CN116144696A

CN116144696A - Virus-induced gene silencing method for rape

Info

Publication number: CN116144696A
Application number: CN202310289784.3A
Authority: CN
Inventors: 刘志文; 杨明煊; 王晨晨; 李芙蓉; 于放; 张悦; 高潇潇; 金朝霞
Original assignee: Dalian Polytechnic University
Current assignee: Dalian Polytechnic University
Priority date: 2023-03-22
Filing date: 2023-03-22
Publication date: 2023-05-23

Abstract

The invention discloses a method for virus-induced gene silencing of rape, belonging to the technical field of biology. The invention firstly obtains the gene conservation area BnBADH gene of rape through PCR amplification, then connects with the virus gene expression vector pTRV2 to obtain pTRV2-BnBADH expression vector plasmid, transfers into competent agrobacterium, verifies correct bacterial liquid retention, mixes pTRV1 with the bacterial liquid according to a certain proportion, centrifugates the bacterial liquid, adds invasion dye liquid into the obtained precipitation bacterial liquid, shake cultures, injects the bacterial liquid from the back of rape seedling leaf between veins, and detects the expression condition of target genes after culturing for a period of time. The method for inducing gene silencing by the pTRV-VIGS virus of the rape, which is established by the invention, can be used for down regulating the relative expression quantity of the target gene of the rape, has the advantages of simplicity, rapidness, high flux and easiness in operation, and provides a new approach and technical support for developing the functional research of the rape gene in the future.

Description

Virus-induced gene silencing method for rape

Technical Field

The invention belongs to the technical field of biology, and relates to a method for researching gene functions of rape, in particular to a virus-induced gene silencing method of rape.

Background

Virus-induced gene silencing (VIGS) is a naturally occurring defense system in plants against the invasion of foreign nucleic acids, which normally protects plants from viral infection. Tobacco embrittlement virus (tobacco rattle virus, TRV) is a virus vector which has wide application range and good efficiency and durability, and can mediate gene silencing without causing virus-induced symptoms. TRV1, which includes genes encoding RNA-dependent RNA polymerase, motor protein and 16KD protein, is a helper virus vector of the VIGS system. TRV2 includes capsid protein genes, multiple cloning sites, etc., for insertion of a gene of interest. The modified virus can promote the insertion of non-viral sequences and the subsequent infection of plants, so TRV has wide application in the functional identification of plant genes.

TRV-VIGS is a transient gene silencing technique, the principle of which is similar to RNA interference, and is mainly used for down-regulating the expression of target genes. The technical principle is as follows: loading the fragment of the target gene into a viral vector, and then infecting plants with the constructed viral vector, thereby causing the phenomenon of homologous gene silencing of the recipient plant. When the viral vector just enters the plant body, RNA of the viral vector is replicated by RNA polymerase of a host to form double-stranded viral RNA, and then the double-stranded viral RNA is recognized and cut by endonuclease to form small interfering RNA. And finally, degrading the target gene into a single strand, wherein the single strand RNA specifically complements the target gene to form local double-stranded RNA combination, and activating a double-stranded RNA degradation mechanism in the plant body, so that the target gene in the host plant is degraded, namely, the target RNA is specifically silenced at the post-transcriptional level.

Compared with the traditional gene function research methods such as transgene, gene knockout, antisense inhibition and the like, the VIGS technology does not need genetic transformation or mutant acquisition of plants, and can simply, quickly, instantaneously, efficiently and specifically identify the gene function. With the continuous development of the VIGS technology, the technology is widely applied to the research and identification of related functional genes such as different plant resistance, growth and development, metabolic regulation and the like, and becomes an effective technology for researching plant gene functions.

However, the current method for researching the gene function of rape has the defects of long time consumption, high cost, complex operation and the like, and the related research for researching and developing a virus-induced gene silencing method of rape and further researching the gene function of rape is not reported yet.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for gene silencing induced by rape virus, which establishes a pTRV-VIGS virus silencing vector system and infects rape through agrobacterium mediation, so as to be expected to be capable of researching the gene function of rape simply, rapidly and in high throughput.

