Functional Study Of DiZF-CCCH1 Gene In Davidia Involucrata

Dove tree(Davidia involucrata Baill.)is a deciduous tree in the Davidia family.It is a relic species surviving the Tertiary period.Now dove tree is listed as the first grade national protected plant in China.The low fecundity of dove tree limited its natural regeneration.Long dormancy period and serious seed abortion are main causes for the low seed fertility of dove tree.Studying the sexual reproduction mechansim of dove tree is critical for improving its natural regeneration capacity in order to better protect and utilize this precious species resources.To reveal the molecular mechanism of seed development in dove tree,we performed htranscriptome analysis in its seeds,and identified a transcript with the code c37849.graph_c0,whose expression levels were high in well-developed seeds,while almost undetected in abortive seeds.In this study,the full-length sequence of the transcript was cloned and identified as a gene encoding a CCCH zinc finger protein transcription factor,thus it was named as DiZF-CCCH1.Expression pattern analysis of DiZF-CCCH1 demonstrated that it was predominantly expressed in seeds.Then DiZF-CCCH1 was transformed into Arabidopsis and tobacco.The biological functions of DiZF-CCCHl gene on plant growth and development were observed.The main results of this study are as follows1.The DiZF-CCCHl gene encoding a CCCH zinc finger protein transcription factor was cloned from dove tree.The sequence of the target gene is 711 bp in length without introns,encoding a 236-amino acid protein.The target protein belongs to the plant-specific RR-TZF type zinc finger protein family,and has the highest similarity to a CsCCCH protein from Citrus sinensis L.2.Expression analysis using qPCR proved that the expression level of DiZF-CCCH1 gene in seeds was significantly higher than those in other tissues.Moreover,the expression level of DiZF-CCCH1 was highest in the seeds at early developmental stage,and it was about twice as high as that in the seeds at late developmental stages,suggesting that DiZF-CCCH1 might play a critical role in the early stage of seed development.3.Phenotypic observations of transgenic Arabidopsis showed that overexpression of DiZF-CCCH1 gene caused inhibition in root growth,delayed flowering,abnormal development in flower organs and pod,and reduced seed yield.Genes involved in Arabidopsis flowering regulation,including AtFT,AtSOC1,AtLFY,AtAP1 and AtFUL were down-regulated,while the flowering suppressor AtFLC gene was up-regulated in transgenic Arabidopsis.The expressions of genes related to floral organ development,including AtAP3,AtAG,AtWUS,AtSPL,AtBPE,AtJAG,AtRBE and AtP1,were also altered in transgenic Arabidopsis.The results showed that DiZF-CCCH1 gene inhibited plant growth and development in Arabidopsis.4.Phenotypic observation of transgenic tobacco indicated that overexpression of DiZF-CCCH1 gene resulted in inhibition in root development,early flowering,shortened internodes,lighter flower color,smaller berries and seeds,less seed yield and delayed seed germination.Genes involved into tobacco flowering regulation,including NtFT,NtSOC1,and NtFLC were up-regulated in transgenic plants.However,the expressions of NtFUL and NtAP1 were not affected.Overexpression of target genes can also lead to down-regulation of the genes involved in anthocyanin biosynthesis in tobacco,including NtDFR,NtLAR,NtCHS,and NtUFGT,but it did not affect the expression of NtANS and NtCH1.The results proved that DiZF-CCCH1 gene also has a certain inhibitory effect on the growth and development in tobacco.Moreover,the target gene can lighten the flower color by inhibiting the expression of anthocyanin-related genes in tobacco.5.The stress tolerance of transgenic tobacco lines were evaluated.The results showed that transgenic tobacco lines were more susceptible to drought and high salt stress than wild type,especially the drought resistance,which decreased significantly.Overexpression of DiZF-CCCH1 reduced the adaptability of tobacco plants to drought and salt stress.According to the results of this study,the DiZF-CCCH1 gene was highly expressed in dove tree seeds,especially at early developmental stage.Therefore,DiZF-CCCH1 was speculated to play a critical role in the regulation of seed development in dove tree.However,overexpression of DiZF-CCCH1 in Arabidopsis and tobacco both showed an inhibitory effect on the plant growth,and reduced seedviability.The target gene inhibited the root development of seedlings and reduced the tolerance of plants to drought and high salt stress.These results indicated that DiZF-CCCH1 is a negative regulatory factor.Whether the gene performs similar functions in dove tree and how it participates in the seed development of dove tree needs further investigation.