CN112210570A

CN112210570A - Method for cultivating lily with pollen abortion and herbicide resistance

Info

Publication number: CN112210570A
Application number: CN202011002187.0A
Authority: CN
Inventors: 郑思乡; 廖晓珊; 荣朵艳; 周俊; 张翔; 谭益民; 汤建新; 宋志伟; 邓雅文; 林庆丹; 吴龙云
Original assignee: Zhuzhou Institute Of Agricultural Science
Current assignee: Zhuzhou Institute Of Agricultural Science
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2021-01-12

Abstract

The invention provides a method for cultivating pollen abortion and herbicide-resistant lily, which mainly comprises the following steps: s1, constructing a recombinant vector, loading a promoter TA29 and a terminator Nos onto Barnase to obtain TA29-Barnase-NOs, inserting the TA29-Barnase-NOs into a vector I, and extracting a recombinant plasmid after propagation; s2, recovering target DNA from the recombinant plasmid obtained in the step S1, performing PCR amplification, and connecting to a vector II to obtain a recombinant vector; s3, transferring the constructed recombinant vector into lily scales by using an agrobacterium-mediated method, obtaining positive plants by using a herbicide screening method, optimizing an agrobacterium-mediated method system, and transferring into a rooting and germination induction culture medium for culture to obtain the positive transgenic lily. The obtained lily plants are successfully transferred into the pollen abortion gene, no pollen is generated during flowering, and the lily plants have the characteristic of herbicide resistance, so that the efficient weeding by using the herbicide in the planting process is facilitated, the use of labor force can be effectively reduced, and the production benefit is improved.

Description

Method for cultivating lily with pollen abortion and herbicide resistance

Technical Field

The invention relates to the field of plant cultivation, in particular to a method for transferring a target gene into a lily plant through genetic engineering and screening the lily plant to obtain pollen abortion and herbicide resistance.

Background

Lily plants are common agricultural garden plants, bulbs of the lily plants can be used as food or medicine, and lily is popular among people as a common ornamental flower. In order to meet the diversified demands, the requirement for cultivating the new lily germplasm is correspondingly improved. The application of the fertilizer and the pesticide in the lily planting process can be carried out by a mechanical device, the production efficiency is high, and the labor cost is relatively low; in contrast, weed removal in the lily planting process cannot be effectively performed mechanically, artificial weeding efficiency is low, and multiple times of weeding are required in the lily planting period, so that labor cost is high. In addition, the transgenic plant formed by genetic engineering is easy to cause gene drift through pollen mediation, so that exogenous genes enter non-transgenic plants to cause biological safety problems such as gene pollution and the like.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for cultivating lily plants, the obtained lily plants are successfully transferred into a pollen abortion gene, and the field identification shows that no pollen is generated and the lily plants have herbicide resistance, so that the method is convenient for using herbicides to efficiently weed in the planting process to replace artificial weeding, can effectively reduce the use of labor force and improve the production benefit.

The technical scheme of the invention is to provide a pollen abortion and herbicide-resistant lily cultivation method, which mainly comprises the following steps:

s1, constructing a recombinant vector

Loading a promoter TA29 and a terminator Nos onto Barnase to obtain a TA29-Barnase-Nos sequence, inserting the TA29-Barnase-Nos into a vector I, and then carrying out amplification to extract a recombinant plasmid; wherein the TA29-Barnase-NOs sequence is shown in SEQ ID NO: 1 is shown in the specification;

s2, recovering target DNA from the recombinant plasmid obtained in the step S1 to be used as a template, carrying out PCR amplification, and connecting to a vector II to obtain a recombinant vector;

s3, transferring the constructed recombinant vector into lily scales by using an agrobacterium-mediated method, obtaining positive plants by using a herbicide screening method, optimizing an agrobacterium-mediated method system, and transferring into a rooting and germination induction culture medium for culture to obtain the positive transgenic lily.

Further, the vector I in the step S1 is a pUC57-simple vector;

further, vector II in step S2 is Pcambia3301 vector;

through the primary transformation of the vector I, the recovered DNA is used as a template to construct a recombinant vector, so that other gene segments influencing the properties of the target gene segment such as ligase activity and the like can be removed, and a purer target gene can be obtained.

Further, the Agrobacterium concentration used in the Agrobacterium-mediated method in step S3 is OD₆₀₀The value is 0.4-1.0, the infection time is 5-15 minutes, and the co-culture time is less than or equal to 4 days.

Further, the herbicide in the step S3 is glyphosate, and the screening concentration is 1-3 mg/L.

