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CN-121991982-A - Cultivation method of Isatis tinctoria plant over-expressing EVM0003075 gene

CN121991982ACN 121991982 ACN121991982 ACN 121991982ACN-121991982-A

Abstract

The invention relates to a cultivation method of a woad plant over-expressing an EVM0003075 gene, belonging to the technical field of plant cultivation. Firstly, transforming an agrobacterium strain through a constructed agrobacterium transformation vector expressing an EVM0003075 gene of woad, then transforming an explant of woad by using the recombinant agrobacterium strain obtained by screening, regenerating a seedling of woad, screening and cultivating the woad seedling with stable over-expressed EVM0003075 gene, and obtaining a woad plant with increased content of indigoid in woad. The expression level of the EVM0003075 gene of the transgenic woad is improved to 5-9 times, the content of the indigoid is obviously improved, and the property of short accumulation period of the indigoid is shown. The method of the invention can increase the content of the indigoid in the woad to 10-66 times. The following is a specific information content of a method patent for increasing the content of indigoid in Isatis tinctoria by over-expressing EVM0003075 gene.

Inventors

  • ZHANG LIBIN
  • HUANG HUI
  • WANG YUHUA

Assignees

  • 中国科学院昆明植物研究所

Dates

Publication Date
20260508
Application Date
20260309

Claims (5)

  1. 1. A method for cultivating a woad plant over-expressing an EVM0003075 gene, the method comprising the steps of: (1) Constructing an agrobacterium transformation vector for expressing an EVM0003075 gene of woad; (2) Transferring the agrobacterium transformation vector constructed in the step (1) into competent cells of agrobacterium GV3101 by using a freeze thawing method to obtain a transformed agrobacterium strain; (3) Taking woad as a receptor material, and carrying out genetic transformation by utilizing the transformed agrobacterium strain obtained in the step (2) to obtain woad seedlings with the EVM0003075 gene stably and excessively expressed; (4) And (3) screening the Isatis tinctoria seedlings obtained in the step (3), and culturing to obtain Isatis tinctoria plants over-expressing the EVM0003075 genes.
  2. 2. The cultivation method according to claim 1, wherein said step (1) is to construct an agrobacterium transformation vector expressing the woad EVM0003075 gene using a pRI101-GFP vector.
  3. 3. The cultivation method according to claim 1, wherein the specific operation of the freeze thawing method in the step (2) is: 1) Placing the agrobacteria GV3101 competent cells preserved at-80 ℃ on ice for thawing; 2) Adding 10 mu L of pRI101-GFP-EVM0003075 plasmid of the step 1) into competent cells of agrobacterium GV3101, gently mixing, and sequentially standing on ice for 5min, liquid nitrogen for 5min, 37 ℃ water bath for 5min and ice bath for 5min; 3) Adding 700 μl of LB culture medium without antibiotics into the step 2), mixing, resuscitating at 28deg.C for 2 hr at 200rpm, absorbing 200 μl of resuscitated bacterial liquid, coating on double antibody solid culture containing 50 μg/mL of Carna and 20 μg/mL of rifampicin with a coating rod, and placing the plate in a 28 deg.C incubator upside down for 2-3 days.
  4. 4. The cultivation method as claimed in claim 1, wherein said genetic transformation in step (3) is performed by agrobacterium-mediated genetic transformation using woad as a receptor material.
  5. 5. The cultivation method according to claim 1, wherein said cultivation in step (4) is carried out by transplanting the rooted woad seedlings directly into the soil through hardening.

