Method for improving hairy root induction efficiency and genetic transformation efficiency of bupleurum chinense, and application thereof

A technology for genetic transformation efficiency and hairy roots, which is applied in the field of improving the induction efficiency and genetic transformation efficiency of Bupleurum chinensis hairy roots, can solve the problems of not getting positively transformed hairy roots, and achieve the effect of simple selection

Active Publication Date: 2017-09-01
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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Problems solved by technology

[0007] In addition, in the article "Agrobacterium Induced Hairy Root System Establishment and UGT Gene Function of Bupleurum chinensis" (Sun Jing et al., Microbiology, 2014), "The genetic transformation of BcUGT10 gene of Bupleurum chinense mediated by Agrobacterium rhizogenes Preliminary research", but the results found: "The induced hairy roots were screened with 25 mgm hygromycin to obtain 3 resistant hairy roots. After PCR detection, no positive transformed hairy roots were obtained"

Method used

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  • Method for improving hairy root induction efficiency and genetic transformation efficiency of bupleurum chinense, and application thereof
  • Method for improving hairy root induction efficiency and genetic transformation efficiency of bupleurum chinense, and application thereof
  • Method for improving hairy root induction efficiency and genetic transformation efficiency of bupleurum chinense, and application thereof

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Embodiment 1

[0084] The embodiment of the present invention provides a method for improving the induction efficiency of Bupleurum hairy roots, comprising the following steps:

[0085] 1. Obtaining of explants

[0086] 1. Dried Bupleurum bupleuri seeds, rinse the floating dust with tap water, and then rinse with sterilized distilled water several times;

[0087] 2. The next step is to operate in the ultra-clean bench: soak in 75% alcohol for 30-50S;

[0088] 3. Seal with 20% sodium hypochlorite (NaClO) solution for 15 minutes, rinse with sterile water for 3 to 5 times;

[0089] 4. Blot the sterilized seeds dry with sterile filter paper. Ordinary MS culture is based on dark culture at 25°C. Every 2-3 days, the infected seeds are transferred out. After 8-9 days, there is basically no bacteria contamination. Culture until 14 In about a day, the seeds begin to germinate;

[0090] 5. Cut off the roots of the seedlings and put them into the subculture medium (MS+6-BA2.5mg / L+IAA2.5mg / L+30g / L su...

Embodiment 2

[0110] In order to further illustrate the beneficial effects of the present invention, the present invention provides the following replacement embodiment 2, replacement embodiment 2 replaces some conditions in steps 1 to 3 of embodiment 1, and the others remain unchanged. For the corresponding test results, see Table 2, 3(d: day; min: minute; h: hour):

[0111] Table 2.

[0112]

[0113] table 3.

[0114]

[0115] It can be seen from Table 2 and 3 that the rate of rooting will be affected if the OD value is too high or too low; the rate of rooting will be affected if the co-cultivation time is too long or too short.

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Abstract

The invention provides a method for improving the hairy root induction efficiency and the genetic transformation efficiency of bupleurum chinense, and application thereof. On the basis of exploration on the conditions of the hairy root agrobacterium strains, the infection bacteria liquid OD value, the co-culture time and the like, the optimum combination condition for improving the infection efficiency of the hairy root agrobacterium is determined; compared with the traditional hairy root induction efficiency being about 10 percent, the hairy root induction efficiency obtained by the technical scheme provided by the invention reaches 70 to 85 percent. The method for improving the hairy root genetic transformation efficiency of the bupleurum chinense provided by the invention has the advantages that the exogenous gene conversion rate being 80 percent or higher can be obtained; better, more efficient, simpler and more convenient selection is provided for the bupleurum chinense functional gene research and / or the industrialization of hairy root metabolites of the bupleurum chinense.

Description

technical field [0001] In the field of biological technology, the invention specifically relates to a method for improving the induction efficiency and genetic transformation efficiency of Bupleurum bupleurum hairy roots and its application. Background technique [0002] Hairy roots, also known as hairy roots, hairy roots, are inserted into the nuclear genome of plants or organs, tissues (including callus), single cells, and protoplasts by T-DNA on the Ri plasmid contained in Agrobacterium rhizogenes , and a large number of by-products are produced at the site of infection. The induced culture of hairy roots can be used as a bioreactor to produce valuable plant metabolites. In addition, exogenous genes can also be introduced by Agrobacterium rhizogenes to regulate the synthesis of target metabolites. [0003] Bupleurum bupleurum is a typical traditional bulk medicinal material in my country, with a history of more than 2000 years of application. It is an essential medicin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06
CPCC12N15/8205
Inventor 隋春吴素瑞魏建和金钺
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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