The invention has the advantages and beneficial effects that: the invention firstly transforms the pollen abortion gene into a recombinant vector through genetic engineering, then the recombinant vector is transformed into lily scales through an agrobacterium-mediated method, transgenic lily is obtained through culture, and simultaneously lily plants with herbicide-resistant genes are screened out through a herbicide screening method, so that the obtained plants have herbicide-resistant characteristics and pollen abortion characteristics at the same time, and the pollen abortion can avoid the biosafety problem caused by the herbicide-resistant genes through the gene drift of pollen as a medium.

Drawings

FIG. 1 is a schematic representation of the recombinant vector TA 29-Barnase-Nos.

FIG. 2 shows the results of electrophoresis of plasmid DNA from Pcambia3301 and Pucs7-bar, where M: DNA Marker; 1-2: pcambia3301 plasmid DNA; 3-4: pucs7-bar plasmid DNA.

FIG. 3 shows the result of the cleavage and identification of Pcambia3301 plasmid, wherein M: DNA Marker; 1: a Pcambia3301 negative control which is not digested; 2: the Pcambia3301 after the enzyme digestion.

FIG. 4 shows the results of PCR amplification of Pucs7-bar at 57-60 ℃ where M: DNA Marker; 1-4: the annealing temperatures of 57 ℃, 58 ℃, 59 ℃ and 60 ℃ respectively to obtain the bands of the amplified Pucs 7-bar.

FIG. 5 shows PCR amplification for recovery detection of Pucs7-bar, M: DNA Marker; 1-2: the product was recovered at 57 ℃ and 58 ℃.

FIG. 6 is a negative-positive control identification colony PCR, where M: DNA Marker; 1-12: positively cloning strains; 13: a positive control; 14: and (5) negative control.

FIG. 7 is a schematic diagram of recombinant vector Pcambia3301-TA 29-Barnase-Nos.

FIG. 8 is a graph showing the effect of preculture time on genetic transformation.

FIG. 9 shows the effect of infection time and bacterial concentration on genetic transformation.

FIG. 10 is a comparison of lily bulb scale infection before and after infection.

FIG. 11 shows the growth of lily in the dark, pre-cultured for 3 days, the 2-week growth after ppt selection, the 1.5-month growth after multiple changes of selection medium, and the 3-month growth after infection.

FIG. 12 shows the results of PCR transgene molecule detection and GUS identification.

FIG. 13 is a diagram showing the alignment of the recovered PCR positive fragment sequence and the target gene sequence.

FIG. 14 shows field cultivation of lily, in which FIG. A shows transgenic lily plants and FIG. B shows non-transgenic lily plants.

FIG. 15 is a photograph of transgenic lilies after one month of growth.

FIG. 16 is a photograph of transgenic lilies after three months of growth.

Detailed Description

The present invention will be further described with reference to the following embodiments.

Example 1

1.1 Synthesis of TA29-Barnase-Nos element

The sequence information of the TA29-Barnase-NOs element is searched in a NCBI database, and SpeI and Pst I enzyme cutting sites are added between a TA29 promoter and a Barnase regulation element, so that the promoter can be conveniently replaced in subsequent experiments. Sending sequence information to Huada Gene company for synthesizing TA29-Barnase-NOs elements, wherein the sequence is shown as SEQ ID NO: 1 is shown. The Huada Gene Co.inserted the synthesized element into a pUC57-simple vector, and the vector obtained after insertion was named pUC57-simple-TA29-bar (Pucs 7-bar for short) as shown in FIG. 1.

1.2 LB Medium preparation

The components of 150mL solid-liquid culture medium are as follows:

TABLE 1.1 LB Medium

1.3 propagation and extraction of plasmids

The plasmid extraction of Pcambia3301 and Pucs7-bar was performed using the Shanghai worker Sanprep column plasmid extraction kit.

1.4 agarose gel electrophoresis detection of target plasmids

The target plasmid was detected by agarose gel electrophoresis, and the results are shown in FIG. 2, where M: DNA Marker; 1-2: pcambia3301 plasmid DNA; 3-4: pucs7-bar plasmid DNA.

1.5 double digestion

The Pcambia3301 plasmid DNA extracted by enzyme digestion with restriction enzymes EcoRI and HindIII is used as a template and reacted in a water bath at 37 ℃ overnight for double digestion.

TABLE 1.250. mu.L double enzyme digestion System

1.6 recovery of double digestion products by rubber cutting

The plasmid DNA of Pcambia3301 after double digestion was recovered using the UNIQ-10 column mini agarose gel DNA recovery kit, as shown in FIG. 3, where M: DNA Marker; 1: a Pcambia3301 negative control which is not digested; 2: the Pcambia3301 after the enzyme digestion.

