Application of the Arabidopsis DNA topoisomerase gene at4g14990 in the regulation of root development
A technology of AT4G14990 and topoisomerase, applied in the field of genetic engineering
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Embodiment 1
[0022] Example 1: Proving that the Arabidopsis AT4G14990 gene is associated with root development
[0023] Seeds of Arabidopsis wild-type (Col) and mutants (SALK-110349, SALK-134773 and SAIL-682-F06) were planted on MS medium with 0.8% agar, and cultured vertically for 7 days to measure the growth of main root.
[0024] The results showed that the main root length of the mutant was significantly smaller than that of the wild type, such as figure 1 As shown, the main root length of SALK-110349 and SAIL-682-F06 in the mutant plants has a significantly short root phenotype compared with the control, indicating that the AT4G14990 (TOPII) gene is involved in the regulation of Arabidopsis root development.
Embodiment 2
[0025] Example 2: Demonstrating the mechanism of Arabidopsis AT4G14990 gene regulating root development
[0026] Existing studies have shown that the disorder of root tip stem cells is an important reason for the short root phenotype of Arabidopsis. To this end, the inventors used T-DNA insertion mutants and overexpression lines of the gene AT4G14990 to study the phenotype of root-tip stem cells. Statistical methods such as figure 2 As shown, wherein SALK-110349 and SALK-134773 are T-DNA insertion mutants of gene AT4G14990 (TOP II), respectively, and 35S:TOP II:GFP is an overexpression line of gene AT4G14990 (TOP II).
[0027] The results showed that the mutant had obvious QC cleavage phenotype, while the phenotype of QC cleavage was restored in the overexpression line. It shows that the Arabidopsis AT4G14990 gene affects root development by regulating root tip QC division.
Embodiment 3
[0028] Example 3: Tissue localization of Arabidopsis DNA topoisomerase II gene AT4G14990 (TOP II)
[0029] Topoisomerase II gene TOP II promoter sequence-GUS fusion expression vector TOP IIp:GUS was constructed, and plasmids were extracted by shaking bacteria. The constructed TOP IIp:GUS promoter recombinant plasmid was transformed into Agrobacterium GV3101 competent cells by freeze-thaw method, and wild-type Arabidopsis was transformed by the Agrobacterium liquid of the sequenced positive clone by flower dipping method, and antibiotics Positive seedlings were screened for GUS staining. The composition of GUS staining solution was 2mM X-Gluc, 0.5mM potassium ferricyanide, 0.5mM potassium ferrocyanide and 100mM phosphate buffer (pH 7.2) containing 0.1% Triton X-100. Take 1mL of the staining solution into a 1.5mL centrifuge tube, intrude the positive seedlings into the reaction solution, incubate at 37°C for 24h, make a pressed sheet, observe and take pictures under a microscop...
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