阅读说明：本技术 一种发根农杆菌介导的中间锦鸡儿毛状根遗传转化体系的建立及优化方法 (Method for establishing and optimizing agrobacterium rhizogenes-mediated genetic transformation system of middle caragana hairy roots ) 是由 万永青 柳金华 杨闯 李国婧 王瑞刚 于 2019-11-13 设计创作，主要内容包括：本发明属于植物基因工程技术领域,具体涉及一种发根农杆菌介导的中间锦鸡儿毛状根遗传转化体系的建立及优化方法,选取生长良好的中间锦鸡儿幼苗,选取下胚轴较粗壮的植株进行注射农杆菌悬浮液进行侵染,侵染后选取中间锦鸡儿毛状根检测GUS基因表达情况。毛状根遗传转化体系使基因在中间锦鸡儿毛状根中过量表达,操作简单,表达水平高,可为开展基因功能验证和筛选功能基因提供技术支持；而且待毛状根长到一定大小后切除主根,这时毛状根取代主根成为该中间锦鸡儿植株的一部分及形成转基因毛状根与原植株茎叶的嵌合体,在研究基因功能时,由于植物是个有机的整体,在处理植物根部时,植物整个植株都可能会发生相关形态及生理生化变化,有利于在整体条件下研究基因功能。(The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for establishing and optimizing an agrobacterium rhizogenes-mediated hairy root genetic transformation system of caragana intermedia. The hairy root genetic transformation system enables the gene to be over-expressed in the hairy root of the caragana intermedia, has simple operation and high expression level, and can provide technical support for developing gene function verification and screening functional genes; and when the hairy root grows to a certain size, the main root is cut off, and the hairy root replaces the main root to become a part of the middle caragana plant and form a chimera of the transgenic hairy root and the stem and leaf of the original plant.)

1. A method of effecting gene expression in the hairy root of caragana intermedia, comprising the steps of:

(1) electrically transforming an expression vector containing a GUS reporter gene into an agrobacterium K599 competent cell;

(2) culturing the agrobacterium obtained in the step (1) and agrobacterium K599 which does not contain any exogenous vector in an LB liquid medium;

(3) selecting well-grown middle caragana seedlings growing for about 6-9 days, and slowly injecting the agrobacterium liquid prepared in the step (2) into the leaf nodes of the middle caragana and hypocotyl parts above nutrient soil of the leaf nodes;

(4) and after transformation, observing the growth condition of the hairy roots of the caragana intermedia and detecting the expression condition of a GUS reporter gene.

2. The method for realizing gene expression in the hairy roots of caragana intermedia as claimed in claim 1, wherein the expression vector containing the GUS reporter gene is pCambia 1305.2.

3. The method for realizing gene expression in the hairy root of caragana intermedia according to claim 1, wherein the LB liquid culture medium of the step (2) is configured as follows: 1g of Tryptone, 0.5g of Yeast extract, 1g of NaCl, and NaOH or HCl to adjust the pH value to 7.0, diluting deionized water to 100mL, and autoclaving at 121 ℃ for 20 min; among them, LB liquid medium contained 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin.

4. The method for achieving gene expression in hairy roots of caragana intermedia as claimed in claim 1, wherein agrobacterium K599 carrying pcambia1305.2 vector and without any exogenous vector is inoculated into LB liquid medium in step (2); shaking at 200rpm and culturing at 28 deg.C overnight to OD₆₀₀The value is about 1.2, 1mL of bacterial liquid is taken and transferred into 20mL of fresh LB liquid culture medium again (the volume ratio is 1:20), and the bacterial liquid is cultured by shaking at the temperature of 28 ℃ and the rpm of 200 until the OD600 value is 1.2; acetosyringone was then added to a final concentration of 100. mu. mol/mL.

