Cloning And Analysis Of CbTPD1 Genefrom Capsella Bursa-pastoris And Its Roles In Carpel Morphogenesis

TAPETUM DETERMINANT1(TPD1),a 176 aa small protein,together with EMS1,a LRR-RLK protein receptor kinase,participates in regulation of pollen formation.Also,they could be ectopic expression in carpel and therefore regulates carpel formation.To investigate the roles of TPD1 in carpel morphoensis of Capsella bursa-pastori,TPD1 gene was cloned,and its biological function was analyzed.The resultsas follows:1.TPD1 was cloned from wild-type Capsella bursa-pastoris through homology-based cloning method and named asCbTPD1.TheCbTPD1 gene was 3000 bp in total,containing a1561 bp of promoter and a 528 bp of cDNA sequence.Bioinformatics analysis showed that the homology of nucleotide sequences between CbTPD1 and AtTPD1 gene was 86.4%,and the homology of amino acid sequence was 81.4%.To further explore the expression differencesofCbTPD1 and AtTPD1 in the development process of carpel,fluorescent quantitative PCR method was performed using the cDNAs as template from the 12,15 and 17 stages of floral organ of Capsella bursa-pastoris and Arabidopsis.Data demonstrated that the expression levels of TPD1 were higher in these three stages than other stages both in Capsella bursa-pastoris and Arabidopsis.Moreover,the expression of Cb TPD1 was significantly higher than AtTPD1.These results indicated that maybe the expression differences of CbTPD1 and AtTPD1 resulting in theextreme variation ofcarpel's morphology.2.Promoter elements analysis data revealed that basic components,such as high transcriptional level regulatory factor,photo-response element and jasmonic acid response element exist both in Capsella bursa-pastoris and Arabidopsis.However,stress response elements such as,endosperm expression element,gibberellin response element and salicylic acid response element were much different.GUS reporter vector systemincludedCbTPD1 pro promoter was constructed and transform into Columbia ecotypeArabidopsis.GUS staining results suggested that TPD1 expression pattern were similar in seedling,filament,stigma,nectary and carpel.Furthermore,the expression ofAtTPD1 was also detected in anther.In contrast,CbTPD1 did notexpression in anther.Therefore,the distinction expressions of TPD1 inanther also contribute to thevariationofcarpel's morphology in Capsella bursa-pastoris andArabidopsis.3.To further explore the function of CbTPD1,35S::CbTPD1 expression vectorandCbTPD1pro::CbTPD1 expression vector were built and then transform into Columbia ecotypeArabidopsis.Observation demonstrated that over-expression of CbTPD1 using 35S promotor leading tothe length and width of carpel obviously less than that of wide type.Similarly,over-expression of CbTPD1 using CbTPD1 promotor also made the length and width of carpel less than that of wide type.However,over-expression of CbTPD1 using 35S promotor causeddysontogenesisof carpels and ovules,and that were normal using CbTPD1 promotor.In addition,there were some petals adhering to carpel and hard to fall off from carpel in the stage 18 and 19 of floral organ development processes of 35S::CbTPD1transgenetic seedling.Therefore,it can be deduced that the difference of promoter results in the difference of expression stages,position and expression level of CbTPD1.In the meantime,it also indicated thatCbTPD1 might participate in morphogenesis of carpel and network regulation of other genes.Thus,it exerts influences on physiological metabolism and development of plant.4.To further investigate the effects of stress response elements between Capsella bursa-pastoris and Arabidopsis on carpel morphogenesis,trans-CbTPD1pro::CbTPD1Arabidopsis were treated using various concentrations of gibberellins and salicylic acid before flowering.Results showed that after gibberellin treatment,the length and width of carpel were changed compared with untreated plants.PIN3 and PIN7,auxin polar transport gene,participated in auxin crossrange transport andlead to the changes of length and width of carpel after feeling exogenous gibberellin stimulation when lacking of GARE-motif gibberellin response elements.The regulation of salicylic acid was affected by photo-response elements.Thenon-salicylic acid response elementCbTPD1 participated in controlling salicylic acid signaling pathway after feeling exogenous salicylic acid stimulation,and finally caused the changed of carpel width.