| Cotton is an important crop in China and it is beneficial to the lives of people. With the development of industry and the improvement of human living standard, industry gradually need high quality textile. Cotton fiber is composed of ovule epidermal differentiation and unbranched single cell hair. The number of projections and cotton fiber length associate with the cotton quality and production efficiency. It is very important to provide the ways to know the molecular mechanism of cotton fiber development. It is significant for improving cotton fiber quality through cloning the gene in the development of cotton fiber.Brassinolide is an important plant hormone. It plays an important role in initiation and elongation of cotton. In order to study the molecular mechanism in brassinosteroid signaling pathway of cotton fiber, we analysised that the interaction between Gh14-3-3and its target protein BZR1. We also studied that the BR signaling pathway had function in regulating the development of cotton fiber. The main results are as follows:1. Identification of Gh14-3-3h gene and its expression profiling in cotton Seven cotton14-3-3cDNA in the lab had been isolated in previously study. We cloned a14-3-3gene which was named Gh14-3-3h from the yeast two-hybrid cDNA library of cotton fiber. We also cloned the gene from the cotton genome. The structure of Gh14-3-3h gene contained six exons and five introns, belonging to the epsilon type14-3-3proteins. Gh14-3-3h with other species of the14-3-3protein amino acid had homology of61%-92%. In addition, Gh14-3-3h had the14-3-3typical structure characteristics and nine conservative alpha helixes. But its N and C terminals were not conserved.Fluorescence quantitative PCR was used to analyze the expression of Gh14-3-3h in cotton organizations. We found the expression of Gh14-3-3h in the fiber was high. However, the expression level of the gene in other organizations was low. Gh14-3-3h gene had high expression level during the early stage of fiber (3-10DPA) and decreased during fiber cell secondary wall development. In addition, the expression level of Gh14-3-3h was up-regulated by addition of BL. These results indicated that Gh14-3-3h might play a role in regulating the initiation and elongation of cotton fiber cell. Moreover, Gh14-3-3might involve in the response to BR signaling pathway.2. Screening the interaction partners of Gh14-3-3proteinsTo study Gh14-3-3involved in regulating the signaling pathway during the growth and development of cotton fiber, we analyzed the interactions between Gh14-3-3f/g/h proteins and their partners by the yeast two-hybrid system. We achieved a total of32kinds of proteins. They were divided into six categories, containing cotton14-3-3proteins, hormones related proteins, metabolic related proteins, signaling related proteins, proteins involved in growth and development, and unknown proteins. We analyzed the sequence of these proteins in the cotton EST database and we found that most of these interaction proteins were presented in cotton fiber. These results indicated that Gh14-3-3proteins might interact with these proteins in the fiber cells to co-regulate the growth and development of cotton fiber.14-3-3is highly conserved protein in eukaryotes, and it usually interacts with other proteins through homogeneous or heterogeneous dimer. In the14-3-3target proteins, we found that2.8%proteins were the14-3-3proteins. It indicated that the14-3-3proteins could form the homogeneous or heterogeneous dimer. Moreover, we checked the interaction between the Ghl4-3-3h and five other Ghl4-3-3s. We found that it had specific interactions between them, and the six Ghl4-3-3s were in the form of heterologous dimers. We speculated that14-3-3played a role as heterodimer to participate in the regulation of cotton fiber elongation.3. Analysis of the interaction of GhBZRl and Gh14-3-3proteins We cloned a target protein of Ghl4-3-3s by yeast two-hybrid. It had high homology comparing with Arabidopsis BZR1and it was named GhBZRl. Analysis of cotton BZR1protein, we found that it had22probable BIN2phosphorylation sites and the PEST area. It also had a motif of interaction with the14-3-3. In addition, GhBZRl located in the cytoplasm and nucleus. When addition of BL, a large number of GhBZRl-GFP proteins accumulated in the nucleus.To study the influence of phosphorylation to the interaction