| Cercidiphyllum japonicum belongs to the deciduous tree of Cercidiphyllaceae and is mainly distributed in the warm temperate and subtropical regions of my country.It has high ornamental and industrial value.The wild Cercidiphyllum japonicum population is mostly male,with few female individuals and low seed setting rate,which is in a state of endangerment.In addition,due to the dioecious flowers of Cercidiphyllum japonicum,the inflorescence structure is extremely simplified,and the special flower structure characteristics become an excellent material for the study of angiosperm sex differentiation.This study systematically explored the regulation mechanism of Cercidiphyllum japonicum gender differentiation from the aspects of morphological observation,hormone regulation,physiological and biochemical and key gene expression.The main results are as follows:1.Cytological observation showed that the morphological changes of male and female flower buds had begun at the end of July,and the stamens of male flower buds were clearly observed.The growth points in female flower buds were significantly expanded and protruded into cylindrical shape,which was the preliminary shape of ovary.In mid-October,the stamens become slender and the ovary in the pistil takes initial shape.The anthers of the male flower buds in December are long columnar,and the closely arranged ovules can be vaguely seen in the ovary in the pistil.At the end of March of the next year,more pollen grains were formed in the anthers of stamens,indicating that the flower buds were mature at this time,and the young ovules were clearly visible in the ovary of pistils,and the arrangement was upright.2.The change trend of soluble sugar content in both female and male flower buds of Cercidiphyllum japonicum increased first and then decreased.The soluble sugar content in the early stage of flower bud differentiation is low,and it rises rapidly and reaches the peak during the dormant period of flower buds,and is significantly different from other differentiation periods.It is speculated that the flower bud differentiation stage plays an important role in sex differentiation,a higher content of soluble sugar is more beneficial to the development of male and female flower buds;the soluble protein in female flower buds is significantly higher than that in male flower buds,so it is inferred that the soluble protein may be Participate in the process of plant sex differentiation;the content of malondialdehyde in female flower buds is significantly higher than that in male flower buds.It is speculated that malondialdehyde may be related to the sex determination of plants;SOD content has been at a high level during the differentiation of Cercidiphyllum japonicum flower buds,which is speculated to be higher The level of SOD can promote the sex differentiation of flower buds.3.Transcriptome sequencing analysis: There were three biological replicates of female and Cercidiphyllum japonicum male flower buds,and the number of clean raw reads obtained from each sample was more than 20 million.A total of 83435 Unigene were assembled,with an average length of 1136 nt.The annotation rate was over 57.75%(48,385entries)in seven databases including GG,NR,Nt,Pfam,COG,Swiss-Prot and KEGG.The results showed that there were 62118 Unigenes in both female and male flower buds,and 11225 Unigenes in female flower buds and 10092 Unigenes in male flower buds.Compared with male flower buds,there were 1511 differentially up-regulated genes and 1829 differentially down-regulated genes in female flower buds.These differentially expressed genes were mainly concentrated in sucrose and protein metabolism,hormone metabolism and hormone signal transduction and other pathways related to sex differentiation of flower buds.Based on GO function and KEGG analysis,five differentially expressed genes related to sex differentiation were screened,respectively:Cluster-28080.0(Ethylene biosynthesis related key enzyme genes,ACS),Cluster-34083.32607(protein phosphatase,negative regulator of ABA signal transduction pathway,PP2C),Cluster-34083.30633(negative regulator of GA signal transduction,DELLA),Cluster-34083.33735(S-adenosylmethionine synthase gene of ethylene biosynthesis),SAM),Cluster R-34083.1098(cytokinin signal regulator,ARR),and then using fluorescence real-time quantitative PCR(q PCR)to verify the accuracy of transcriptome sequencing results.The results showed that the experimental data of q PCR in this experiment were consistent with the experimental data of transcriptome sequencing,so the reliability of transcriptome sequencing results was further confirmed.4.The results showed that IAA,ABA,GA,JA and SA were closely related to sex differentiation in Cercidiphyllum japonicum female and male flower buds,and there were significant differences between Cercidiphyllum japonicum male and female flower buds in most periods.combined with the number of transcriptome analysis,found that: DELLA protein may promote the development of female flower buds of Cercidiphyllum japonicum by regulating GA;PP2C may negatively regulate ABA signal transduction,thereby promoting the differentiation of male flower buds of Cercidiphyllum japonicum.Except that ABA content in male flower buds was higher than that in female flower buds,the content of ABA in the other four endogenous hormones was higher in female flower buds than that in male flower buds.It was speculated that IAA,GA,JA and SA could promote the differentiation of female flower buds more,while ABA promoted the differentiation of male flower buds. |
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