CN114480480A - Method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes - Google Patents

Method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes Download PDF

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CN114480480A
CN114480480A CN202210238928.8A CN202210238928A CN114480480A CN 114480480 A CN114480480 A CN 114480480A CN 202210238928 A CN202210238928 A CN 202210238928A CN 114480480 A CN114480480 A CN 114480480A
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王彩云
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Huazhong Agricultural University
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Abstract

The invention discloses a method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes, belonging to the technical field of plant tissue culture, and the technical scheme is characterized by comprising the following steps: s1, cutting off pyrethrum tissue culture seedling leaves, and infecting the leaves with bacterial liquid; s2, filtering out leaves, inoculating in MS minimal medium, culturing in dark for 2 days; s3, moving the leaves into the solid culture medium I for culturing for 20 days to form leaf roots; s4, cutting off the root tip of the leaf root, placing the cut leaf root tip on a solid culture medium II with the root tip upward, and culturing for 7 days to obtain a hairy root; s5, selecting a hairy root without geotropism, cutting off the root tip of the hairy root, placing the cut hairy root on a solid culture medium III, and culturing for 20 days to obtain a resistant root; s6, selecting resistant roots, cutting into multiple sections, horizontally placing on a solid culture medium IV, and then carrying out subculture. The invention is mainly used for obtaining transgenic pyrethrum hairy roots, establishes a high-efficiency and rapid pyrethrum hairy root transformation system, and can provide effective technical support for scientific research and breeding work of pyrethrum.