1.7 PCR amplification to obtain TA29-Barnase-NOs target sequence

The PCR amplification system is shown in Table 1.3:

TABLE 1.3 Pucs7-barPCR

The PCR reaction procedure is shown in table 1.4:

TABLE 1.4 PCR reaction procedure

The PCR amplification was performed by extracting pUC57-simple-TA29-bar as a template and configuring a 50. mu.L system, and the results are shown in FIG. 4, in which M: DNA Marker; 1-4: an annealing temperature of 57 ℃, 58 ℃, 59 ℃, 60 ℃. The result of the recovery using UNIQ-10 column type micro agarose gel DNA recovery kit is shown in FIG. 5, M: DNA Marker; 1-2: the product was recovered at 57 ℃ and 58 ℃.

1.8 ligation of recombinant products

The TA29-Barnse-Nos obtained in step 1.7 were ligated to the Pcambia3301 plasmid, as shown in Table 1.5:

TABLE 1.5 connection systems

1.9 double digestion of Pcambia3301-TA29-Barnase-NOs (EcoRI and Spe I) and colony identification

Preparing 50 mu L of enzyme cutting system, carrying out double enzyme cutting on the Pcambia3301 vector connected with TA29-Barnse-Nos, wherein the enzyme cutting system is shown in a table 1.6:

TABLE 1.6 enzyme digestion System

After transformation into escherichia coli and colony culture, single colonies numbered 1-12 were picked and colony PCR was performed, and the results are shown in fig. 6, where M: DNA Marker; 1-12: positively cloning strains; 13: a positive control; 14: and (5) negative control. According to the colony identification result, the number 10 of twelve selected colonies 1 to 12 shows positive clones, the comparison 13 is a certain Pucs7-bar positive control capable of amplifying the result and a negative control 14 without a carrier for analysis, the positive clones and the positive control bands are both located at about 1000bp, and the negative control band has no band. Therefore, the ligation was successful, and the recombinant vector Pcambia3301-TA29-Barnase-No was obtained, as shown in FIG. 7.

Example 2 Agrobacterium-mediated System establishment and optimization

2.1 preculture

Selecting healthy lily seedlings, and cutting lily scales into pieces

Size, inoculated in M1 medium. The scale which is not directly pre-cultured is used for pre-culturing for 1d, 2d, 3d and 4d under the dark condition of 25 ℃, the co-culture time is the same, and the treatment concentration of the agrobacterium is the same as the screening culture condition. After 40 days, it was confirmed by counting and comparing the regeneration rates of the different resistant shootsThe pre-incubation time is determined. The results of the study showed that three days of preculture were most favorable for transformation (see FIG. 8).

2.2 preparation of Agrobacterium

200uL of the bacterial solution containing the vector (referring to the obtained recombinant vector shown in FIG. 7) was added to 5mL of LB liquid medium and cultured at 28 ℃ for 20-25 hours on a shaker at 180 r/min. 1mL of the bacterial solution was added to 50mL of LB liquid medium and cultured at 28 ℃ for 18 to 24 hours on a 180r/min shaker.

2.3 Agrobacterium inoculation and Co-cultivation

The bacterial solution containing the carrier was centrifuged in a centrifuge (4000r/min, 10 min) and the supernatant was discarded on a clean bench. The centrifuged cells were added to the MS liquid medium for mixing, and the cut lily scales were immersed. OD600 values were 0.4, 0.6, 0.8 and 1.0, respectively. The explants were immersed in the bacterial solution for 5 min, 8 min, 10 min and 15 min, respectively, and shaken. In order to bring the bacterial suspension into full contact with the scale, 200r/min was used. Shaking the shaking table. Then, it was placed flat on sterile filter paper, the bacterial liquid on the surface was blotted dry, and the scales were placed on AS-containing medium for 0 days, 1 day, 2 days, 3 days, and 4 days. From FIG. 9, it can be seen that the bacterial liquid OD is 0.8 at the highest transformation efficiency when the infection is carried out for 10 minutes. As can be seen from Table 2.1, the effect was the best in the co-cultivation for 3 days.

TABLE 2.1 Effect of Co-cultivation time on transformation efficiency

Co-cultivation time/day	Transformed plant	Conversion rate/%
			0	101	0
1	95	0.3
			2	108	2.5
3	104	3.1
			4	105	2.8

2.4 screening of Glyphosate concentration

Well-growing, uniformly-growing tissue-cultured seedlings of oriental lily are used as explants and inoculated in culture media containing glyphosate at different concentrations. The culture medium is 0.5 mg/L6 BA +1.0mg/L NAA. The concentration of glyphosate was set at 1, 1.25, 1.5, 1.75, 2.0, 2.5, 3mg/L, 7 levels, and the concentration of the blank was 0, cultured for 30 days to observe the differentiation and differentiation state of tissue culture seedlings and calculate the browning rate. From Table 2.2 it can be seen that the optimum screening concentration for ppt is 2.0 mg/L.

