Complementation Of Arabidopsis Mutant And Influence On Cotton Bolls Development By Gossypium Hirsutum GhTIR1

Auxin is an important phytohormone, and it can regulate the whole growth and development process by auxin signaling transduction pathway. TIR1receptor as a vital member of this pathway, perceives and transmits auxin signals, and influences the expression of genes downstream, so as to affect the physiological status of plants. A cotton boll, one of the primary propagative organs, mainly composed of seeds and fiber that both show important values in scientific researches and economic aspects. So, there is great significance in studying the influence of TIR1on bolls development.In order to get a more knowledge about cotton auxin receptor gene GhTIRl, we analyzed by experiments as complementation of the Arabidopsis tirl-1mutant by GhTIRl, and expression characteristics analysis of GhTIRl promoter. Besides, we screened and analyzed some relative economical traits of T3generation transgenic cotton plants that up and down-regulated the expression level of GhTIRl driven by Cauliflower mosaic virus promoter (CaMV35S) and/or by seed-specific promoter (PV). The main results were as followed:1. Complementation of Arabidopsis tirl-1mutant by GhTIR1The constructed plant expression vector p5-35S::senseGhTIR1were transformed into Arabidopsis tir1-1mutant by Agrobacterium-mediated method, then by DNA extraction and PCR tests, the T1generation of transgenic plants were obtained. We found that lateral roots number of transgenic plants increased to a level similar to wild-type plants’. When treated with0.085μM2,4-D, the primary roots of the former ones reduced to a length as that of the latter ones. The results above suggested that when GhTIRl expressed in tirl-1mutant, the phenotype of which could be restored. Besides, TIR1from Gossypium hirsutum had the similar functions with that from Arabidopsis thaliana.2.Expression characteristics of GhTIRl promoterTo understand better the functions of GhTIRl, a pair of specific primers was designed in order to clone a fragment (PGhT1R1) of1.9kb in the upstream regulatory region of this gene. Followed, the constructed plant expression vector pBI-PGhT1R1::GUS was transformed into Arabidopsis and transgenic plants were obtained, both with the same methods as above. When analyzed the expression characteristics of this promoter, we found that uidA reportor gene drived by PGhT1R1was expressed mainly in osculums of rosette leaves and cauline leaves, petiolar vascular bundles, as well as petal, stem and lateral roots. But in leaves of different developmental phases and locations, there were some differences. For example, the expression of uidA was evident in osculums of leaves, while in the other parts weaker and incontinuous, and sometimes showed in a form of dots. Another difference was that GUS signal could’t be detected in rosette leaves until the cauline leaves appeared. Interestingly, GUS signal also showed in epidermal hairs of young cauline leaves.3. Influence on cotton bolls by up and down-regulated the expression level of GhTIRlWith the segregated non-transgenic plants as control, we selected four transgenic plant lines from the T2generation transgenic cotton plants whose the expression levels of GhTIRl were significantly up-regulated with the gene driven by CaMV35S promoter, and another four transgenic plant lines that the expression levels of GhTIRl, driven by CaMV35S promoter or PV promoter, were sharply down-regulated. Analysis of free IAA content in ovules at ODPA by LC-MS indicated a decline in the level of endogenous IAA in T3generation GhT1R1overexpression plant lines, while a increase in suppression GhTIRl expression plant lines. Meanwhile, we found that seeds of per boll of the overexpressing GhTIRl transgenic plants increased, on the contrary, when antisense suppressed, decreased.