A kind of breeding method of glyphosate-resistant gene and transgenic glyphosate-resistant tobacco
A technology of glyphosate resistance and cultivation method, applied in the directions of genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problem of less glyphosate-resistant EPSPS, and achieve the effect of low price and ensuring biological safety.
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Embodiment 1
[0060] The target gene I.variabilis-EPSPS used in the present invention is obtained by cloning from the bacteria Isoptericola variabilis by the research group of Professor Liu Ziduo of Huazhong Agricultural University. The vector pAroAI.va* containing the aroAI.va* gene optimized by chloroplast codons was digested with Nde I and Xba I, and the structure diagram of the vector pAroAI.va* is shown in figure 1 , the sequence length of the basic vector pUC57 is 2710bp, including LacZ promoter, ampicillin resistance site, replicon rep (pMB1), and the target gene aroAI.va* with a sequence length of 1384 inserted in the multi-cloning site MCS. After digestion of the vector pAroAI.va*, the aroAI.va* gene fragment was isolated and connected to the intermediate vector pZF4, and then the aroAI containing the tobacco chloroplast-specific strong promoter Prrn and the specific terminator TrbcL was double-digested with Sac I and Hind III The .va* gene expression cassette was finally connected...
Embodiment 2
[0088] Transgenic glyphosate-resistant tobacco plant I.variabilis-EPSPS* site-specific integration and homogeneity detection obtained in Example 1
[0089] I.variabilis-EPSPS* analysis was performed on I.variabilis-EPSPS* families by Southern hybridization. For the extraction of total DNA from plant leaves and the analysis methods of Southern hybridization, refer to the operation manual of the laboratory. 10 μg of tobacco genomic DNA was digested with Bgl II, and psaB synthesized by digoxin-labeled PCR was used as a probe for Southern blotting detection; the upstream primer psaB-F sequence of the psaB probe was shown in Seq ID No.10; The downstream primer psaB-R sequence of the psaB probe is shown in Seq ID No.11. It is expected that the hybridization band size of the wild-type tobacco is about 3.5kb, and the hybridization band size of the chloroplast transformant of the pLSZ1 vector successfully integrated with the exogenous gene is about 6.8kb, and the homogenized transgeni...
Embodiment 3
[0090] Example 3: Germination and homogenization verification of transgenic glyphosate-resistant tobacco plants in Example 1
[0091] In order to further verify whether the transformed plants have achieved homogeneity, the seeds of the transformed plants were taken for a germination test. Theoretically, the homogeneous transformed plants can germinate on the antibiotic medium without segregation of traits. Germination results show: control wild type, transfected with aroA I.va* The T1 seeds of the chloroplast-transformed plant tobacco pLSZ1#2 germinated normally on the medium without resistance, but some albino seedlings germinated on the medium containing spectinomycin, indicating that pLSZ1#2 was a heterogeneous plant. Turn to aroA I.va* The T1 generation seeds of the chloroplast transformed plant Nicotiana pLSZ1#1 can normally germinate into green seedlings on the medium containing 500mg / L of spectinomycin and no resistance, and there is no segregation of traits (such as ...
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