The Mechanism Of Ethylene Function During Mulberry Female Formed

Flower sex regulation has alaways been the focus of attention of biologists. It’s different to flower sex requirements for development of mulberry varieties regarding to the different uses, for example, to get the fruit to eat, to get the leaf to silkworm or to beautify and protect the environment. In the traditional way, the new mulberry germplasm resources were bred by the method of selecting the superior traits of the plant to carry out the asexual reproduction. Exploring different flowers of mulberry decision mechanism contributes to the development of molecular breeding. Therefore, the study of the sex regulation of mulberry flower has greattheoretical economic value.Ethylene gas is an important plant hormone, widely being involved in the regulation of plant growth, development and response to an external signal, such as seed germination, seedling development, ripening, leaf senescence, resistance to biotic and abiotic stresses, differentiation of flowers. Ethylene as sex hormone regulates the sex differentiation of plants, Two sex determination loci (F, M) of cucumber were identified as the rate limiting enzymes ACS family genes of ethylene biosynthesis.The sex of mulberry flowers is very complex, such as female, male, hermaphrodite, hermaphroditic. Genetic factors have an mainly influence on the sex of mulberry flower. Plant hormones, in addition, also play an important role in the sex regulation of mulberry flowers. Partial male plants treated with ethephon can induce more female, while gibberellin can induce the formation of male flowers. Detecting gene expression of mulberry flower bud development process will contribute to study the relationship of ethylene and female flowers and provide rich reasearch data for screening the sex determining genes and construction the genotype of dioecious breeding materials. These will accelerate the breeding rate.First, we identified ethylene synthesis and signaling related genes in mulberry by the method of biological information analysis. Then we analyzed the gene structure, conserved domains and phylogenetic relationships. Six different tissues (root, bark, leaves, male flowers, female flowers, fruit) were used to detect the expression pattern of these genes. We selected Zhenzhubai cultivation mulberry, which open female flowers, as experimental material. Morphological changes during the flower buds development were observed by paraffin section method. Six consecutive developmental time point of flower buds were chosen to transcriptome sequencing. We did several analysis in many aspect. The differentially expressed genes were identified. Then we clustered these genes by hierarchical method and did KEGG significantly enrich analysis. Hormone-related genes, AP2/ERF genes and MADS-box genes were identified. We also did a co-expression analysis. The expression location of a gene code the ethylene rate-limiting enzyme were detected by in situ hybridization. Expression changes of AP2/ERF genes were detected in flower buds by treating AVG and ethephon. The results obtained in this studv are as follows:1. Identification and bioinformatics analysis of ethylene biosynthesis and signal transduction related genes in MulberryWe identified ethylene biosynthesis and signal transduction related gene in mulberry by the method of bioinformatics analysis. Total 29 ethylene-related genes and 116 AP2/ERF genes were identified. MnEIN3 and MnEIL1, MnEIL4 and MnEIL5 were all located on the same scaffold in the mulberry genome. The gene structure, conserved domains and phylogenetic relationships of four family (ACS, ACO, ETR and EIN3) genes were analyzed. All the genes had conserved gene structure, except MnEIL4 which had an intron. MnACS family genes all contain conserved functional motifs, except for MnACS5, which lacked the corresponding phosphorylation sites due to the absence of C-terminal. MnACO family proteins all contain conserved functional motifs. MnEIN4 contained a signal peptide structure at the N-terminal, while MnERS1 was missing a signal receiving domain at the end of the C-terminal. MnEIN3 and MnEILl contained two conserved regions found in the mung bean gene, which were rich glutamic acid and aspartate acid. Six different tissues (root, bark, leaves, male flowers, female flowers, fruit) were used to detect the expression pattern of these genes. We found some high expression gene and tissue-specific expression genes, MnACS5 displayed specific expression in female flowers. MnACO1 and MnAC02 were specifically expressed in the fruit, MnEIL1 and MnEIN3 showed higher expression in root and fruit。According to the number of AP2 domains and their structural features, AP2/ERF proteins were divided into five subfamilies. There were 58 genes in the ERF subfamily,33 genes in the DREB subfamily,21 genes in the AP2 subfamily,3 genes in the RAV subfamily, and 1 gene in the Soloist subfamily. Phylogenetic analysis revealed all proteins were separated into 15 groups. Groups I-IV represent the DREB subfamily, and V-X represent the ERF subfamily. There were 11 genes in the V group of mulberry and 5 genes in the V group of Arabidopsis. There are three types of EAR motifs in mulberry plants, DLNXXP, LXLXL, and LDLNLXPP. Sequence analyses revealed that 19 genes that bear LXLXL motif found in mulberry proteins, the MnERF subfamily genes containing EAR motifs were mainly concentrated in the MnERF-B 1 subgroup. These genes had different expression patterns. Several genes were also found to be expressed in a tissue-biased manner. For example, MnERF-B3’-21 was expressed solely in male flowers. MnDREB-A4-7 was more abundantly expressed in leaves than in other tissues. MnAP2-5 had high levels of expression in male flowers. However, the expression of 9 genes was not detectable in any tissue.2. Morphological changes