Expression And Purification Of Two LBD Transcription Factors And Their DNA-binding Domains

LBD (Lateral Organ Boundaries Domain) genes defined a novel class of plant-specifictranscription factors. LBD proteins contain a conserved LOB domain in the N-terminus.According to sequence similarity, phylogenetic analyses subdivide the LBD family into twoclasses: class I and class II. Research results show that class I members play a crucial role indefining organ boundaries and controling initiating of lateral organs and are involved inalmost all aspects of plant development, including root, leaf, inflorescence, embryo anddevelopment. class II members are involved in the regulation of anthocyanin biosynthesis andnitrogen metabolism.Crop production is directly influenced by panicle morphological structure, thus to clarifythe morphogenesis mechanisms of panicle is crucial for crop genetic improvement. Erik etal.(2005) found that the mutation of a maize class I LBD gene ramosa2would resulted tostrongly branched spike in ears and tassels. In recent years, Our research group has focusedon genetic and molecular biology study on wheat branched-spike, and we have cloned thehomologous gene of ramosa2from Triticum turgidum L (TtRa2), which is speculated to beinvolved in spike morphology controlling. To further clarify interaction mechanism betweenLBD family transcription factors and target DNA, we used TtRa2and AtLBD37as therepresentatives of class I and class II LBD members, and established an efficient expressionand purification system for them and their DNA binding domains. It will facilitate the furtherstructural and functional research on LBD family tanscription factors. Based on the early genemapping results of wheat branch-spike, we developed new molecular markers and laid afoundation for fine mapping and map-based cloning of wheat branch-spike genes. The resultsobtained in this paper are as follows:1. Using leaf total RNA of Arabidopsis thaliana as template, the full coding region ofclass Ⅱ LBD geneAtLBD37(753bp) was cloned by RT-PCR, and then its DNA bindingdomain AtLBD37-BD（1-431bp） was cloned. Using classⅠ L BD geneTtRa2codingsequence （774bp）as template, its DNA binding domain TtRa2-BD（1-450bp）was obtainedtoo.2. Based on expression vecotor pET21a, we designed and constructed a pHMT vecotor,which successively contains the coding sequence a His-tag, a MBP and a TEV protease site in upstream of target gene. Finally we obtained four fusion expression vector by insertingAtLBD37, TtRa2, AtLBD37-BD and TtRa2-BD into pHMT respectively.3. The expression conditions for fusion protein were optimized. The optimum inducingtemperature determined ultimately was28℃. Under this temperature, quantity of targetprotein is very high and almost all exist in a soluble form. The optimum inducing time is5h,at this time the quantity of recombinant protein is the best and the amount of miscellaneousprotein is the least.4. TtRa2, AtLBD37and their DNA-binding domains were expressed efficiently, they allexisted as suluble form and accounted for about50~70%of total protein. Before the targetprotein was obtained fusion protein was purified by amylose affinity column, His6-MBPdouble tags were cut off by TEV protease, then TEV and double tags were removed byNi-NTA affinity chromatography. Following above process, we could get15mg TtRa2-BDwith98%purity from a liter of culture.5. To mapping branched-spike genes in common hexaploid wheat a F2population ofChinese Spring×FEN33was employed. A SSR marker wmc503and a SNP marker werenewly selected, the genetic distance with branched-spike gene was23.2cM and28.8cMrespectively. Integrating the new and previous results we found that two branched-spike geneswas located on chromosome2AS and2D and the marker Xwmc522（8.8cM）and gwm484（15.0cM）flanked this gene proximally.6. To mapping branched-spike genes in tetraploid wheat a F2population of GAN-A1582×GAN-A631F2population was employed. Two SSR markers wmc407and wmc198wereselected, the genetic distance with branched spike gene was34.2cM and41.1cM respectively.We established an efficient expression and purification process for LBD familytranscription factors and their DNA binding domains. It will provide adequate high purityprotein for spatial structure analysis of LBD family transcription factors. We found twomolecular markers linked to branched-spike genes in a hexaploid and a tetraploid F2populations respectively. It will lay a foundation for subsequent map-based cloning of ebranched-spik genes.