TABLE 2.2 screening of ppt (Glyphosate) concentration

2.4 selection and regeneration of transformed plants

The inoculated scales were screened from the co-culture in ppt selection medium. Media and treatment as shown in Table 2.3, and cultured at 25 deg.C, subcultured every 14 days, and grown to 3-4cm for transfer to M4 medium.

TABLE 2.3 media formulation and treatment protocol

FIG. 10 shows a comparison of lily bulb scale infestation before and after infection; FIG. 11 shows the growth of lily in culture, wherein panel a shows the growth of lily pre-cultured for 3 days in the dark, panel b shows the growth of lily after ppt selection for 2 weeks, panel c shows the growth of lily after 1.5 months after multiple changes of selection medium, and panel d shows the growth of lily after 3 months of infection.

Example 3 detection of resistant plant molecules

3.1 PCR detection

From the screening medium of step 2.4, the results of selecting the plants with normal growth for PCR detection and GUS identification are shown in FIG. 12, wherein a is a PCR detection result graph, and b is a GUS identification result graph, wherein M: marker DL 2000; p: a positive plasmid; 0: (ii) an untransformed lily; 1-16 in FIG. a: transforming lily; 1-13 in FIG. b: and (5) converting the lily. PCR identification results show that the detected plants (No. 1-16) are all positive, GUS identification shows that the detected plants (No. 1-13) are all positive, and the preliminary proof shows that the target gene is transformed into the genome of the lily plant.

3.2 recovery of PCR-positive fragments for sequencing

And (3) recovering the PCR positive fragment, wherein the sequencing result of the PCR positive fragment is completely consistent with the target gene sequence as shown in figure 13, so that the target gene is fully proved to be successfully transferred into the genome of the lily plant.

Example 4

FIGS. 14 to 16 show the field cultivation of lily, wherein FIG. 14 shows a diagram A of transgenic lily plants and a diagram B of non-transgenic lily plants, wherein three days after spraying glyphosate with a concentration of 2mg/L, the transgenic lily in the diagram A can grow normally, and the common lily plants in the diagram B are yellow and die; FIG. 15 shows transgenic lilies grown for one month; FIG. 16 shows the growth of transgenic lilies for three months. The field identification result shows that after herbicide spraying, all non-transgenic plants die, the survival rate is 0%, and the survival rate of the transgenic plants is 100%. The identification result in the further flowering process shows that the pollen of the non-transgenic plant is normal and the pollen quantity is large. Whereas transgenic plants do not produce pollen. The cultivation method of the invention successfully obtains new germplasm of the herbicide-resistant and pollen-free lily.

Materials, reagents and experimental equipment related to the embodiment of the invention are all commercial products meeting the field of biomedical engineering if no special description is provided.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, modifications and decorations can be made without departing from the core technology of the present invention, and these modifications and decorations shall also fall within the protection scope of the present invention. Any changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Sequence listing

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<120> method for cultivating pollen abortion and herbicide-resistant lily

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Claims

1. The method for cultivating the lily with pollen abortion and herbicide resistance is characterized by comprising the following steps:

s1, constructing a recombinant vector

Loading a promoter TA29 and a terminator Nos onto Barnase gene to obtain TA29-Barnase-Nos, inserting the TA29-Barnase-Nos into a vector I, and then carrying out amplification to extract recombinant plasmids; wherein the TA29-Barnase-NOs sequence is shown in SEQ ID NO: 1 is shown in the specification;

s3, transferring the constructed recombinant vector into lily scales by using an agrobacterium-mediated method, obtaining positive plants by using a herbicide screening method, optimizing an agrobacterium-mediated method system, transferring the positive plants into a rooting and germination induction culture medium for culture,

obtaining the positive transgenic lily.

2. The method of claim 1, wherein the vector I is pUC57-simple vector.

3. The method for pollen abortion and herbicide-resistant lily cultivation of claim 1, wherein the vector II is Pcambia3301 vector.

4. The method of claim 1, wherein in step S1, SpeI and Pst I cleavage sites are inserted between the promoter TA29 and Barnase gene.

5. The method for cultivating pollen abortion and herbicide-resistant lily as claimed in claim 1, wherein the herbicide in step S3 is glyphosate, and the screening concentration is 1-3 mg/L.

6. The method for pollen abortion and herbicide-resistant lily culture of claim 1, wherein the agrobacterium-mediated method in step S3 is performed at an agrobacterium concentration OD₆₀₀The value is 0.4-1.0, the infection time is 5-15 minutes, and the co-culture time is less than or equal to 4 days.

7. The method of claim 5, wherein the glyphosate screening concentration is 2.0 mg/L.

8. The method of claim 6, wherein the concentration of Agrobacterium used in the Agrobacterium mediated method of step S3 is OD₆₀₀The value was 0.8, the infestation time was 10 minutes and the co-cultivation time was 3 days.