5. The method for realizing gene expression in the hairy root of caragana intermedia as claimed in claim 1, wherein the step (3) is specifically as follows: under the normal growth condition, selecting well-grown middle caragana seedlings growing for 6-9 days, and selecting plants with stout hypocotyls for injection. And (3) sucking the agrobacterium suspension which is added with the acetosyringone and then stands for 24h by using a 1mL needle tube, and pricking a hole at the hypocotyl part to inject the bacterial liquid. 1 injection is carried out every 24h, 3 injections are carried out totally, and dark treatment is carried out for 24h after each injection is finished and 3d totally.

6. The method for realizing gene expression in the hairy roots of caragana intermedia, as claimed in claim 1, wherein after infection, the hairy roots of caragana intermedia are subtracted from the hairy roots of caragana intermedia within 21-28 days to detect the GUS gene expression condition by keeping the culture humidity at about 75% and the temperature at about 25 ℃, and then the leaves are placed into a GUS staining solution for staining.

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for establishing and optimizing an agrobacterium rhizogenes-mediated intermediate caragana hairy root genetic transformation system.

Background

Compared with traditional crops or transgenic crops planted in outdoor fields, the hairy root tissues are always in an artificially controllable closed environment, and the risk of gene drift is greatly reduced; can grow rapidly and can proliferate at high speed in the culture process; agrobacterium rhizogenes is a gram-negative soil bacterium of the genus Agrobacterium of the family Rhizobiaceae, which is capable of infecting most dicotyledonous plants and a few monocotyledonous plants as well as individual gymnosperms. Compared with agrobacterium tumefaciens, hairy roots generated by agrobacterium rhizogenes have stable genetic property, and a plurality of dicotyledonous plants obtain heritable transgenic plants through a rooting system; in addition, the hairy roots can produce metabolites in the culture medium, providing convenient conditions for extraction and isolation. The agrobacterium rhizogenes is already applied to various plants because most of the plants can root after infecting the plants, but the agrobacterium rhizogenes is only applied to precious medicinal materials such as ginseng and the like because the rooting condition is strict.

Middle Caragana (Caragana intermedia Kuang) belongs to leguminous shrubs and is mainly distributed in arid and semiarid desert regions in inner Mongolia, Ningxia and northern Shaanxi in China. The caragana plant has the characteristics of cold resistance, drought resistance, salt and alkali resistance, barren resistance and the like, has strong adaptability to desert regions, not only maintains water and soil, prevents wind and fixes sand, but also has higher feeding value. Therefore, the research on the gene function is urgently needed to discover the stress-resistant mechanism. However, unlike some model plants such as arabidopsis thaliana, tobacco, rice, etc., which already have mature genetic transformation systems, there is an urgent need to establish a gene function verification method based on transgenic hairy roots in middle caragana due to the lack of regeneration systems and transgenic technologies in middle caragana.

At present, the research on the hairy root genetic transformation system of leguminous plants is relatively few, the number of successfully constructed examples is not many, the leguminous plants which are researched more are soybean, pigeon pea and the like, and no one has conducted systematic research on the hairy root genetic transformation system of caragana intermedia at present. At present, the genetic transformation method of the hairy roots of the plants is mainly combined with the tissue culture technology, the genetic transformation efficiency is relatively high, but the operation process has many careless matters, the operation is relatively complicated, the possibility of pollution is also provided, and the application of the successful practices has certain difficulty; the method for directly infecting agrobacterium rhizogenes on the plant is simple and easy to implement, avoids the problem of bacterial infection, and improves the possibility of application of transgenic hairy roots. In addition, hairy roots generated by the plant which is directly infected by agrobacterium rhizogenes are verified to be transgenic positive roots, and can be used for related researches on gene functions, metabolites and the like.

Disclosure of Invention

The invention aims to establish a method for realizing gene expression in the hairy root of caragana intermedia, which is used for functional verification research of genes. The method is realized by the following technical scheme:

(1) electrically transforming an expression vector containing a GUS reporter gene into an agrobacterium K599 competent cell;

(2) the Agrobacterium obtained in (1) and Agrobacterium K599 without any exogenous vector were cultured in LB liquid medium to the desired concentration (expressed as OD value).