Description

Cultivation method of Isatis tinctoria plant over-expressing EVM0003075 gene Technical Field The invention relates to the technical field of plant cultivation, in particular to a cultivation method of a Isatis tinctoria plant over-expressing an EVM0003075 gene. Background Isatis tinctoria (Isatis tinctoria L.) is a two-year old herb of Brassicaceae, has at least 2000 years of cultivation history, and is an important industrial raw material for medicines, dyes, eating and cosmetics. But different varieties or farmhouse cultivars are formed in the long-term cultivation and artificial breeding processes, and the quality of the cultivars is often greatly different, so that the production and the application of the woad are severely restricted. The research shows that the indigotin (indigo) and the indirubin (indirubin) are main medicinal components of the woad, the indigotin has the pharmacological effects of resisting inflammation, protecting liver, improving body immunity and the like, and the indigotin is a natural organic pigment and is widely applied to industries of printing and dyeing, foods, cosmetics and the like, and the indigotin is an antitumor drug and can be used for effectively treating chronic granulocytic leukemia clinically. However, in the early cultivation process, the variety breeding of the Isatis tinctoria mainly adopts a conventional method to mainly breed varieties with high yield and insect resistance, such as Ji-lan No. 1, tetraploid radix isatidis and the like, but aiming at improving the varieties or strains of indigo and indirubin, the variety breeding of the Isatis tinctoria has not been achieved. So the content of indigo and indirubin in woad is low at present only 3.4% and 0.6% respectively. Therefore, in order to solve the above problems, a method for cultivating woad plants is needed, which can improve the quality of woad, increase the content of indigo and indirubin in woad, and has important significance for the production and application of woad. Disclosure of Invention In order to solve or partially solve the problems in the related art, the method for cultivating the woad plant provided by the invention not only ensures that the survival rate of the positive seedlings of the woad is high, the growth condition of the seedlings is good, but also the content of the indigoid is obviously improved, and the accumulation period is shortened. The application provides a cultivation method of a woad plant over-expressing an EVM0003075 gene, which comprises the following steps: (1) Constructing an agrobacterium transformation vector for expressing an EVM0003075 gene of woad; (2) Transferring the agrobacterium transformation vector constructed in the step (1) into competent cells of agrobacterium GV3101 by using a freeze thawing method to obtain a transformed agrobacterium strain; (3) Taking woad as a receptor material, and carrying out genetic transformation by utilizing the transformed agrobacterium strain obtained in the step (2) to obtain woad seedlings with the EVM0003075 gene stably and excessively expressed; (4) And (3) screening the Isatis tinctoria seedlings obtained in the step (3), and culturing to obtain Isatis tinctoria plants over-expressing the EVM0003075 genes. Further, in the step (1), an agrobacterium transformation vector expressing the woad EVM0003075 gene is constructed using a pRI101-GFP vector. Further, the specific operation of the freeze thawing method in the step (2) is as follows: 1) Placing the agrobacteria GV3101 competent cells preserved at-80 ℃ on ice for thawing; 2) The pipette sucks 10 mu L of pRI101-GFP-EVM0003075 plasmid in the step 1), adds the plasmid into competent cells of agrobacterium GV3101, lightly mixes the plasmid and stands on ice for 5min, liquid nitrogen for 5min, 37 ℃ water bath for 5min and ice bath for 5min; 3) Adding 700. Mu.L LB culture medium without antibiotics into the 2), mixing uniformly, resuscitating for 2h at 28 ℃ at 200rpm, absorbing 200. Mu.L resuscitated bacterial liquid, coating the liquid on double-antibody solid culture containing 50. Mu.g/mL of Carna and 20. Mu.g/mL of rifampicin by using a coating rod, and placing the flat plate in a 28 ℃ incubator reversely for culturing for 2-3 days. Further, the genetic transformation in the step (3) is performed by using agrobacterium-mediated genetic transformation, and using woad as a receptor material. And (3) cultivating in the step (4) is to transplant the rooted woad seedlings into soil directly through hardening off. Advantageous effects 1. The method for cultivating the woad plant provided by the application can directionally induce the increase of the content of the indigoid and shorten the accumulation period of the indigoid, so that the content of the indigoid in the woad of the transgenic over-expressed EVM0003075 gene is increased to more than 10-66 times. 2. According to the measurement, when the method provided by the application is used for cultivating the woad,