Cloning And Characterization Of Three Genes Related With Auxin Signal Transduction In Gossypium Hirsutum L.

Since cultivated widely, cotton plays an important role in the national economy and people's lives.The phytohormone, auxin, is central to myriad of aspects of plant growth, developmental and physiological processes. Auxin regulates cell division, extension and differentiation, apical dominance, adventitious root formation, tropisms, fruit development, embryogenesis and so on. So, it is significant to do research in depth on auxin signal transduction and find out the critical genes. Discovering the functions of genes involved in auxin signal transduction in cotton, would elaborate role of auxin in cotton development and morphology formation, and illustrate the molecular mechanism of auxin signaling. Auxin activates expression of numerous genes whose products probably perform most developmental responses. Most of these genes are primary/early response genes, and they are activated rapidly after auxin treatment and protein synthesis is not required for their activation. Primary/early response genes include Aux/IAAs (auxin/indole acetic acids proteins), SAURs (small auxin-up RNAs) and GH3 genen families.The filter arrays of cDNAs were performed to identify the differentially expressed genes between the Upland cotton cultivar TM-1 and the Sea island cotton cultivar Hai7124 in the early developing stages of 5DPA,10DPA,15DPA,20DPA, and 25DPA. Three critical genes related to the auxin signal transduction in cotton were isolated, and they have been verified by Q-PCR.(1) Cotton auxin-binding protein\(GhABP, GenBank Accession number:AAO92740), the insert fragment of the cDNA clone was 897bp, its open reading frame was 618bp, and encoded a polypeptide containing 206 amino acids; (2) Two primary auxin responsive genes, one was auxin/indoleacetic acid protein, whose cDNA sequence obtained 1014bp, and its open reading frame was 726bp, and encoded a polypeptide containing 242 amino acids. Blastx analysis indicated this gene had homologies with the IAA16 genes of Arabidopsis thaliana, Ricinus communis and Ipomoea nil, so this gene was designated as GhIAA16a; the other cDNA clone was small auxin-up RNA(SAUR), its insert fragment was 794bp, its open reading frame was 366bp, and encoded a polypeptide containing 122 amino acids. Blastx analysis indicated this gene had identity with SAURs of Arabidopsis thaliana and Zea mays, so designated this gene as GhSA UR2.We predicted their signal peptide via signalP process. GhABP had typical signal peptide but GhIAA16a and GhSAUR2 did not. We also analyzed their conserved domain.The three genes have been verified by QRT-PCR, and the results were in accordance with the cDNA microarray. The expression of GhABP was opposite to the GhIAA16a expression, both in TM-1 and Hai7124. GhABP was preferentially expressed in elongating fiber cells of 10 DPA while GhIAA16a had the weakest expression. After 10DPA, the expression of GhABP gradually decreased but the expression of GhIAA16a decreased on the contrary. It implied that GhABP may promote the cotton fible development, while GhIAA16a may be related to the initiation of the cotton fiber.We have used the BC1 mapping population derived from the hybridization between the Upland cotton cultivar TM-1 and the Sea island cotton cultivar Hai7124, using TM-1 as recurrent parent. GhABP and GhIAA16a were localized on the chromosome 11, while GhSAUR2 was located on chromosome 8.The genes fusion expression vector of GhABP, GhIAA16a and GhSAUR2 were constructed with GFP respectively.Using gene gun bombardment transformation method to bombard the onion epidermis.The subcellular localization revealed GhABP gene was located in the cell membrane, while GhIAA16a was located in the karyon.Sense expression vector containing 35S promoter using pBI121 plasmid were constructed for GhABP, GhIAA16a and GhSAUR2.These sense expression vectors will be transferred into Gossypium hirsutum L.