| This study used male and female 20-30 year-old Fraxinus mandshurica Rupr,which were in reproductive phase as experiment materials.The F,mandshurica were from Qingshan seed orchard of Wei He forestry bureau in Heilongjiang Province.By two years of consecutivily follow-up survey,we observed and record the flowering phenology of male and female plants in the level of group,individual,inflorescence and single flower systematically.Through anatomical and morphological study of male and female flowers,we obtained the most detailed anatomical and morphological analysis of the inflorescence and single flower of the male and female flowers during organ development,and we also got the time point and the morphological characteristics in different flower developmental stages.Difference analysis of physiology and biochemistry,hormones,flowering related genes expression was made between male and female flowers in different flower developmental stages.In addition,we selected two typical periods,one was the male flowers coming pollination and the other was female flower coming megasporogenesis,low temperature stress experiment was done to investigate differences of physiological,biochemical and genes expression in response to low temperature stress between two genders of floral organs.We cloned the full-length genes of CO(CONSTANS),AP2(APETALA2),PI(PISTILLATA)and FT(FLOWERING LOCUS T).Bioinfomatic analysis was done of these four genes were constructed.The main conclusions are as follows:1.Study on flowering phenology of F.mandshurica Rupr:(1)the male flowers blossom earlier than the female.The flowering stage of male flower was from the end of April to early May and the female was from early May to the late May.The early flowering stages of male was 1-2 developmental stages earlier than female,and about to converge pollination at late stage.(2)The relative frequency of flowering intensity was 68.86%in male and 77.16%in female,and skewness was right-oblique;(3)After collecting,the pollens were stored at low temperature for 3-4 days,the germination rate reached 65.60%;(4)Within observation,the highest seed-setting rate of female plants was 62.78%and the lowest was 11.88%;(5)The length of flowering stages and fruit-setting rate showed a negative correlation,and the flower number and fruit-setting rate showed a positive correlation.2.Study on developmental morphology of flower of F.mandshurica Rupr:(1)In male organs,there were 4 anthers,meiosis of microsporocyte were simultaneous,and most tetrads observed were regular tetrahedron.Pollen maturity was two cell type pollen grain.Pollen has three germinal apertures.(2)In female organs,the ovule was anatropous,and the integument was single layer.Ovary had 2 rooms,and each room had 2 ovules;Abnormal developmental stamen or inflorescence which both had pinnate compound leaf were observed in few female flowers.After meiosis,the megasporocytes divided into four linearly arrayed megaspores,and the embryo sac was polygonum type.(3)Female unisexual flower development belongs to type I,and male unisexual flower development belongs to type II.3.Gender differences of F.mandshurica Rupr in flower development and under chilling stress:(1)Male flower subjected to more severe damage than female,and female flower had a higher adaptability to the environment.(2)During the development of male and female flowers,high-level ZR and low-level GA,IAA promoted gender differentiation.Unstable ABA showed its own state of balance,which indicated the existence of mutual restraint between hormones during flower development.4.Changes in gene expression during flower development of F.mandshurica Rupr:(1)Trubulin was suitable as the reference gene for male and female flowers.(2)At the second stage of male flower,as the meiosis stage;The fourth development stage of female flower,as the two nuclear embryo sac-eight nuclear sac stage,the expression of genes related to flowering reached the highest level.5.Cloning and analysis of key genes in flower development in F.mandshurica Rupr:Full-length cDNA sequences of flowering related genes CO,AP2,PI and FT were obtain by RT-PCR.The open reading frames(ORFs)of these four genes were predicted as 1032bp,1506bp,645bp and 525bp;Gene Homology between Darwin cotton CO gene,grapes AP2 gene,sesame PI gene and apple FT gene reached 59%66%,87%and 88%respectively,and coverage reached more than 99%;Amino acids which these genes encoding were hydrophilic unstable proteins and the main component of their secondary structure were random coil.Innovation and significance of this study:as the F.mandshurica Rupr is the typical large dioecious woody plant.This study started from flowering phenology of male and female plants and we found the differences between the male and female flowers development,such as different flowering times.And thereby further research and analysis were conducted on morphology,physiology and biochemistry,gene expression between two gender flowers of male and female plants.And full-length sequence of flowering key genes,CO,AP2,PI and FT,were obtained and preliminarily analyzed.Study above clarified the flowering phenological development regularity,and summed up the organ development characteristics of male and female flowers.We discussed gender differences in the process of male and female development and in response to low temperature stress and obtained the key genes of flowering,which promoted process of flower developmental molecular biology for the future research on F.mandshurica Rupr.Foundation has been laid for research on flower development and flowering network regulatory mechanism,which helps us to understand changes of flower development traits to adapt to environment in the natural state.This study would work as a theoretical guidance while in practice,and ultimately lay the theoretical foundation for protecting germplasm of F.mandshurica and accelerating the genetic improvement of the species. |
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