| Rubber tree(Hevea brasiliensis Muell.Arg.)is an important economic crop in tropical regions of China,and the latex produced by it is the most important source of natural rubber.Rubber tree female flower abortion varieties are important germplasm resources for selecting parents in hybrid breeding of rubber trees.However,the cytological characteristics of the female flower abortion of rubber trees are unclear,and there are few research reports on the mechanism of female flower abortion of rubber trees.In this study,by observing the morphological and cytological characteristics of normal female flowers(Reyan 88-13)and aborted female flowers(PR107)during growth and development,key nodes of female flower abortion were discovered.Using high-throughput sequencing technology(RNA-seq)to compare and analyze the transcriptome data of normal female flowers and aborted female flowers,we screened out the differentially expressed genes related to female flower abortion.Through functional annotation and pathway enrichment analysis of the differentially expressed genes,the candidate genes related to female flower abortion were further screened,q PCR technology was used to verify the transcriptome data,and gene cloning and expression analysis of some candidate genes and their gene family members were performed.This study initially revealed the molecular mechanism of the abortion of the female flowers of rubber trees,and provided a scientific basis for the utilization of hybrid breeding of rubber trees.The main results were as follows:1.The morphological and cytological observations showed that the external morphology of aborted female flowers was not significantly different from fertile female flowers.The ovary of aborted female flowers was narrower than fertile female flowers in the same development period.Before the formation of mononuclear embryo sacs,the development of ovules of aborted female flowers is basically the same as that of normal female flowers;after the formation of mononuclear embryo sacs,the abnormality of the functional megaspores in the embryo sac mitotic process leads to the inability to form normal embryo sacs,which eventually leads to female flowers of abortion.2.Using transcriptome sequencing technology combined with gene function annotation,we selected 56 candidate genes related to female flower abortion at the gene expression level.These candidate genes could be divided into five categories:includes four key regulatory factors homologous genes AGAMOUS(AG)for the development of flower organs(LOC110666153,LOC110669076,LOC110640008 and LOC110641968);two BEL1 homologous genes(LOC110659796 and LOC110635038);four WUSCHEL(WUS)homologous genes(LOC110638249,LOC110655693,LOC110669207 and LOC110633278);two LEUNIG(LUG)homologous genes(LOC1106474666 and LOC11065503)and 44 candidate genes associated with ethylene response factors.3.Using GO enrichment results combined with the cytological characteristics of aborted female flowers,31 candidate genes related to female flower abortion were screened.These candidate genes are functionally involved in germ cell mitosis,development process,flower development process,cell cycle regulation,cell separation,cell death regulation process and cell regulation process.These candidate genes may play an important role in the formation of the female flower abortion of rubber trees.4.Using transcriptome sequencing technology combined with the KEGG pathway enrichment results,four pathways related to the abortion of rubber tree female flowers were screened.The four pathways are alpha-linolenic acid metabolism,plant signal transduction pathway,endoplasmic reticulum protein processing and galactose metabolism.21 candidate genes related to alpha-linolenic acid metabolism were screened from normal and aborted flowers at same developmental stages.109 candidate genes related to plant signal transduction pathways were selected in different developmental stages of aborted female flowers;70 candidate genes related to endoplasmic reticulum protein processing were selected in different developmental stages of aborted female flowers;33 candidate genes related to galactose metabolism were selected in different developmental stages of aborted female flowers.These genes may play a key role in the formation of the female flower abortion of rubber trees.5.Three mitochondrial differentially expressed genes of rps3 A,cox3 and ycf3 A were screened by transcriptome sequencing technology.Abnormal expression of these genes may lead to cell energy deficiency,resulting in the abortion of the female flowers of rubber trees.6.Successfully cloned three growth-regulating factors in rubber trees.They are named Hb GRF1,Hb GRF2 and Hb GRF3,respectively,and their login numbers are:MN396751,MN396752 and MN396753.Bioinformatics analysis and expression analysis were conducted on them,which lays the theoretical foundation for the study of the function and function mechanism of the rubber tree Hb GRF genes,which also provides the theoretical basis for the molecular auxiliary breeding of rubber tree. |
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