| Cotton is one of the most important economic crops. The research of cotton somatic embryogenesis (SE) is a basic for biotechnology applied in breeding. Meanwhile, as a typical example of cell totipotency, Plant SE is a good model for life research. To study the mechanism of this process in depth, improving the cotton SE system effectively were valuable for research and application of cotton breeding.In order to find the crucial genes in cotton SE, four members of Gossypium hirsutum high mobility group protein (GhHmgB) family were identified. The embryo-specially expressed gene GhHmgB3 was selected for further research. The main results of our study were as follow:1. Four GhHmgB genes were identified using the RACE PCR technology. The expression patterns of these genes were determined by real-time PCR in root, stem, leaf, zygotic embryos and somatic embryos. The result showed that they could be divided into three subgroups. Referred to the naming convention in mouse, we named the two ubiquitously expressed genes as GhHmgBl (HM449081) and GhHmgB2 (HM449078), the embryo-preferentially expressed gene as GhHmgB3 (HM449079) and the GhHmgB4 (HM449080) for the remained one which had a high expression level in non-embryo tissues. The GhHmgB3 was selected for functional research.2. Sequence analysis showed the novel protein had a high conserved Hmg-box domain followed by a 36-amino acids acidic tail. The detail expression pattern of GhHmgB3 during embryo development was examined by Real-time PCR and Northern blotting. It expressed preferentially in SE calli and zygotic cotyledon embryos.3. A 184-bp (207-391) region containing a part of the GhHmg-box domain and acids acidic tail was used for RNAi vector construction. The vector was transformed to the hypocotyls and embryo callus of Coker 201 respectively via Agrobacterium tumefaciens. Isolation and identification the RNAi cell lines for further research.4. In different stages of cotton SE, the phenotypes of dedifferentiation and differentiation were recorded. The GhHmgB3-deficient somatic cells of hypocotyls dedifferentiated more vigorously than the control cells, but they failed to differentiate to ECs. In another case, the proliferation and differentiation of GhHmgB3-deficient ECs were significantly improved, but failed to form plantlets.5. The different expression genes between the control and GhHmgB3-deficient ECs were identified by Solexa sequencing technology. The bioinformatics analysis and real-time PCR verification of some typical genes revealed series ofβ-catenin-related signaling during cotton SE. First, GhHmgB3 inducedβ-catenin expression to promote the proliferation. The ectopic high-level growth factorβ-catenin induced the stress-induced MAPK/p38 pathway, which regulates the canonical TCF/LEF pathway negatively leading to the lack of growth. On the other hand, moreβ-catenin proteins were biased in favor ofβ-catenin/Actin interaction that depended on calcium, which promoted the differentiation.6. The cell cycle analysis were performed on control and GhHmgB3-deficient ECs after treatment with 5-fluorouracil to test the cell cycle progress. The results reflected the fast cell cycle progress of GhHmg-B3-deficient ECs. Meanwhile, the result of MTS assay suggested the high proliferation of GhHmgB3-deficient ECs.7. Referred to the expression pattern of someβ-catenin downstream genes, cell cycle analysis were performed on the ECs in normal culture. The increased numbers of GhHmgB3-deficient ECs arrested at the Gi phase compared with controls in cell cycle analysis suggested that the nuclei signal ofβ-catenin were repressed.8. The expression patterns of the cytoskeletalβ-catenin-related genes were examined by real-time PCR. The higher expression level of Actin10 and SERK in GhHmgB3-deficient ECs suggested a high differential ability. This result was confirmed by TEM scanning of control and GhHmgB3-deficient ECs under normal culture in the similar development stage.In summary, the GhHmgB3 plays an important role during the cotton somatic embryogenesis.
|
PDF Full Text Request