The invention aims at realizing the following steps:

the invention provides a virus-induced gene silencing method for rape, which mainly comprises the following steps:

(1) Obtaining a gene conservation region BnBADH gene of rape through PCR amplification, performing gel cutting recovery after verification to obtain a target gene fragment, adding A reaction to the target gene fragment, and connecting the obtained target gene fragment with a viral gene expression vector pTRV2 through ligase to obtain a pTRV2-BnBADH expression vector plasmid;

(2) Preparing competent agrobacterium, adding the pTRV2-BnBADH expression vector plasmid obtained in the step (1), freezing in liquid nitrogen for 3-10min, standing at 35-39 ℃ for 3-10min, adding LB culture medium, shake culturing at 25-30 ℃ for 1-4h, screening, performing PCR colony verification, and verifying that the correct bacterial liquid is reserved for later use;

(3) Culturing the bacterial liquid in the step (2) to OD ₆₀₀ Uniformly mixing pTRV1 and the obtained bacterial liquid according to the volume ratio of 1:2-2:1 between 0.6 and 0.8, centrifuging, removing the supernatant, adding an invasion dye solution into the precipitated bacterial cells, and carrying out shake culture at 25-30 ℃ for 2-5h;

(4) And (3) injecting the bacterial liquid obtained in the step (3) from the back of the rape seedling leaf between veins, wherein the leaf cannot be crushed during the injection, and detecting the expression condition of the target gene after culturing for a period of time.

Based on the above technical scheme, further, the PCR amplification conditions in step (1) are: 94℃for 5min,94℃for 30s,60℃for 30s,72℃for 15s and 72℃for 10min.

Based on the above technical scheme, further, the nucleotide sequence of the primer amplified by PCR in the step (1) is shown as SEQ ID NO: 1-2.

Based on the technical scheme, the specific process of glue cutting and recycling in the step (1) is as follows: adding 600 μL of sol Buffer into EP tube filled with gel block, standing for 3min after gel block is melted, centrifuging at 12000rpm for 1min, adding into adsorption column, centrifuging at 12000rpm for 1min, pouring centrifugate, adding 600 μL of Washing Buffer, standing for 3min, centrifuging for 1min, changing adsorption column into 1.5mL EP tube, adding 50 μL of ddH ₂ Eluting the DNA by O, and spin-drying the eluent on a vacuum spin-drying instrument for 20min to obtain the DNA.

Based on the technical scheme, further, the conditions of the reaction of adding A in the step (1) are as follows: 60min at 72℃and 10min at 65 ℃.

Based on the above technical scheme, further, the ligase in the step (1) is T4 ligase.

Based on the above technical scheme, further, in the step (2), the agrobacterium is specifically agrobacterium GV3101.

Based on the technical scheme, the preparation process of competent agrobacterium in step (2) is specifically as follows: inoculating Agrobacterium into LB culture medium, and culturing overnight at 28deg.C in shaking table for activationAbsorbing 500. Mu.L of bacterial liquid in 50mL of LB culture medium containing antibiotics, wherein gentamicin is 40. Mu.g/mL and rifampicin is 20. Mu.g/mL, and shake culturing at 28 ℃ to OD ₆₀₀ About 0.8, centrifuging the bacterial liquid at 4000rpm for 10min, removing the supernatant, adding 60mM CaCl ₂ 10mL of the solution was mixed and centrifuged for 10min, the supernatant was removed, and 1mL of 60mM CaCl was added ₂ The solution and 100. Mu.L of dimethyl sulfoxide are mixed uniformly to prepare competent Agrobacterium.

Based on the technical scheme, the centrifugation condition in the step (3) is that the centrifugation is carried out for 3-10min at 3000-5000rpm at 4 ℃.