and transcriptional analysis of early flower bud development in MulberryWe selected Zhenzhubai cultivation mulberry, which open female flowers, as experimental material. Morphological changes during the flower buds development were observed by Paraffin section method. Six consecutive developmental time point of flower buds, here named as FB1-FB6, were chosen to transcriptome sequencing. A total of 118584 contigs and 87719 unigenes were identified. The mean length was 381bp, the lagest length was 16738 bp, N50 was 1307 bp and N90 was 282 bp in unigenes. Functional annotation showed 45.44% gene can be identified at least in a database. In the NR database obtained the most annotation genes (39.64%), only 6.78% of the genes in all databases can get comments.35536 (40.5%) and 26726 (30.4%) sequences were predicted to encode proteins by blast and estscan, respectively. We compared the transcriptome at a time point against its predecessor as type 1 DEGs and compare a time point against FB1 as type 2 DEGs. They have 808,1037 different expression genes respectively. Cluster analysis showed that most of the differentially expressed genes were up-regulated in FB2 and FB3, and then were down regulated. We speculate that in this period,Zhenzhubai flower buds has changed dramatically.KEGG enrichment analysis showed that between FB1 and FB2 differential genes were significantly enriched in plant hormone signal transduction, phosphatidylinositol, signaling system, alpha-Linolenic acid metabolism and inositol phosphate metabolism and Flavonoid biosynthesis. The differential genes between FB3 and FB4 were also enriched in the four pathways except for flaconoid biosynthesis. The KEGG pathway of the plant hormone pathway showed that the EBF1/2 genes of ethylene signaling pathway was down regulated between FB 1 and FB2. It is suggested that the EIN3 transcript may be accumulated, and its’downstream of the corresponding factors were activated.We extracted the FPKM of hormone related genes and detected the changes of expression. MaACS2 gene encode ethylene biosynthesis rate limiting enzyme was increased expression in FB2. Several Auxin genes also had high expression in FB2. The brassinosteroid genes were mainly expressed in FB3-FB4. The genes of gibberellin were mainly expressed in FB4-FB6. While, the genes of abscisic acid were mainly expressed in FB6.Further, AP2/ERF transcription factor family genes and MADS box genes were identified. They exhibited different patterns of expression. Some of AP2/ERF genes expressed higher in FB2 and FB3. However, nearly half of MADS-box genes’expression were gradually increased over time. We used these two group genes as the characteristic expression pattern to analyze the co-expression patterns with all the genes with FPKM value over five at one or more of the time points. All the genes were divided into 27 expression patterns, as P1-P27. P19 corresponds to AP2/ERF genes and P24 corresponds to MADS-box genes. Co-expression network showed that there were more than 200 genes in P19 showed strong correlation. P24 showed a relatively loose structure. More than 40 links were detected for one gene, there were 2 genes have 20-40 links with other genes and 10 genes had 10-20 links with other genes. These results suggested that these genes in the development of flower bud may play an important role in different aspects.3. Functional analysis of MaACS2 and 4 AP2/ERF genes in ZhenzhubaiThe expression location of MaACS2 in the flower bud was detected by in situ hybridization. The results showed that the peripheral of the inflorescence could be able to observe significant signal. Morphological data showed that flower organ began to differentiate in this stage. It is speculate unisexual flower formation in Zhenzhubai belongs to the first flower sex differentiation model, bisexual primordium initiation in early development of floral organ, then stamen primordia development is inhibited, the process may be the results ethylene were formation in the early stage of floral organ differentiation. TENUL detection showed a significant green fluorescence signal on the inside of the calyx, it’s suggest that ethylene inhibits the development of stamens by causing apoptosis.The flower buds at FB2 period were treated by AVG and ethephon. The expression of AP2/ERF genes were detected. The expression of MaDREB-A1 was significantly increased at seventh day by treating Ethephon. However, the expression of related genes was not consistent with previous studies after AVG treatment. Heterologous expression of AP2/ERF genes in tobacco resulted in abnormal development of tobacco flowers. Petals hypoplasia, prominent pistil, the number of stamens were reduced and some of stamens were abnormal development.In this study, we identified 29 ethylene biosynthesis and signal transduction related genes in mulberry by bioinformatics methods. Sequence analysis showed that it is highly conserved in the sequence structure, conservative structure domain and phylogenetic. It’s suggested that these genes may play the same function with their homologs. Morphological changes of early stage of flower bud development and the analysis of the transcriptional showed that there were a large number of differentially expressed genes during flower bud differentiation. The expression of MnACS2 increased significantly in FB2 and the expression of several auxin genes also were increased. This suggested that ethylene in female flower development play a certain role. In situ hybridization of MaACS2 showed that it location in inflorescence periphery. TENUL detection displayed apoptosis occurs in the inner of the calyx. The flowers of transgenic tobacco had abnormal development. These results implied Zhenzhubai female flower development process, ethylene is likely by causing apoptosis inhibiting stamens development and promote pistil development.