Function Of The Three Conserved Cysteine In GhABP1

Auxins are plant growth hormones that cause a lot of plant developmental processes,including embryogenesis,vascular differentiation,organogenesis,tropic growth,and root and shoot architecture. AUXIN BINDING PROTEIN 1（ABP1） binds auxin at physiologically relevant levels,that it can discriminate between physiologically active and inactive forms of auxin.Therefore,we took a transgenic approach to examine the function of three cysteines and C-terminal of the GhABP1 in tobacco.The pattern of GhABP1 gene expression during the fiber development were also investigated.The main results are as follows.1.The sequences analysis of GhABP1The cDNA sequence of GhABP1 is 630 bp in size and encoded a polypeptide of 191 amino acid residues with significant homology to the ABP1 in other plant.After removed the 24 amino-acid residues signal peptide at N- terminal,the three conservative cysteines residues locate on the site of 3,63,157 in sequence respectively.These cysteine might be sensitively to the structure of GhABP1.2.GhABP1 show auxin-specific response in tobaccoInterveinal strips excised from growing transgenic tobacco leaves,which GhABP1 were drived by CaMV35S promoter,have stronger auxin-specific,epinastic growth response treated with 10μm NAA than that from wildtype.The function of ABP1 is conserved.Without being treated by 10μm NAA,the curvature of GhABP1 overexpressed plant leaf tissue is similar with wildtype.It suggests that ABP1 is not sensitive to auxin of low concentration.3.The function of theα-helix in C-terminal of GhABP1To investigate the function of theα-helix in C-terminal of GhABP1,the GhABP1-NC without C-terminal were introduced into tobacco drived by CaMV35S promoter.The same curvature of transgenic and wildtype strips treated with NAA were observed and the transgenic tobacco showed normal morphology and growth.It suggested that the GhABP1 would lost its function after removing theα-helix in C-terminal,furthermore GhABP1-NC didn’t show the competing reaction to the docking site with wildtype ABP1.4.The function of three cysteine of GhABP1To confirm the function of three conservative cysteines in GhABP1,the GhABP1-C3S GhABP1-C63S and GhABP1-C157S mutant were obtained via converting cysteine into serine respectively.The leaves from the transgenic tobacco overexpressed with GhABP1-C3S and GhABP1-c63s showed the significant same auxin-specific response compared with that of overexpressed GhABP1 after treated with NAA.But the reaction of epinastic growth overexpressed GhABP1-c157s was not observed.It demonstrated that the GhABP1 would lost its function after mutanted the 157^th cysteine to serine.By contraries after mutanted the 3^rd or 63^rd cysteine to serine, the GhABP1 will hold its epinastic growth.But,it is interesting that there are different observation between the GhABP1-C3S and GhABP1-C63S overexpressed plants.Without treated by 10μM auxin,the curvature measured in epinastic growth test of ABP1-C63S is much larger than anyone else.But,the curvature of the leaf strips form GhABP1-C3S overexpressed plants is the same as wildtype.It conclude that,3^rd cysteine is not enssential in the function of GhABP1.But,mutating the 63^rd cysteine will makes GhABP1-C63S be easy to enter active phase,even in low concentration of NAA.5.Effection of highly active GhABP1 in plantsABP1-C63S overexpressed plants show high activity with low level of NAA.And the plants are very different from the wildtype plants.They have smaller size,slower development.And the leaf is much different from normal leaf.The leaf is much smaller in size,with tough and less luster surface.It suggests that GhABP1 play an important role in development of plant treated by excess auxin. Highly active GhABP1 may effects the cell cycle of plants,slow the development and apolexis of plants.The GhABP1-C63S overexpressed plants may act as model on research in the field of function of ABP1.6.relationship between the ABP1 and development of cotton fiber.We found that in the period between 9DPA and 20DPA,when cotton fiber elongating fast,the cotton fiber has the highest expression level of GhABP1.It suggest that GhABP1 may involve in the development of cotton fiber.