Studies On The Expression Of Arabidopsis Thaliana ADF4 In Transgenic Tobacco

Actin that constitutes cytoskeleton microfilament system is an important protein and ubiquitous among eukaryotes. The actin cytoskeleton microfilament system performs essential functions in the morphogenesis, especially in the plant cell such as pollen tube, root hair, leaf hair, guard cell. In addition, the study in single-cell system demonstrated that F-actin appears to play key roles in heigher plant morphogenesis of cell division and cell enlogation. However the in vivo studies concerning the faction of plant actin is very few. In vitro studies demonstrated that the function of actin is regulated by many actin-binding proteins in animal and yeast. Among these proteins, actin-depolymerizing factor (ADF) is an essential regulative factor for F-actin. Few studies about ADF have been done during plant growth and develoment. To date, there are some reseach reports on Arabidopsis thaliana ADF1, Lily ADF1 (LIADF1) and Zea mays ADF3 (ZmADF3) which indicated that they are in relation to plant morphogenesis. ADF4 is one of the members in Arabidopsis thaliana actin-binding protein family. At presnt, the function of ADF4 hasn't been known yet. To explore the function of ADF4 in plant growth and develoment, we cloned ADF4 gene from Arabidopsis thaliana and expressed it in tobacco. According to the experimental data, we roughly deduce that ADF4 affect plant morphogenesis. The data obtained provide the experimental proofsfor the research of ADF function. This studies is constituted of cloning the ADF4 gene, constructing plant-expression-vector pBADF4, transforming the tobacco by Agrobacterium tumefaciens mediated transformation method, molecular detecting transgenic plants and observing morphological phenotype with TI lines .The results of the study are as follows:1. The AtADF4 gene was cloned from Arabidopsis thaliana genomic DNA by PCR. Comparing the sequence of AtADF4 gene and AtADF4 mRNA, there is a 91bp intron in sequence 251-341bp. The nucleic acid sequence and the derived protein sequence have completely indentity with the report.2. Based on pBI121, the plant-expression-vector containing AtADF4 was constructed, named pBADF4.3. At ADF4 gene was transformed into tobacco by Agrobacterium tumefaciens and was integrated into the genome of tobacco assayed by PCR. AtADF4 gene was expressed in transgenic tobaccos assayed by RT-PCR.4. The results of ADF4 affecting in transgenic tobaccos growth are as follows: (1) The hypcotyl of TI line growing in the light and in the dark respectively are wavy, especially in the dark condition, whearas, the control are straight. (2) The number of the root hairs in TI line are less than the control, and they are also wavy. (3) TI line cotical cells are larger than the control and the arrangement of vascular bundle is out of order. (4) The stomatal guard cells ofTI line are closure. (5) The flowering time of TI line is at least 7day earlier thanthe control. (6) Comparing with control, the pollen tubes of TI line are short andlarge.