| Moso bamboo(Phyllostrachys edulis)is the most widely distributed bamboo species in China which has a long history of cultivation.It accounts for more than 74% of the total area of bamboo forest,possessing important economic,culture and social value.P.edulis mainly has an asexual clonal reproduction based on powerful rhizome system extension.In the long term of production and cultivation,this mode of reproduction was gradually strengthened by human beings.Compared with the traditional asexual propagation method,the seedling cultivation of moso bamboo has the advantages of low cost,high survival rate,easy management and high stress resistance of seedlings.However,the seed setting rate of moso bamboo is low,and the differentiation of seedlings is serious,which hinders the application of sexual reproduction.Although the predecessors have carried out some work,the mechanism of regulating seed setting rate is not clear.Anther is one of the most important reproductive organs,playing a decisive role in the fertility of plants.The research on the anther fertility can lay an important foundation for improving the breeding of moso bamboo.In this paper,the differential expression level of mi RNA in main reproduction organs was analyzed and the miRNA related to fertility of moso bamboo was identified.Moreover,the regulatroy role of PhemiR159 in anther development was analyzed.A detailed analysis of the bioinformatics and expression patterns of the R2R3 MYB gene family was made,and two important target GAMYB genes of mi R159 were screened.Finally,the the potential function and regulation mechanism of moso bamboo miR159 and target GAMYB genes in development anthers and pollens were preliminary explored.The main results are as follows:1.Mining and indentification of miRNAs and their target genes related to reproductive organs of moso bambooThe small RNA sequencing was carried out using the leaves,stamens,pistils and immature embryos as main materials.A total of 96 known miRNAs and 152 novel miRNAs were identified and 332 targets genes were predicted,including 243 target genes for known miRNAs and 113 targets for novel miRNAs.Some highly conserved mi RNA families,such as miR156 and mi R159 and so on were involved in at least two more reproduction organs development process.In stamens,the targets related to DNA-templated transcription and cell specification process were most enriched,and the most enriched pathway is plant pathogen interaction pathway.Therefore,the main stress response process of pathogen may be involved in anther development of P.edulis.On the basic of degradome sequencing data,this paper optimized the target genes of the conservative miRNAs and identified the targets of the differential expressed miRNAs,such as miR159.The differential expressed known mi RNA and its target genes were selected and analyzed by qRT-PCR,showing that target genes were negatively regulated by corresponding miRNAs,participating in plant development process.2.Effects of PhemiR159 on anther development regulationThe miR159 was selected and two Phe-PremiR159 sequences were acquired and cloned in this paper.Ovexpression vector containing different Phe-PremiR159 genes was transformed in to the Arabidopsis mir159 ab double mutants,respectively.As a result,the phenotype of mutant was successfully restored.Therefore,the miR159 precursor gene of moso bamboo can be correctly processed into miR159 mature sequence in Arabidopsis to play a regulatory role.Combined the results of phenotypic analysis and qRT-PCR,the endogenous AtMYB33 was the main target of PhemiR159 and platy a decisive role in the recovery of mutant phenotypes.The ovexpression of Phe-PremiR159 in wildtype(WT)Arabidopsis could induce abortion in some high-expression transgenic lines.The results of body anatomy and electron scanning showed that the overexpression of Phe-PremiR159 could cause anther faile to dehisce,the mature pollens could not be dispersed and further reduce fertility.Semi-thin section result showed that anther endothelial layer of Phe-PremiR159 overexpressing lines was lack of secondary thickening,resulting in lack of the force required for opening.The expressions of MYB26,NST1,NST2 realted to anther dehiscence in ovexpression lines were significantly verified,indicating the Phe-mi R159 should regulated these secondary thickening related genes expression throught negative regulation of AtMYB33,thus affecting the anther dehiscence.3.MYB gene family and expression pattern analysis in moso bambooGAMYB is the most important target of miR159,and it belong to R2R3 MYB transcription factor(TF).In plant kingdom,R2R3 MYB family functions are diverse and play an important role in the regulation of anther development,the occurrence process of microsporogenesis and the occurrence of male gametophyte.Therefore,a total of 114 R2R3 MYB genes were screened and preliminarily studied,including the foundation of a phylogenetic tree,gene structure and gene evolution analysis.According to the expression pattern in four flower development stages and variety tissues of stamens,pistils,glumes,palea in moso bamboo,the important regulatory MYB genes related to flower development and reproductive organs were identified.As studied by Gao et al.,(2014),the MYB TF might be involved in GA-signaling pathway in moso bamboo flowers.Thus,the GA-stimulated transcriptional family(GAST)was also selected and analysed using RNA-seq data.The co-expression network based on expression pattern showed that the four PheGAST hub genes were connected with 44 PheR2R3 MYB genes with significate positive correlation(PPC > 0.85),and thirty of them were extremely significant positive correlated(PPC > 0.9),including one of the miR159 target genes PheMYB2R-98.It is preliminarily conjectured that PheR2R3 MYBs can participate in the process of flower organs development of P.edulis through the coordination with PheGASTs,integrating GA-pathway and flower development and sexual reproduction process of P.edulis.4.Effects of PheGAMYBs on anther development regulationTwo GAMYBs were identified in moso bamboo(PheMYB2R-42 and PheMYB2R-98)as the main target genes of miR159.They significantly differential expressed in stamens and might be involved in anther development of moso bamboo.The PheMYB2R-42 and PheMYB2R-98 both had the conserved GAMYB structure characteristic and localized in the nucleus.However,the PheMYB2R-98 had no transcriptional activation activity which was differ from PheMYB2R-42.The ovexpression of PheMYB2R-42 and PheMYB2R-98 both resulted in pollen malformation,reduction of pollen activity and germination rate of pollen tube in transgenitic Arabidopsis.In this paper,the PheMYB2R-42 ovexpressed lines were focused and detailed analyzed.qRT-PCR analysis showed that the PheMYB2R-42 overexpression could inhibit the expression of tapetum PCD and sporopollenin synthetise related genes and possiblely down-regulated the upstream early anther development related genes through a negative feedback regulation,thus affected the pollen development and reduced the male gametophyte fertility of transgenic Arabidopsis.Because of the lack of transformation system,the regulatory role of mir159-GAMYB regulation in anthers of moso bamboo can not be verified.In this paper,a purtative downstream target gene of PheMYB2R-42,PheGPAT3 was screened according to the homologous comparison and yeast one-hybrid experiments.It preliminarily speculated that the PheMYB2R-42 could regulate the expression of other anther development genes by regulation of PheGPAT3 under the negative regulation of miR159.Overall,this study identified the known and novel miRNAs in reproduction organs of P.edulis,mining the “miRNA-target” regulation pathway related to the fertility of moso bamboo.the GAMYB targets of miR159 were indentified from R2R3 MYB gene family.The functional roles of “miR159-GAMYB” pathway involved in anther and pollen development were preliminarily verified.This paper will provide experimental basis for the study of anther development and sexual reproduction,and make some contributions to the study of genetics breeding and germplasm resources research in moso bamboo... |
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