Description

Method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes.
Background
Pyrethrum (Tanacetum cineraiifolium, syn. chrysantheum cineraiifolium) is perennial flowers of the family asteraceae and is the only plant for intensive cultivation for extracting natural insecticide pyrethrin. Because the pyrethrin has strong activity of killing pests and low toxicity to human bodies, the pyrethrin is easy to degrade into nontoxic substances in the environment and is widely accepted by agricultural producers. The emerging biological technology provides a new method for scientific research and breeding of pyrethrum. However, the molecular studies of pyrethrum have been difficult to develop due to the lack of a rapid and efficient indigenous transformation system.
Introduction of foreign genes into cells and subsequent induction of transgenic cells to regenerate into plants is an essential operation for stable transformation of plants, which involves cell dedifferentiation, integration of T-DNA, redifferentiation of cells, and selection of transgenic plants. Because the screening and regeneration are carried out simultaneously, the processes are long and tedious, the regenerated plants do not grow to a sufficient size, the explants die under the stress of antibiotics, and the released phenolic substances poison the regenerated plants. Currently, only one study reported transformation of pyrethrum, which utilizes Agrobacterium tumefaciens to transform pyrethrum leaves to obtain stable transgenic plants. However, this method is not suitable for molecular studies of pyrethrum because of its long experimental period and low transformation efficiency.
To date, relatively few studies have been made on pyrethrum transgenes, and the classical transformation methods available today are time consuming, laborious and inefficient.
In order to solve the above problems, there is provided an Agrobacterium rhizogenes-mediated method for obtaining transgenic pyrethrum hairy roots based on the prior art.
Disclosure of Invention
The invention aims to provide a method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes, which has the advantages of easily obtained materials, short experimental period, rapid amplification of transgenic materials, low requirement on pyrethrum genotype, low cost, high transformation efficiency and capability of rapidly and stably expressing foreign genes in pyrethrum; the invention establishes a high-efficiency and rapid pyrethrum hairy root transformation system for the first time, and can provide effective technical support for scientific research and breeding work of pyrethrum.
The technical purpose of the invention is realized by the following technical scheme:
a method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes comprises the following steps:
s1, cutting leaf from petiole of asexual W99 pyrethrum, and applying OD6000.5 of agrobacterium rhizogenes MSU440 bacterial liquid infects the leaves;
s2, filtering the leaves in the step S1, sucking bacterial liquid on the surfaces of the leaves by using filter paper, inoculating the leaves with the front faces upward into an MS minimal medium, and culturing in dark for 2 days;
s3, transferring the leaves in the step S2 to a solid medium I for culturing for 20 days, and inducing the leaves to form leaf roots;
s4, cutting off the root tips of the leaf roots, horizontally placing the root tips side by side on a solid culture medium II, vertically placing the solid culture medium II with the tip ends of the root tips facing upwards, and culturing for 7 days to obtain hairy roots;
s5, selecting a hairy root without geotropism, cutting off the root tip of the hairy root, horizontally placing the cut hairy root on a solid culture medium III side by side, and culturing for 20 days to obtain a resistant root;
s6, selecting an extensible resistant root, cutting the resistant root into multiple sections, horizontally placing the sections on a solid culture medium IV, and then carrying out subculture to obtain the transgenic pyrethrum hairy root.
Further, in step S3, the solid medium I comprises MS minimal medium and 400mg/L cefotaxime; in step S4, the solid medium II comprises MS minimal medium and 400mg/L cefotaxime.
Further, in step S5, the solid medium III comprises MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin; in step S6, the solid medium IV comprises MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
Further, the MS culture medium, the solid culture medium I, the solid culture medium II, the solid culture medium III and the solid culture medium IV all contain 0.7% of agar and 3% of sucrose.
Further, the MS culture medium, the solid culture medium I, the solid culture medium II, the solid culture medium III and the solid culture medium IV are all cultured under the dark condition at the temperature of 24-26 ℃.
Through the technical scheme, the agrobacterium rhizogenes can simultaneously carry pRi and pTi plasmids, the two plasmids can be simultaneously transferred into plant cells together, and transgenic hairy roots can be formed after the cells are simultaneously transferred into the pRi and pTi plasmids; wherein, the pTi plasmid carries the target gene, and the pRi plasmid is the plasmid carried by the agrobacterium rhizogenes and has the related gene for synthesizing auxin, so that the hairy root of the stably transformed pRi plasmid grows very rapidly; also, pRi-transformed roots are less sensitive to gravity than normal roots because the genes involved in the synthesis of auxin carried interfere with the pluripotency of hairy roots; therefore, by using gravity and double screening of antibiotics, hairy roots co-transformed by pRi and pTi plasmids can be obtained.
In conclusion, the invention has the following beneficial effects: the invention has the advantages of easily obtained materials, short experimental period, rapid amplification of transgenic materials, low requirement on pyrethrum genotype, low cost and high transformation efficiency, and can quickly and stably express exogenous genes in pyrethrum; the invention establishes a high-efficiency and rapid pyrethrum hairy root transformation system for the first time, and can provide effective technical support for scientific research and breeding work of pyrethrum.
Drawings
FIG. 1 is a schematic flow chart of the genetic transformation experiment in example 1 of the present invention;
FIG. 2 is a drawing showing induction of hairy roots of pyrethrum according to example 1 of the present invention, wherein the scale in the drawing represents 1.0 cm;
FIG. 3 is a drawing showing Agrobacterium rhizogenes (carrying pBI121 vector) mediated transformation of pyrethrum according to example 1 of the present invention, the scale in the drawing representing 5.0 mm;
