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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

Active Publication Date: 2015-02-18
MAANSHAN SHENLUKERUI PHARMA
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Problems solved by technology

[0006] Aiming at the problems of high cost and immature methods for increasing the content of acteoside in traditional Chinese medicinal materials in the prior art, the present invention provides a method for improving the content of acteoside in Rehmannia glutinosa by transfecting the RgPAL1 gene. The gene RgPAL1 is cloned from Rehmannia glutinosa by using genetic engineering methods. A plant expression vector containing RgPAL1 was constructed, mediated by Agrobacterium tumefaciens, RgPAL1 was transformed into Rehmannia glutinosa and the plants were regenerated, and a transgenic Rehmannia glutinosa line with significantly increased verbascoside content was obtained

Method used

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  • Method for improving content of verbascoside in rehmannia by virtue of transformation of RgPAL1 gene
  • Method for improving content of verbascoside in rehmannia by virtue of transformation of RgPAL1 gene
  • Method for improving content of verbascoside in rehmannia by virtue of transformation of RgPAL1 gene

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Experimental program
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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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Abstract

The invention discloses a method for improving the content of verbascoside in rehmannia by virtue of transformation of RgPAL1 gene, and belongs to the field of biotechnology. The method comprises the following steps: cloning gene RgPAL1 from rehmannia, constructing a plant expression vector containing the RgPAL1, transforming the RgPAL1 into the rehmannia by virtue of mediation of agrobacterium tumefaciens and promoting regeneration of a plant, detecting integration of an exogenous target gene RgPAL1 through polymerase chain reaction (PCR), quantitatively analyzing the content of verbascoside in the transgenic rehmannia by virtue of HPLC (High Performance Liquid Chromatoraphy)-PDA, and screening to obtain a transgenic rehmannia plant having improved verbascoside content. By virtue of the method disclosed by the invention, the content of verbascoside in the transgenic rehmannia is significantly improved, and the content of verbascoside in the RgPAL1 transgenic rehmannia is 2.57 times as much as that in non-transgenic rehmannia, so that a feasible method is provided for scaled production of verbascoside from the transgenic rehmannia. The method disclosed by the invention provides an innovative and practical related method and technology for improving the verbascoside content.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for improving the content of verbascoside in rehmannia glutinosa by transfecting RgPAL1 gene. Background technique [0002] Rehmannia glutinosa Libosch. belongs to the Scrophulariaceae family and is a perennial herb with the functions of clearing away heat and cooling blood, regulating menstruation and detoxifying. Rehmannia glutinosa is a large traditional Chinese medicinal material in China. According to statistics, its frequency of use in traditional Chinese medicine prescriptions ranks among the top ten. Verbacoside is the main medicinal substance of Rehmannia glutinosa and a representative compound of phenylethanoid glycosides (PeGs, phenylethanoid glycosides), which has anti-fatigue, anti-oxidation, and anti-aging effects. "Pharmacopoeia of the People's Republic of China" 2010 Edition Part 1 Rehmanniae Radix (REHMANNIAE RADIX) content determination, th...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 谢峻崔平陈启厚
Owner MAANSHAN SHENLUKERUI PHARMA
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