between GhBZR1and Ghl4-3-3protein, we cloned the cotton BIN2gene. The gene expression pattern was the same as expression pattern of GhBZR1. It had the preferential expression in elongation stage of cotton fiber. We found GhBIN2could interact with GhBZRl protein by the experiments of yeast two-hybrid and pull down. GhBIN2phosphorylated GhBZR1in vitro and phosphorylation GhBZRl could interact with Gh14-3-3protein. In addition, GhBZRl interacted with Gh14-3-3protein by conservative Mode I motif and phosphorylation of S (163) was important to the interaction between them. It indicated that GhBIN2phosphorylated GhBZR1to promote the interaction between GhBZR1and Gh14-3-3proteins and the response played an important role in regulating elongation of cotton fiber.4. Analysis of the interaction of GhMYB73and Gh14-3-3proteinsIn the yeast two-hybrid system, it had an interaction partner of Ghl4-3-3.The amino acid of GhMYB73had homology with Arabidopsis thaliana AtMYB73and it was named GhMYB73. The expression of GhMYB73had the highest amount in elongation of cotton fiber and subcellular localization was in the nucleus. GhMYB73interacted with the transcription factor AtBES1in BR pathway of Arabidopsis thaliana. The results indicated that GhMYB73might interact with AtBESl to participate in BR signaling pathway.GhBIN2could interact with GhMYB73in yeast two-hybrid and pull down experiments. GhBIN2could phosphorylate GhMYB73in vitro. In addition, phosphorylated GhMYB73could interact with Gh14-3-3. It indicated that GhMYB73might involve in14-3-3regulating the BR signaling pathway of fiber development.5. Gh14-3-3proteins function in fiber cell development of cottonTo study the regulation of Gh14-3-3proteins in the differentiation and development of cotton fiber cell, we constructed the vectors of Gh14-3-3L/e/h RNAi and Gh14-3-3L overexpression.When the vectors were introduced into cotton by Agrobacterium-mediated transformation, TO generation and their later generations T1-T4were obtained. Analysis of transgenic cotton (T1-T4generation), the number and length of cotton fiber initiation significantly suppressed in35S promoter driven Gh14-3-3L/e/h RNAi transgenic plants. The number of Gh14-3-3L RNAi fiber decreased and the length got shorter. The length of mature fiber in T1-T4generation Gh14-3-3L/e/h RNAi was shorter than wild type, while the length of cotton fiber in overexpression Gh14-3-3L was longer than wild type. It indicated that Gh14-3-3had the important function in the development of cotton fiber.In vitro culture of fiber, the length and TFU of Gh14-3-3L RNAi decreased comparing to wild type. But overexpression Gh14-3-3L increased. When applying with BL, Gh14-3-3L RNAi fiber length and TFU increased compared with in normal condition, but they had not recovered to wild type levels. Expression of GhCPD in Gh14-3-3L RNAi in cotton fiber and the expression level GhDWF4decreased significantly. But expression of GhCPD in overexpression plants was no significant difference and the expression of GhDWF4was up-regulated comparing with wild type. After BL treatment, only GhDWF4expression level was slightly down-regulated in Gh14-3-3L RNAi transgenic plants, compared with control (without BL). But the expression of two cotton BR synthesis genes in overexpression Gh14-3-3L comparing with wild type were rapidly down-regulated. Above results showed that Gh14-3-3might involve in BR signal feedback regulation in the cotton fiber elongation stage and it affect cotton fiber elongation and TFU production.To study the relationship between Gh14-3-3transgenic cotton fibers phenotype and brassinosteroid signal transduction, we studied the genes associated with BR signal transduction and cotton fiber initiation and elongation related gene expression in transgenic cotton fiber. The results showed that GhBZR1, GhXTH1, GhEXP, GhTUB1and GhCesA5in Gh14-3-3L/e/h RNAi transgenic cotton fiber and Gh14-3-3L overexpression of cotton fiber had different degree of changes in the expression. Analysis of T1-T4generation, the expression of these genes were changed in different generations and the hereditary was stable. The results indicated that Gh14-3-3proteins participated in regulating BR signaling pathways to influence the initiation and elongation of cotton fibers through regulating the expression levels of these genes. |
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