Plant transgenic visual tracking expression system and application thereof
A plant expression vector and gene technology, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problem of transgenic plants purple and other problems, achieve the improvement of accuracy and reliability, strong purpose, and reduce the complexity of work. Effect
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Embodiment 1
[0035] Embodiment 1, the synthesis of maize anthocyanin regulation gene and the construction of plant expression vector
[0036] Considering the safety of antibiotics in biolistic transformation, in order to facilitate the deletion of antibiotic genes in subsequent experiments, the pGreen vector was transformed, and two loxP sites in the same direction were inserted on both sides of the Kan gene expression frame, which were identified by sequencing. The vector was named pBAC814. Insert the EPSP gene (EPSP gene expression cassette) driven by the Intron1 of the CaMV 35S promoter and the maize Adh1 gene at the NdeI restriction site before the pBAC814 vector multiple cloning site afterwards, and the EPSP gene is used as a selection marker gene for screening transgenic plants . The correct vector identified by sequencing was named pBAC823.
[0037] Digest the above pBAC823 vector with DraIII, add AgeI / PacI linker, then insert the expression cassettes (sequence 4) of the two trans...
Embodiment 2
[0039] Embodiment 2, transient expression of anthocyanin gene
[0040] Take wheat leaves and wheat Baofeng 104 15-17 days after flowering (Wang Junwei, Yang Fengping, Zhang Xiaodong, etc., Induced expression of DREB transcriptional factor and study on its physiological effects of drought tolerance in transgenic wheat.Acta Genetica Sinica, 2006, 33( 5): 468~476) immature embryos, after removing the top part of the growth, inoculate the scutellum upside down on MS solid medium, and use the purified concentration of 1 μg μL -1 The pBAC9008 vector, according to Zhang Xiaodong et al. (Zhang Xiaodong, Liang Rongqi, Chen Xvqing, Yang Fengping, Zhang Liquan, Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat. 48 (8): 771-776.) The method described in the preparation of gene gun microprojectiles, PDS-1000 / He type gene gun bombards scutellum and blade, after 48h, observe anthocyanin gene in different tissues under a Leic...
Embodiment 3
[0042] Embodiment 3, corn biolistic transformation
[0043] Induction medium: N6 basic medium + 10mg L -1 AgNO 3 +100mgL -1 Inositol+2mg L -1 2,4-D+30g L -1 Sucrose+7g L -1 agar.
[0044] Differentiation medium: N6 basic medium + 0.5mg L -1 AgNO 3 +100mgL -1 Inositol+1.5mg L -1 KT+30g L -1 Sucrose+7g L -1 Agar+0.5mgL -1 glyphosate.
[0045]The ears of corn elite inbred line 501 about 12 days after pollination were taken, and the immature embryos were picked and inoculated on the induction medium after surface disinfection with 75% ethanol, and embryogenic callus was induced after dark culture for 3-5 days. Adjust the concentration of the purified pBAC9008 vector to 1 μg μL -1 , according to Zhang Xiaodong et al (Zhang Xiaodong, Liang Rongqi, Chen Xvqing, Yang Fengping, Zhang Liquan, Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat. ): 771-776.) The method carries out the preparation of gene...
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