Method for improving content of verbascoside in rehmannia by virtue of transformation of RgPAL1 gene
A technology of acteoside and transgene, which is applied in the direction of genetic engineering, plant gene improvement, botanical equipment and methods, etc., can solve the problems of immature method and high cost of acteoside content, and achieve the effect of mature method, low cost and increased content
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Embodiment 1
[0054] Cloning of Rehmannia glutinosa RgPAL1 gene
[0055] 1. Extraction of total RNA from Rehmannia glutinosa
[0056]Use TaKaRa MiniBEST Plant RNA Extraction Kit (Code No.9769) to extract total RNA from the young leaves of Rehmannia glutinosa. The steps are as follows: take a small amount of young leaves of Rehmannia glutinosa (collected from Huaihua, Henan Province, and replanted in Putang Botanical Garden, Maanshan City, Anhui Province) , after liquid nitrogen quick-freezing, quickly grind into powder with a mortar, take about 50mg of powder and add it to a 1.5mL sterile centrifuge tube containing 450μL Buffer RL for lysis, and repeatedly blow and beat with a pipette until there is no obvious precipitation in the centrifuge tube to be lysed solution; put the lysate at 12000rpm and centrifuge at 4°C for 5 minutes, then pipette the supernatant into a new 1.5mL sterile centrifuge tube; add absolute ethanol to the 1.5mL sterile centrifuge tube containing the supernatant Mix w...
Embodiment 2
[0062] Construction of Plant Expression Vector pCAMBIA1305.1-RgPAL1 Containing Rehmannia glutinosa RgPAL1 Gene
[0063] Using pCAMBIA1305.1 as the expression vector, use NcoI and SpeI to double digest the pMD obtained in Example 1 TM 19-T-RgPAL1 and pCAMBIA1305.1, recovered pMD TM The 19-T-RgPAL1 gene was digested to obtain the RgPAL1 gene fragment, which was connected with the digested linear pCAMBIA1305.1 large fragment, transformed, single clone was picked, and the plasmid was extracted for PCR detection and enzyme digestion verification, PCR detection The result is as figure 2 As shown in the figure, it can be found that the four single clones picked have bands at 2.2kb, and the enzyme digestion verification results are as follows image 3 As shown, a pCAMBIA1305.1 vector backbone with a length of 11.6 kb and a RgPAL1 gene band with a length of 2.2 kb can be found in the figure; the above results show that the RgPAL1 gene has been successfully constructed into the plant...
Embodiment 3
[0066] Genetic transformation of Rehmannia glutinosa mediated by Agrobacterium tumefaciens with RgPAL1 gene to obtain transgenic Rehmannia glutinosa plants
[0067] 1. Acquisition of plant expression vector Agrobacterium tumefaciens containing RgPAL1 gene
[0068] With the plant binary expression vector containing RgPAL1 gene in embodiment 2, by freezing-thawing method, transfer into Agrobacterium tumefaciens EHA105 (the biological material that the market publicly sells), and carry out PCR verification, PCR verification result is as follows Figure 4 As shown, there is an obvious band at 2.2kb from the figure; the results show that the plant expression vector containing the RgPAL1 gene has been successfully constructed into the Agrobacterium tumefaciens strain.
[0069] 2. Transformation of Rehmannia glutinosa mediated by Agrobacterium tumefaciens with RgPAL1 gene
[0070] 2.1 Pre-cultivation of explants
[0071] The leaves of Rehmannia glutinosa were washed with running wa...
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