Cloning And Functional Characterization Of Transcriptional Factors WRI1 And WRI4 From Cyperus Esculentus

Yellow nutsedge(Cyperus esculentus)is a perennial herb mainly distributed in tropical and temperate regions.Its underground tubers are rich in nutrients,including 25-30% of oil,and the quality of extracted oil is excellent.The aboveground part of nutsedge can be used as feed,with the yield as high as 12 tons/hectare.In recent years China imports huge quantities of soybeans for edible oil because of its large population.Therefore it is very important to ensure the edible oil safety to improve the oil content and drought resistance in nutsedge.In this thesis we cloned genes for CeWRI1 and CeWRI4 from yellow nutsedge and characterized their functions in transgenic Arabidopsis.The main results are as that follows:1.Amino acid sequence analysis indicated that both CeWRI1 and CeWRI4 contained two AP2 domains,therefore belonging to AP2/ERF superfamily.Evolutionary analysis showed that CeWRI1 and CeWRI4 were conserved to some extent with the corresponding WRI1 and WRI4 transcription factors of other plant species,respectively.CeWRI1 and CeWRI4 retained essential amino acids and domains required for their functions,and sequences outside of AP2 domains were quite different from other species.2.The expression level of WRI1 in tuber from different time points was significantly higher than that in leaves and roots;The expression of WRI4 was the highest in leaves,followed by that expressed in roots,and the lowest in tubers.3.In situ imprinting of yeast assay suggested that WRI1 could specifically bind to Arabidopsis fatty acid synthesis related genes BCCP2 and PIPK-β1 promoter sequence.q PCR analysis showed that the expression level of genes involved in fatty acid biosynthesis pathway in ce WRI1 transgenic Arabidopsis was significantly up-regulated,which was significantly different from that of wild type.The leaf oil content of ce WRI1 transgenic Arabidopsis was 1.3-1.6 times higher than that of wild type.There was no significant change in fatty acid composition,but the proportion of different fatty acids changed due to overexpression of WRI1.4.The drought resistance of ce WRI4 transgenic Arabidopsis was significantly better than that of wild type: 1)the growth of transgenic lines was better than that of wild type under 5%PEG simulated drought stress and real water deprivation stress;2)Under drought stress,stomatal conductance and transpiration rate of transgenic lines were significantly lower than those of wild type,and water use efficiency was 30%-50% higher than that of wild type;Under the same stress level,the contents of free soluble sugar,proline and malondialdehyde in the leaves of transgenic lines were significantly lower than those of wild-type Arabidopsis.3)The leaf water loss rate and chlorophyll extraction rate of transgenic lines were lower than that of wild type,which indicated that the capacity of holding water in leaf from transgenic lines was higher than that of wild type.5.Under normal conditions,the expression of fatty acid and wax synthesis genes in ce WRI4 transgenic Arabidopsis was higher than that in wild type.Under drought stress,the expression levels of the above three genes in transgenic lines were significantly increased;The wax content in leaves and stems of ce WRI4 transgenic Arabidopsis was significantly higher than that of wild type.Under non stress conditions,the wax content of transgenic lines increased by 22%-26% compared with that of wild type.Similarly,the wax content of the transgenic plants was 24%-31% higher than that of the control.Under drought stress,the wax content in stems and leaves of transgenic lines were 55%-57% and 36%-43% higher than that of wild type,respectively.It was found that there was more cuticular wax on the leaf surface of transgenic lines than that of wild-type plants.6.The 5 ’upstream promoters of ce WRI1 and ce WRI4 were cloned.Promoter element analysis showed that WRI1 pro and WRI4 pro contained common plant promoter elements.WRI4 pro contains two MYB binding sites.WRI1 pro has no binding sites for lipid regulatory factors,but there are some stress resistance factor binding sites.The ce MYB36 like transcription factor binding to WRI4 pro was screened by yeast one hybrid,and the binding was specific.Mutation of the binding site would destroy their interaction.Treatment with deacetylase inhibitor could improve the expression level of ce WRI1 to a certain extent.Chromatin immunoprecipitation showed that deacetylase could bind to the chromatin region of WRI1 pro,and the binding sites covered some stress resistance factor binding sites.These results suggest that ce WRI1 may be involved in some metabolic processes in addition to lipid regulation.In conclusion: CeWRI1 and CeWRI4 have transcription factor functions.They could improve oil content or drought resistance in transgenic Arabidopsis by up-regulating expressions of multiple downsteam genes.CeWRI4 may be regulated by Ce MYB36,and the regulation of CeWRI1 is possibly related to chromatin acetylation.