(3) Selecting well-grown middle caragana seedlings which grow for about 6-9 days, and slowly injecting the agrobacterium liquid prepared in the step (2) into hypocotyl parts above the middle caragana cotyledon node and nutrient soil thereof.

(4) And observing the growth condition of the hairy roots of the caragana intermedia and detecting the GUS reporter gene expression condition within 21-28 days after transformation.

The step (1) is specifically as follows: the expression vector containing GUS reporter gene used was pCambia1305.2 (laboratory preservation), Agrobacterium K599 (Shanghai Weidi Biotechnology, Inc.). K599 Agrobacterium rhizogenes contains pRi2659 agropine Ri plasmid, has wide host range and simultaneously has streptomycin resistance.

The step (2) is specifically as follows: inoculating agrobacterium K599 carrying pCambia1305.2 vector and no exogenous vector to 4mL LB liquid medium (Tryptone 1g, Yeast extract 0.5g, NaCl 1g, NaOH or HCl to adjust pH value to 7.0, deionized water to 100mL, autoclaving at 121 ℃ for 20 min); among them, LB liquid medium contained 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin. Shaking at 200rpm and culturing at 28 deg.C overnight to OD₆₀₀The value is about 1.2, 1mL of the bacterial liquid is taken and transferred into 20mL of fresh LB liquid culture medium again (volume ratio is 1:20), the bacterial liquid is cultured at 28 ℃ and 200rpm with shaking until the OD600 value is 1.2, and then acetosyringone with the final concentration of 100 mu mol/mL is added.

The step (3) is specifically as follows: under the normal growth condition, selecting well-grown middle caragana seedlings growing for 6-9 days, and selecting plants with stout hypocotyls for injection. And (3) sucking the agrobacterium suspension which is added with the acetosyringone and then stands for 24h by using a 1mL needle tube, pressing the needle tube with proper force after the needle tube is fully sucked, and pricking holes at the hypocotyl part to inject the bacterial liquid, so that the bacterial liquid is ensured to be injected into the pricked holes and the pricked holes are vertical to the hypocotyl as much as possible. 1 injection is carried out every 24h, 3 injections are carried out totally, and dark treatment is carried out for 24h after each injection is finished and 3d totally.

The step (4) is specifically as follows: after infection, the culture humidity is kept at about 75%, the temperature is kept at about 25 ℃, and the hairy roots of caragana intermedia are selected within 21-28 days to detect the GUS gene expression condition. The leaves were then stained in GUS staining solution.

Compared with the prior art, the invention has the following advantages:

1. according to the invention, the gene is over-expressed in the hairy root of the caragana intermedia by injecting K599 agrobacterium infection, the method is simple to operate, high in expression level and reliable in result, and can provide technical support for developing gene function verification and screening functional genes; and when the hairy root grows to a certain size, the main root is cut off, and the hairy root replaces the main root to become a part of the middle caragana plant and form a chimera of the transgenic hairy root and the stem and leaf of the original plant.

2. The method takes bacterial liquids with different concentrations as initial conditions for exploration, selects acetosyringone with the best effect and the concentration of 100 mu mol/mL from acetosyringone with different concentrations to assist the infection of agrobacterium, increases the infection efficiency of agrobacterium, and further improves the rooting efficiency of hairy roots by increasing the injection times. In conclusion, the optimal genetic transformation efficiency of the hairy roots is finally determined through different condition exploration.

Drawings

FIG. 1: schematic structure of plant expression vector pCambia 1305.2.

FIG. 2 shows the results of PCR verification of pCambia1305.2 vector, in which 1-10 are the amplification results of primer ① and 11-20 are the amplification results of primer ②.

FIG. 3: growing 7-day-old middle caragana seedlings and infecting the hypocotyls of the middle caragana by an injection method, wherein a is the 7-day-old middle caragana seedlings and b is the process of infecting the hypocotyls of the middle caragana by the injection method.

FIG. 4: the difference between the hypocotyl after injection and the hypocotyl before non-injection.

FIG. 5: injecting middle caragana that grows for different time after infection.

FIG. 6: GUS staining condition of caragana intermedia after infection.

Detailed Description