Cloning And Analysis Of Genes PoWUS And PoAGL15 Relevant To Somatic Embryogenesis In Paeonia Ostii

In this research,mature zygotic embryos in Paeonia ostii were used as explants to realize the direct somatic embryogenesis.Tissue materials of somatic embryos in P.ostii were obtained and divided into 6 periods and 10 stages:embryo stage（0 d,5 d,10 d,15 d,20 d）,cotyledon growth,somatic embryogenesis,secondary embryo germination,secondary embryo growth and maturation.These tissue materials were studied from the aspects of histocytology,physiology,biochemistry and molecular biology.Paraffin sections were made to observe the cell structure and explore the origin of embryonic cells.At the same time,the contents of IAA,ABA,GA3 in different stages of somatic embryogenesis were detected,and the dynamic changes of these hormone and the ratios of IAA/GA3 and ABA/（IAA+GA3）were analyzed.Three generations of full-length transcriptome were sequenced during somatic embryogenesis.Po WUS and Po AGL15 were cloned and identified by bioinformatics and expression analysis.The purpose of this study is not only to reveal the changes of cell morphology and endogenous hormones in different stages of the direct embryogenesis of tree peony somatic cells to provide theoretical basis for establishing important time in the culture of tissue culture and optimizing the hormone ratio of culture medium,but aslo to cloning the genes related to the somatic embryogenesis to provide a basis for subsequent analysis of its expression,transcriptional expression pathway and regulatory mode and in order to laid a foundation for the establishment of efficient and stable plant in vitro regeneration system.The main results are as follows:（1）The histological and cytological sections of tree peony were observed in different stages of somatic embryogenesis.It was found that the somatic embryos induced by mature zygotic cotyledons,directly originated from the pre-embryogenic determined cells（PEDC）at the edge of cotyledon epidermis or near the hypocotyl.（2）The changes of three endogenous phytohormones inclouding IAA,ABA,GA3 and the ratios were explored in eight stages.It was found that the highest content of IAA was 0.947ng·g^-1at 0 d,then decreased significantly at 10 d and increased at 20 d.With the development of somatic embryogenesis,IAA content decreased gradually and reached the lowest value during the growth of secondary embryos.The content of ABA remained at a higher level than the other two hormones at all stages.A"double peak"showed that the highest values of ABA were 11.353 ng·g^-1at the 10 d and 11.300 ng·g^-1at the maturation stage,respectively.The content of GA3 decreased gradually in the early stages and increased again in the maturation stage.In the 8 stages,the value of IAA/GA3 rised first and then declined.The maximum value was 27.778 when the cotyledons turned green.Then,the ratio of IAA/GA3 began to falled,it were 1.053 and 1.066 at secondary embryo growth period and maturation stage,respectively.The value of ABA/（IAA+GA3）increased gradually after culturing with light,and reached the maximum value of 175.900 at the time of secondary embryo formation.Then it decreased rapidly to 89.920 and still maintained a high value.（3）Total RNA was extracted from the culture of somatic embryogenesis at different stages,two homologous sequences were cloned and named Po WUS9-13 and Po WUS9-14,respectively.The lengths of these two coding sequences were all 861 bp and encoded 286 amino acids.Homology analysis showed that the average similarity of WUS protein sequence between Po WUS and other known WUS in 15 plants was 64.85%,the highest was 66%with Vitis vinifera.The phylogenetic relationship was the closest between Po WUS and Camellia sinensis.The physical and chemical properties,secondary structure and tertiary structure of the two Po WUS proteins were predicted.The expression patterns of two Po WUS genes were analyzed by q RT-PCR,and it was found that they were different,but they were highly expressed at the10th day and maturation stage.It was speculated that they might play an important role in regulating embryogenic cell formation and organogenesis of stem and leaf during somatic embryo maturation.（4）Two homologous coding sequences of AGL15 gene were isolated,named Po AGL15-1and Po AGL15-2 respectively.It had a coding region of 768 bp which coded 255 amino acids,respectively.They had two conserved domains of MADS-box gene,MADS domain and K domain.Homology analysis showed that the average similarity of AGL15 protein sequence between Po AGL15 and other 15 species was 84.65%,among which the highest similarity with the amino acid sequence of Jatropha carcas,reaching 88%.Phylogenetic analysis showed that two AGL15 protein in Paeonia ostii probably had the closest relationship with Vitis vinifera.The physical and chemical properties,secondary and tertiary structures of two Po AGL15proteins were predicted.The expression patterns of Po AGL15-1 and Po AGL15-2 genes are similar,and they are highly expressed at the 5th,10th and somatic embryo maturation stages.It can be inferred that AGL15 gene accumulates and expresses in the early embryo and plays an important role in regula Paeonia ostiiting the later stage of somatic embryo maturation.