity,and CoFLA1,CoFLA2,CoFLA3,CoFLA4 and CoFLA6 all could promote pollen germination,and the CoFLA6 protein could significantly promote pollen tube elongation.5.Phenotypic observation of CoFLAs overexpression in Arabidopsis.The seven overexpression vectors pCAMBIA1304-CoFLAs were successfully constructed by T4 ligase.Wild-type Arabidopsis was infected by the pollen tube channel method,and the seven CoFLA genes all were screened for over-expressing Arabidopsis by identifications,and three lines of each gene all received T3 generation seeds.Through observation,it was found that the overexpressing Arabidopsis of CoFLA2 and C0FLA6 were significantly different from Wild-type in carobs.The average length of carobs of each line of these two genes was both lower than Wild-type.After segmenting the carobs length,it was found that the proportions of carobs above 1.0 cm in length all had a different degree of decline,while the proportions of carobs shorter than 1.0 cm all had a different degree of increase,and ovules abortion appeared in carobs of different lengths.