Cloning And Reciprocal Transformation Of The Carple Development Related Gene CRABS CLAW CDNA Of Arabidopsis Thaliana And Capsella Bursa-posteris

Arabidopsis(Arabidopsis thaliana)and Shepherd's-purse(Capsella bursa-pastoris L.)are all the weeds in Cruciferae family.Their genetic background and biological traits share high homologous but with an identical carpel that the Arabidopsis is in a rod carpel while Shepherd's-purse in a heart shape.A CRC gene is recovered in Arabidopsis that is related to its carpel morphology.In this study the Shepherd's-purse are used as materials in order to clone the homologous gene with Arabidopsis and study its function by reciprocal transformation of the two homologous cDNA recombinants.Both CRC cDNA of Arabidopsis and the homologous CRC cDNA of Shepherds-purse are cloned by RT-PCR with homologous sequence primers according to reported Arabidopsis CRABS CLAW(CRC)gene cDNA.Sequence blast and homologous analyses and reciprocal transformation are carried out after constructing the two CRC cDNA into plant expression vector.The following results are obtained:1.Cloning the Arabidopsis and Shepherds-purse CRABS CLAW(CRC)cDNA;Primers are designed according to GenBank CRC sequence of Arabidopsis.CRC gene cDNA of both Arabidpsis and Shepherds-purse are amplified by RT-PCR and cloned.The CRC cDNA that is amplified from ecotype of Colambia Arabidopsis has the same sequence in GenBank and the sequence of Shepherd's-purse CRC has a homology of 94%with the reported sequence in GenBank.It idicates the CRC gene cDNA of Shepherd's-purse is successfully cloned.The gene sequence has been submitted to GenBnak and the the access number is DQ119055.2.CRC cDNA recombinants for plant expression are constructed for both Arabidosis and Shepherds-purse;The CRC cDNA of both Arabidopsis and Shepherds-purse are inserted into a Ti plasmid vector pWM101 downstrand of the 35S promoter.Thus the constant high level expression recombinant vectors are constructed.We design it as pWM-atCRC and pWM-cbCRC respectivly.3.The reciprocal transformation of Arabidopsis and Shepherd's-purse with the partner CRC cDNA.Several methods of Agrobacterium tumefaciens transformation are used to transform Arabidopsis and Shepherds-purse.The methods of infiltration, flower-dripping,and callus co-culture are tried and compared.Several transformants are obtained both in Arabidopsis transforms with pWM101-cbCRC and Shepherd's-purse with pWM101-atCRC Molecular analysis showes the transformed gene have been integrated into the genome.The two transformed plants varied both in size and the shape of carpel.It is concluded that the CRC is involved in axial growth of the carpel and may affecting the size of the organ.4.The transformation methods are compared and concluded.The efficiency of different methods of transformation is compared.The results show that:Flower dripping and infiltration is a more targeted method that is easier to operated and with minor damnification.The transformed plants are also recovery faster.While calli co-cultured with Agribacterium is a hard task to contrail the bacterium during screening and the antibiotics restrain the development of the transformed calli result a low rate of differentiation.Therefore the flower dripping or infiltration maybe a selected method for Cruciferae plants transformation like in Arabidopsis and Shepherd's-purse.