| Carboxyl-terminal Proteases (CTPs) are family proteins with unknown fuctions inplant cell. The different CTPs share a highly conserved domain named DUF239 attheir carboxyl terminal of amino acid sequences. The CTP proteins are distributed indifferent subcellular regions respectively, including nucleolus, cell membrane,mitochondria, chloroplast, endoplasmic reticulum, extracellular space, etc. Therefore,the CTP family proteins might play significant fuctions in plant developmentprocesses. In this research, the antisense gene and GhCtp-GFP fusion gene expressionvectors of a cotton Ctp gene were constructed, and then transformed into the tobaccoby Agrobacterium-mediated method, respectively, to investigate the function ofGhCtp gene through analyzing the effects of its suppressive- or over-expression ontobacco growth and development. The results are followings:1. Transformation of GhCtp gene into tobaccoThe leaf explants from sterile plantlets of tobacco K326 were infected byAgrobacterium tumefaciens LBA4404 with the constructed antisense GhCtp gene andGhCtp-gfp fusion gene expression vectors, respectively. After explants cultured in theco-medium (MS+BA 1.0 mg·L-1+NAA 0.1mg·L-1+AS 10 mg·L-1) for 1～2d, thenwere transfered into the differentiation medium (MS+BA 1.0 mg·L-1+NAA 0.1 mg·L-1+Hyg 20 mg·L-1+Cef 500 mg·L-1) to directly induce resistant shoots, andthen the resistant shoots transfered into rooting medium (MS+BA 1.0 mg·L-1+NAA0.1 mg·L-1+Hyg 20 mg·L-1+Cef 500 mg·L-1). Seventy plants from 15 linestransformed with antisense gene and thirty-five plants from 8 lines with fusion genewere obtained, respectively, in which 12 antisense gene transformed lines and 6 fusiongene transformed lines were detected as the transgenic lines by PCR andPCR-Southern analyses. 2. Preliminary function analysis of GhCtp geneThe morphology between the transgenic tobacco plants with fusion gene and thecontrols had no difference, but the morphological variations of transgenic plants withanti-GhCtp gene were observed.①Leaf shapeCompared to the control, the leaf width of transgenic tobaccos with anti-GhCtpgene increased, so that their leaf shapes were also changed. There was a extremelysignificant difference between 4 transgenic lines (A, B, C, E) and the control in theratio of length to width, with a leaf shape of wide ellipse or ellipse, and a significantdifference between 2 transgenic lines (F, H) and the control, with a leaf shape ofellipse.②Flowering timeThe flowering stage of transgenic plants with anti-GhCtp gene was advanced by 10d to 47 d compared to that of the control. But such change of flowering stage wasobviously different from the early folwering of tobacco plants, the latter referring tothat the plants begin to flower in advance when having 10 or so leaves.③Corolla shapeThe corolla length and diameter of 4 transgenic lines (A, C, D, H) with anti-GhCtp geneshowed a extremely significant difference from that of the control. The corolla lengthof transgenic lines was 5.38～5.90 cm, the diameter 2.48～3.06 cm at full flowering stage,however the length and diameter for the control was less than 5.0 cm and 2.4 cm,respectively.④Flower colorThe flower color of all transgenic lines with anti-GhCtp gene had changed into a lightpink comparied to a pink color of the control.Therefore, we concluded that the expression of GhCtp gene might accelerate theplant developmental processes or its epidermal cell growth, but a further research isrequired. |
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