Functional Analysis Of A Villin Gene In Cotton

Cotton fibers are single-celled seed trichomes that originate from the outer integuments of the ovule and undergo four distinct but overlapping developmental steps including initiation,elongation,secondary wall deposition,and maturation.During this process,fiber initials elongate by a factor of 1000 to 3000.It is well-known that microfilament cytoskeleton dynamic change provide axial orientation,framework support,substance transport and signal transduction for fast cell expansion.Therefore,microfilaments cytoskeleton is one of the most important factors affecting cotton fiber development.There have been more than 160 actin binding proteins(ABP),which can bind actin monomers,or polymers,or both,to be involved in actin's polymerization,depolymerization,stability,organisation in bundles or networks,fragmentation and destruction,thereby to regulate plant development and stress tolerance.The regulatory roles of some plant ABP,scuh as actin actin depolymerizing factor(ADF),profilin,fimbrin,etc.in microfilament cytoskeleton have been reported.Recently,a novel palnt ABP of villin has been found to play regulatory roles in plant cell elongation and morphogenesis in Arabidopsis,rice,and tobacco,but liitle is known of that for cotton.In a differential proteome analysis between the fibers of 4 and 8 days postanthesis(DPA)from a lintless cotton fiber developmental mutant of Li-1 and its isogenetic wild type of TM-1(Texas Marker-1)conducted in our lab several years ago,a cotton villin protein was found to down-regulated in Li-1,and the encoding cDNA was molecularly cloned and nominated as GhVLN.GhVLN was transformed into fission yeast,and the results showed that overexpression of GhVLN can significantly increase length and length/width ratio of yeast cells,therefore,cotton villin was proposed to be in volved in cell elongation that possibly play regulatory roles in cotton fiber elongation.The present study was performed to verify the effects of GhVLN on the cytoskeleton and development of cotton fiber,and main results are as follow:1.The GhVLN was inserted into the plant expression vector of pBI121 driven by the cauliflower mosaic virus(CaMV)35S promoter,and subsequently transformed into Arabidopsis by using floral-dip method.5 positive transgenic GhVLN Arabidopsis plants showing normal plant phenotypes were obtained by kanamycin resistance screening and PCR validation.The phenotype of the plants of the GhVLN transgenic Arabidopsis was optically inspected.GhVLN transgenic Arabidopsis plants exhibited increased length and number of roots and root hairs.The results indicated that GhVLN significantly promoted root and root hair growth and development.In addition,under 150mmol/L NaCl treatment,the inhibition rate of root growth and development was significantly alleviated in GhVLN-overepressed Arabidopsis plants commpared to that in Col0 Wild type,suggesting that GhVLN can improve plant salt tolerance.2.A recombinant plant overexpression vector of PBI121-GhVLN was constructed and transformed into cotton by using agrobacterium-mediated method.The growth and development of callus at different stages were documented.The transgenic cotton T0 generation positive regenerated plants seedlings have been obtained and the positive seedlings cuttings have been grafted onto sea island cotton stems,this work will be helpful for further studies on the molecular mechanisms and roles of microfilament cytoskeleton and GhVLN in regulation of cotton fiber development.3.The 6×His Tagged GhVLN was cloned into the pET30a prokaryotic expression vector.GhVLN was higely expressed under 0.5mM IPTG induction.The molecular weight the expressed proteins was about 110kD determined by SDS-PAGE analysis,which is consistent to the theoretically calculated value.The induction condition of GhVLN protein was optimized to obtain high concentration of soluble GhVLN protein.The recombinant protein was purified with Ni-NTA affinity chromatography resin.GhVLN was able to bundle F-actin in vitro.