| Paeonia suffruticosa,paeoniaceae Paeonia suffruticosa is a traditional famous flower of China,which is known as "national beauty and heavenly fragrance" and "kao" and has high ornamental and medicinal value.In recent years,great progress has been made in the studies on in vitro tissue culture and direct regeneration pathway of peony,but there are still problems such as difficult rooting of peony test-tube seedlings,low quality of root system and low survival rate of transplanting,which seriously restrict the rapid propagation of in vitro culture of peony seedlings.At present,studies on indirect regeneration of peony tissue culture mainly focus on callus induction,but related studies have not been able to establish a set of efficient and stable regeneration system of peony somatic embryo.This study aims to provide molecular biological theory and technical support for the somatic embryo induction process of peony and the establishment of efficient plant regeneration system of peony by observing the embryo development process and transcriptome analysis.The main research results are as follows:1.Determination of critical period of peony embryo developmentObserved in this experiment by making paraffin section embryo development along the proembryo a globular embryo a heart-shaped embryo,torpedo embryo cotyledon embryo developmental embryo developmental process is complete,a second nuclear proembryo time 3d to 5d after pollination,about 60 days after pollination of ovule that can be observed in the globular embryo,heart-shaped embryo were made around mid 65 days after pollination ovule,75 days after pollination of ovule that can be observed in the heart-shaped embryo into torpedo embryo,cotyledon embryo appears more in the ovule of about 90 days after pollination.After the formation of cotyledon embryo,the shape of the embryo body will no longer have obvious change,but the embryo body keeps increasing.However,the embryo generally enters dormancy after 130 days of pollination.2.Transcriptome Sequencing of Embryos in Key Stages of Paeonia suffruticosaIn this study,a transcriptome database of different developmental stages of tree peony embryos was preliminarily established by high throughput sequencing technology.Through transcriptome sequencing of 15 samples of proembryo,globular embryo,heart-shaped embryo,torpedo embryo and cotyledon embryo,167.57 Gb of data were obtained.134,800 Unigenes were obtained after assembly.134800 Unigenes were assembled and annotated in seven functional databases(KEGG,GO,NR,NT,Swiss Prot,fam P and KOG).A total of82,162 Unigenes were finally obtained.Article Unigenes has successfully obtained functional annotations.3.Analysis of different genes in different stages of embryo development of peony speciesIn this study,transcriptional databases of peony embryo at different developmental stages were preliminarily established by high-throughput sequencing technology.Transcriptome sequencing was carried out on 15 samples of the five development stages of peony embryo,including proembryo,spherical embryo,heart-shaped embryo,torpedo embryo and cotyledon embryo.A total of 167.57 gb of data was obtained.After assembly,a total of 134800 Unigenes were obtained.Through the embryo in different periods of DEGs expression were analyzed,and found that the globular embryo and the original embryonic stage(GE/PE),in the comparison of the appraisal to 69986 differentially expressed genes,heart-shaped embryo and globular embryo(HE/GE),in the comparison of the appraisal to 38760 differentially expressed genes,torpedo embryo and a heart-shaped embryo(TE/HE),in the comparison of the appraisal to 36839 differentially expressed genes,cotyledon embryo and torpedo embryo(CE/TE),in the comparison of the appraisal to 34775 differentially expressed genes,according to the difference of gene distribution,the number of from proembryo development to globular embryo in the process,Many gene expressions changed significantly,indicating that the physiological activities in the formation process of the spherical embryo of peony were more intense than that in the later stage of embryo development,and its GO function was mainly concentrated in DNA binding,translation and ribosome structure composition.The differentially expressed genes were mainly concentrated in ribosomal pathway,plant hormone signal transduction,metabolism of starch and sucrose,and MAPK signal transduction pathway.4.Analysis of endogenous hormone-related genes in different stages of embryo development of peony speciesIn this experiment,genes related to endogenous hormones IAA and ABA were found in different stages of embryo development of peony species.A large number of iaa-related genes were highly expressed in proembryo stage,but significantly down-regulated in spherical embryo stage,and partially up-regulated in heart-shaped embryo stage,indicating that iaa-related genes could promote early development and differentiation of embryos.A large number of genes associated with ABA were significantly up-regulated during the formation of spherical and heart-shaped embryos,suggesting that endogenous ABA may play a regulatory role in the normal development and maturation of embryos.5.Gene analysis of antioxidant enzyme activity in different stages of embryo development of peony speciesIn this experiment,genes related to POD,SOD,APX and other antioxidant enzyme activities were found in different stages of embryo development of peony species.The expression level of POD related genes was higher in the proembryo stage,and a large number of genes were significantly down-regulated in the process of spherical embryo formation,and then increased in the heart-shaped embryo and torpedo embryo formation stage,suggesting that the maintenance of POD high activity was conducive to the formation and development of early embryo.The expression level of SOD related genes was higher in the proembryo stage,and a large number of genes were significantly down-regulated in the process of the formation of spherical embryo,and then increased in the heart-shaped embryo stage,indicating that the emergence of the spherical embryo of peony could be judged according to the decrease of SOD enzyme activity.The expression level of apx-related genes increased from the period of spherical embryo to the period of torpedo embryo,and then began to be down-regulated in the period of cotyledon embryo formation,suggesting that APX enzyme activity is conducive to the whole normal development process of embryo.6.Analysis of key genes during embryo developmentPeony species by Swiss Prot annotation embryo development stage(proembryo,globular embryo,heart-shaped stage embryo,torpedo embryo and cotyledon embryo period)of key genes SERK,FUS3,ABI3,AGL15,BBM and CLV gene fragments,the development way of somatic embryogenesis,similar to the zygote embryo development stage has experienced the proembryo,globular embryo,heart-shaped stage embryo,torpedo embryo and cotyledon embryo period,such as stage,the aim of this study is to clone the embryo key genes and preparing for embryo into peony body system.In order to provide molecular biology theory and technology support for the somatic embryo induction process and the establishment of efficient plant regeneration system of peony. |
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