FIG. 4 is a molecular characterization map of TcDXS overexpression transgenic hairy roots of example 2 of the present invention, wherein the scale represents 1.0 cm;
FIG. 5 is a gene editing analysis chart of transgenic hairy roots after one month of subculture in example 3 of the present invention, wherein the scale in the chart represents 1.0 cm;
FIG. 6 is a graph showing the measurement of the change in the E- (b) -farnesene content of TcEbF gene editing hairy root by GCMS in example 3 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the following figures and embodiments:
example 1: transformation of pyrethrum hairy root marker gene GUS, as shown in FIG. 1, the procedure was as follows:
s1 culturing pBI121 vector-containing MSU440 Agrobacterium rhizogenes to OD using YEP liquid medium600At 0.5, the Agrobacterium was resuspended in liquid MS minimal medium containing 100uM acetosyringone and the OD600 was adjusted to 0.5.
Under aseptic conditions, leaves of a clone W99 tissue culture seedling of pyrethrum grown for nearly one month after subculture were cut from petioles with a scalpel, and OD was used6000.5 of agrobacterium rhizogenes MSU440 bacterial liquid to infect the leaves; the agrobacterium rhizogenes MSU440 bacterial liquid is enabled to overflow the leaves and slightly shake for 5 min.
S2, filtering the leaves in the step S1, drying bacterial liquid on the surfaces of the leaves by using filter paper, inoculating the leaves with the front faces upward into the MS minimal medium, and culturing for 2 days in the dark.
S3, transferring the leaf blade of the step S2 to a solid medium I for culturing for 20 days, and inducing the leaf blade to form a leaf blade root, as shown in figure 2a, wherein in figure 2a, after 20 days of transformation, a leaf blade explant generates a root; the solid culture medium I comprises MS minimal medium and 400mg/L cefotaxime.
S4, cutting off the root tip of the leaf root by using a scalpel, wherein the length of the root tip is about 5mm, horizontally placing the root tips side by side on a solid culture medium II, vertically placing the solid culture medium II, placing the tip of the root tip upwards, and culturing for 7 days to obtain a hairy root, as shown in figure 2b, placing the root tip on the culture medium in figure 2b, and vertically placing a culture dish to ensure that the root tip is upwards; the solid culture medium II comprises MS minimal medium and 400mg/L cefotaxime.
S5, selecting hairy roots without geotropism, as shown in figure 2c, wherein the figure 2c shows that after 1 week of culture, two different roots appear, one has geotropism and the other does not have geotropism; DNA was extracted and PCR-checked using rolB gene specific primers to determine that these roots were hairy roots stably transformed with pRi, as shown in FIG. 2d, which is a PCR-test for the presence of rolB gene in both roots (1: bacterial plasmid DNA as positive control; 2, 3, 4: roots with geotropism; 5, 6, 7: non-geotropism).
Meanwhile, cutting off the root tip of the pRi stably transformed hairy root with the length of about 5mm, horizontally placing the root tip on a solid culture medium III side by side, and culturing for 20 days to obtain a resistant root; solid medium III included MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
S6, selecting resistant roots with the length more than 3cm, as shown in figure 3a, wherein figure 3a shows that Carna resistant roots appear after culturing on a selection culture medium containing 10mg/L kanamycin for 20 days; the resistant roots were cut into multiple sections and placed horizontally on solid medium IV, followed by subculture to obtain large quantities of transgenic pyrethrum hairy roots, as shown in FIG. 3b, where FIG. 3b shows the transfer of the resistant hairy roots to selection medium containing 10mg/L kanamycin for 60 days.
The solid culture medium IV comprises MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
DNA of kanamycin-resistant roots was extracted and PCR detection was performed using primers specific to NPTII, as shown in FIG. 3d, where FIG. 3d is the detection of NPT II gene in resistant roots after 60 days of culture using PCR (M: marker +: bacterial plasmid DNA as positive control, -: wild type plant as negative control).
Histochemical staining was performed simultaneously with X-gluc reagent, as shown in FIGS. 3c and 3e, FIG. 3c stained transgenic roots with X-gluc reagent; FIG. 3e is a stain magnification of line 5 (numbers representing different transgenic lines, 1: line 1, 2: line 2, 3: line 3, 4: line 4, 5: line 5), determining these roots as hairy roots stably transformed with pTi.
Example 2: the pyrethrum is transformed into 35S, wherein the obtaining of the hairy root of the TcDXS gene comprises the following steps:
s1 culturing the Agrobacterium rhizogenes MSU440 containing pBI121-35S (TcDXS vector) to OD using YEP liquid medium600At 0.5, the Agrobacterium was resuspended in liquid MS minimal medium containing 100uM acetosyringone and the OD600 was adjusted to 0.5.
Under aseptic conditions, leaves of a clone W99 tissue culture seedling of pyrethrum grown for nearly one month after subculture were cut from petioles with a scalpel, and OD was used6000.5 of agrobacterium rhizogenes MSU440 bacterial liquid to infect the leaves; the agrobacterium rhizogenes MSU440 bacterial liquid is enabled to overflow the leaves and slightly shake for 5 min.
S2, filtering the leaves in the step S1, drying bacterial liquid on the surfaces of the leaves by using filter paper, inoculating the leaves with the front faces upward into the MS minimal medium, and culturing for 2 days in the dark.
S3, transferring the leaves obtained in the step S2 to a solid medium I for culturing for 20 days, and inducing the leaves to form leaf roots; the solid culture medium I comprises MS minimal medium and 400mg/L cefotaxime.
S4, cutting off the root tip of the leaf root by using a scalpel, wherein the length of the root tip is about 5mm, horizontally placing the root tip side by side on a solid culture medium II, vertically placing the solid culture medium II, enabling the tip end of the root tip to be upwards placed, and culturing for 7 days to obtain a hairy root; the solid culture medium II comprises MS minimal medium and 400mg/L cefotaxime.
S5, selecting hairy roots without geotropism, extracting DNA, carrying out PCR detection by using rolB gene specific primers, and determining the roots to be the hairy roots stably transformed by pRi.
