Cloning Of Ginkgo Biloba L BTF3 Gene And Its Expression Pattern During Embryonic Postharvest Development

After the seeds of woody plants such as Cotoneaster multiflorus Bge.,Davidia involucrata Baill.,and Taxus mairei.are detached from the mother,the embryos often germinate after sowing if they have not experienced post-harvest stimulation.The phenomenon of low rates and irregular seedlings has seriously affected the rate of seedling growth.In the seed ripening stage,the spatiotemporal expression of phytohormones related synthesis and metabolism genes changed.By adjusting the ratio of endogenous hormones ABA/GA,the embryos were morphologically differentiated and transitioned to the germination stage.Ginkgo biloba.L has abundant seed sources,large seed kernels,easy exfoliation of embryos and endosperm,and easy to observe the development of embryo.It is the preferred woody plant material for studying the mechanism of seed ripening.This experiment established an optimized system for RNA extraction from Ginkgo biloba.L tissues.The BTF3 proteins with high protein abundance were screened from the differential proteomics of Ginkgo biloba.L development.By RACE cloning Ginkgo biloba.L BTF3 gene,the development of BTF3 in embryo development was investigated.During the process of expression,the main findings are as follows:1.The modified CTAB method and the modified TRIzol method in this paper can extract high-quality Ginkgo biloba.L embryos and endosperm RNAs in a short period of time.It was also confirmed that the modified CTAB method was also applicable to other Ginkgo biloba.L tissues.The test cost of the method was much lower than that of the kit.The obtained RNA had good stability and high quality and can be used for conducting downstream transcriptome sequencing,downstream molecular tests such as RT-PCR and qRT-PCR.2.Based on the EST-Ginkgo biloba database,the GbBTF3 gene was cloned using RACE technology.Sequence analysis showed that the cDNA sequence of GbBTF3 was 855 bp in length and contained 465 bp open reading frame,encoding 154 amino acids.The molecular weight of the protein encoded by GbBTF3 was approximately 17.022 KDa and the theoretical isoelectric point was 6016.Homologous analysis showed that the Picea sitchensis had the highest similarity with 92%.The transcription factor GbBTF3 encoded the ORF between its88-552 bases,and this region has a typical NAC superfamily structure.The analysis of NAC-superfamily phylogenetic tree indicated that GbBTF3 belongs to the BTF3 protein group III branch of terrestrial plants and has a close genetic relationship with PsBTF3 of spruce of North America.These genes may have certain similarities in function,but At present,this branch of the BTF3 gene does not have in-depth functional studies.3.During the development of ginkgo seed,GbBTF3 gene was mainly expressed in the embryo,and the expression level in the endosperm was relatively low.The embryos of stage III,IV,and V were inoculated in a medium supplemented with ABA.It was found thatexogenous ABA impeded the development of explant embryos,making it unable to obtain germination capacity;the sensitivity of ex vivo embryos of stage III-V embryos to exogenous ABA.Gradually increased,and ABA negatively induced GbBTF3 expression.In the I-V stage of embryo development,the expression level of GbBTF3 showed a "V" shaped curve.The high expression of GbBTF3 in stage I may be related to its activation of DNA and RNA synthesis,which can promote early embryonic development.The expression level of GbBTF3 in stage II has been reduced to a minimum.At this time,the genes related to ABA synthesis were abnormally active,which leads to the passivation of GbBTF3 and positively regulates the accumulation of substances such as starch and protein.It was a critical period for embryos to enter the germination stage and lay the material foundation.In the III-V stage of embryo development,the expression of GbBTF3 was constantly increased,which promoted the embryos to gradually complete differentiation and germination.During the development of Ginkgo biloba.L embryos,the ABA content showed an inverted “V” shape,the GbBTF3 gene expression showed a “V” shape,and the GbBTF3 protein showed an inverted “V” shape,indicating that GbBTF3 does not participate in the ABA regulatory pathway from the transcriptional level.It controls the traits of embryos from the protein level.4.Ginkgo biloba.L seeds were soaked in distilled water and 400?M ABA solution and transferred to the sand for ripening treatment.It was found that the endogenous ABA content of the embryos increased during the continuous water absorption of the seeds,and the GbBTF3 gene was down-regulated in response to water stimulation;while the 400?M ABA solution was used for pretreatment.Seed,due to the stimulation of both water and exogenous ABA,the embryonic GbBTF3 was more markedly down-regulated.After the seeds were transferred to the sediment layer,the expression level of GbBTF3 in the ABA-pretreated group quickly rebounded to the peak value,which was significantly higher than that in the clear water group.Subsequently,the expression of GbBTF3 in the ABA-pretreated group began to decrease,and was always lower than that of the clear water group.This change is likely to hinder the development of Ginkgo biloba.L embryos.