Meanwhile, cutting off the root tip of the pRi stably transformed hairy root with the length of about 5mm, horizontally placing the root tip on a solid culture medium III side by side, and culturing for 20 days to obtain a resistant root; solid medium III included MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
S6, selecting resistant roots with the length of more than 3cm, cutting the resistant roots into a plurality of sections, horizontally placing the sections on a solid culture medium IV, and then carrying out subculture to obtain three hairy root strain lines LineB (shown in figure 4a), LineJ (shown in figure 4b) and LineK (shown in figure 4c) with TcDXS over-expression.
The solid culture medium IV comprises MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
Extracting DNAs of LineB, LineJ and LineK, and performing PCR by using specific primers of NPTII and rolB, wherein the results show that LineB, LineJ and LineK all contain T-DNA insertions of pRi and pTi plasmids, as shown in FIG. 4d and FIG. 4e, and FIG. 4d is the detection of NPT II genes in resistant roots by using PCR; FIG. 4e is the detection of rolB gene in resistant roots using PCR.
The fluorescent quantitative detection of the expression level of TcDXS in LineB, LineJ and LineK shows that the expression level of TcDXS in the three transgenic lines is obviously higher than that of wild plants, as shown in FIG. 4f, FIG. 4f is the expression level of TcDXS in the transgenic lines tested by RT-PCR.
Example 3: the editing capacity of a ribozyme CRISPR/Cas9 vector is verified by utilizing a pyrethrum hairy root transformation system, and the steps are as follows:
s1, culturing MSU440 Agrobacterium rhizogenes to OD with YEP liquid medium containing Ceribozyme CRISPR/Cas9 vector6000.5, resuspend Agrobacterium with liquid MS minimal medium containing 100uM acetosyringone and place the OD600Adjusted to 0.5.
Under aseptic conditions, leaves of a clone W99 tissue culture seedling of pyrethrum grown for nearly one month after subculture were cut from petioles with a scalpel, and OD was used6000.5 of agrobacterium rhizogenes MSU440 bacterial liquid to infect the leaves; the agrobacterium rhizogenes MSU440 bacterial liquid is enabled to overflow the leaves and slightly shake for 5 min.
S2, filtering the leaves in the step S1, drying bacterial liquid on the surfaces of the leaves by using filter paper, inoculating the leaves with the front faces upward into the MS minimal medium, and culturing for 2 days in the dark.
S3, transferring the leaves obtained in the step S2 to a solid medium I for culturing for 20 days, and inducing the leaves to form leaf roots; the solid culture medium I comprises MS minimal medium and 400mg/L cefotaxime.
S4, cutting off the root tip of the leaf root by using a scalpel, wherein the length of the root tip is about 5mm, horizontally placing the root tip side by side on a solid culture medium II, vertically placing the solid culture medium II, enabling the tip end of the root tip to be upwards placed, and culturing for 7 days to obtain a hairy root; the solid medium II comprises MS minimal medium and 400mg/L cefotaxime.
S5, selecting hairy roots without geotropism, extracting DNA, carrying out PCR detection by using rolB gene specific primers, and determining the roots to be the hairy roots stably transformed by pRi.
Meanwhile, cutting off the root tip of the pRi stably transformed hairy root with the length of about 5mm, horizontally placing the root tip on a solid culture medium III side by side, and culturing for 20 days to obtain a resistant root; solid medium III included MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
S6, selecting resistant roots with the length of more than 3cm, cutting the resistant roots into multiple sections, horizontally placing the sections on a solid culture medium IV, and then carrying out subculture to obtain hairy root Line A and Line B edited by two TcEbF genes, as shown in figures 5a, 5B and 5c, wherein figure 5a is a control group (CK); FIG. 5b is Line A; FIG. 5c is Line B.
The solid culture medium IV comprises MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
Extracting DNA of Line A and Line B, and carrying out PCR by using ribozyme vector sequence specific primers and rolB specific primers, wherein the results show that both Line A and Line B contain the T-DNA insertion of pRi and ribozyme CRISPR/Cas9 plasmids, as shown in figure 5d and figure 5e, wherein figure 5d is PCR detection RGR sequences (M: marker, +: bacterial plasmid DNA as positive control,: wild type plant as negative control, 1: strain 1, 2: strain 2;); FIG. 5e is the PCR/RE detection of the target sites (M: marker, 1-: TcEbFS fragment of Control (CK), 1+: TcEbFS fragment of Control (CK) digested with BstBI, 2-: TcEbFS fragment of Line A, 2+: TcEbFS fragment of Line A digested with BstBI, 3-: TcEbFS fragment of Line B, 3+: TcEbFS fragment of Line B digested with BstBI).
Sequencing is carried out on the gene sequences of TcEbF in Line A and Line B, and the result shows that the gene sequences of TcEbF in Line A and Line B have base insertion and deletion near the target point, as shown in figure 5f, and figure 5f shows that DNA sequencing confirms TcEbFS gene mutation in pyrethrum hairy roots. The net length of the base insertion or deletion is indicated in the left column, and the number in parentheses indicates the number of clones sequenced E.coli.
The E- (B) -farnesene content of the control group (CK), the Line A and the Line B is measured by GCMS, the (E) -beta-farnesene content of the Line A and the Line B is found to be significantly lower than that of the control group (CK), as shown in figure 6, the E- (B) -farnesene content change of the TcEbF gene editing hairy root is measured by GCMS in figure 6; wherein, FIG. 6a is 1ng/ul E- (b) -farnesene standard substance; FIG. 6b is a control group; FIG. 6c is Line A; FIG. 6d is Line B; FIG. 6e is the (e) - β -farnesene content in Control (CK), Line A and Line B.
By adopting the technical scheme, the invention has the advantages of easily obtained materials, short experimental period, rapid amplification of transgenic materials, low requirement on pyrethrum genotype, low cost and high transformation efficiency, and can quickly and stably express exogenous genes in pyrethrum; the invention establishes a high-efficiency and rapid pyrethrum hairy root transformation system for the first time, and can provide effective technical support for scientific research and breeding work of pyrethrum.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications without inventive contribution to the present embodiment as required after reading the present specification, but all of them are protected by patent law within the scope of the present invention.

Claims (5)

1. A method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes is characterized by comprising the following steps:
s1, cutting leaf from petiole of asexual W99 pyrethrum, and applying OD6000.5 of agrobacterium rhizogenes MSU440 bacterial liquid infects the leaves;
s2, filtering the leaves in the step S1, sucking bacterial liquid on the surfaces of the leaves by using filter paper, inoculating the leaves with the front faces upward into an MS minimal medium, and culturing in dark for 2 days;
s3, transferring the leaves in the step S2 to a solid medium I for culturing for 20 days, and inducing the leaves to form leaf roots;
s4, cutting off the root tip of the leaf root, horizontally placing the root tip side by side on a solid culture medium II, vertically placing the solid culture medium II, placing the tip of the root tip upwards, and culturing for 7 days to obtain a hairy root;
s5, selecting a hairy root without geotropism, cutting off the root tip of the hairy root, horizontally placing the cut hairy root on a solid culture medium III side by side, and culturing for 20 days to obtain a resistant root;
s6, selecting an extensible resistant root, cutting the resistant root into multiple sections, horizontally placing the sections on a solid culture medium IV, and then carrying out subculture to obtain the transgenic pyrethrum hairy root.
2. The method for Agrobacterium rhizogenes-mediated obtaining of transgenic pyrethrum hairy roots according to claim 1, wherein: in step S3, the solid culture medium I comprises MS minimal medium and 400mg/L cefotaxime; in step S4, the solid medium II comprises MS minimal medium and 400mg/L cefotaxime.
3. The method for Agrobacterium rhizogenes-mediated obtaining of transgenic pyrethrum hairy roots according to claim 1, wherein: in step S5, the solid medium III comprises MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin; in step S6, the solid medium IV comprises MS minimal medium, 400mg/L cefotaxime and 10mg/L kanamycin.
4. The method for Agrobacterium rhizogenes-mediated obtaining of transgenic pyrethrum hairy roots according to claim 1, wherein: the MS culture medium, the solid culture medium I, the solid culture medium II, the solid culture medium III and the solid culture medium IV all contain 0.7 percent of agar and 3 percent of cane sugar.
5. The method for Agrobacterium rhizogenes-mediated obtaining of transgenic pyrethrum hairy roots according to claim 1, wherein: and the MS culture medium, the solid culture medium I, the solid culture medium II, the solid culture medium III and the solid culture medium IV are all cultured under a dark condition at the temperature of 24-26 ℃.
CN202210238928.8A 2022-03-07 2022-03-07 Method for obtaining transgenic pyrethrum hairy roots mediated by agrobacterium rhizogenes Pending CN114480480A (en)

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Non-Patent Citations (2)

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Title
党小琳;: "除虫菊愈伤组织和发状根中除虫菊酯杀虫活性测定", 中国农学通报, no. 23 *
公冶祥旭等: "除虫菊发状根的诱导及培养条件优化", 植物科学学报, vol. 35, no. 3, pages 428 - 429 *

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