Based on the technical scheme, further, the preparation process of the infection liquid in the step (3) comprises the following steps: 0.205g of MgCl was added sequentially to 100mL of deionized water ₂ 0.4275g MES, 100 mu L of 200mmol acetosyringone, regulating the pH value to 5.7-5.8, adding 20 mu L of surfactant, and stirring uniformly.

Based on the above technical scheme, further, the culturing conditions in the step (4) are as follows: culturing at 25deg.C for 7-14d under light for 15 hr and dark for 9 hr.

Compared with the prior art, the invention has the following beneficial effects:

1. the virus-induced gene silencing (VIGS) technology directly produces an RNA interference effect on plants, greatly saves time and cost, is not limited by gene elements such as promoters on vectors, and can silence a plurality of genes at the same time.

2. The method avoids the defects of the traditional transgenic system research, and is particularly suitable for immature plants of a transformation system and varieties limited by genotypes, thus being widely used for the gene function research of rape.

3. The method for inducing gene silencing by the pTRV-VIGS virus of the rape, which is established by the invention, can be used for down regulating the relative expression quantity of the target gene of the rape, has the advantages of simplicity, rapidness, high flux and easiness in operation, and provides a new approach and technical support for developing the functional research of the rape gene in the future.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.

Fig. 1 shows a leaf blade after the injection of a bacterial liquid by a syringe, wherein the left half part of the leaf blade is a control group, and the right half part of the leaf blade is an experimental group.

FIG. 2 shows the relative expression of BnBADH in the leaves after the VIGS treatment, the error bars represent the standard error of three biological replicates, representing significant p <0.05 levels;

fig. 3 shows the relative expression levels of saline-alkali resistance related genes in VIGS treated leaves, error bars indicate standard error of three biological replicates, and x indicates significant p <0.05 levels.

Detailed Description

The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.

Example 1

1. Rape planting

The Hua-za 62 rape clone tissue culture Miao Xian which is obtained from the same rape seed and through rapid propagation is subjected to cover opening and seedling hardening for 5-7 days, and then is transplanted from an MS solid culture medium to a culture medium with the components of vermiculite and nutrient soil with the volume ratio of 5:1, the rape leaves grow for 15 to 20 days in the soil at 25 ℃ under the conditions of 15 hours of illumination and 9 hours of darkness, and the infection is carried out when the growth state of the rape leaves is good.

2. Construction of viral gene silencing expression vector

The NCBI is used for searching a target gene conserved domain, and a Primer Premier 5.0 is used for designing a BnBADH gene (restriction sites: xbaI and KpnI) silencing fragment Primer (shown in Table 6) with a restriction site, wherein the fragment size is 447bp. The reaction system of the silencing fragment BnBADH (PCR reaction conditions: 94 ℃,5min, 94 ℃,30s,60 ℃,30s,72 ℃,15s,72 ℃ 10 min) cloned by PCR reaction after primer synthesis is shown in Table 1. Cloning gene fragments by using high-fidelity enzyme, performing agarose gel electrophoresis verification on the products, cutting off target strips after verification of correctness, and performing gel recovery, wherein the method comprises the following specific steps: 600. Mu.L of sol was added to an EP tube containing a gel blockBuffer, after the gel block is melted, adding the gel block into an adsorption column, standing for 3min, and centrifuging for 1min at 12000rpm of a centrifuge. Centrifuging under the same conditions, pouring the centrifugate, adding 600 μl of Washing Buffer, standing for 3min, centrifuging for 1min, and changing the adsorption column to 1.5mLEP tube with 50 μl of ddH ₂ Eluting the DNA by O, spin-drying the eluent on a vacuum spin-drying instrument for 20min, and obtaining the DNA by 16 mu L. The eluted target gene was subjected to an A-addition reaction (PCR reaction conditions: 72 ℃,60min,65 ℃ C., 10 min) and the reaction system was as shown in Table 2.

The BnBADH gene fragment after A addition is connected with pGM-T vector by T4 ligase, 16 ℃ is connected in a PCR instrument for 16 hours, the reaction system is shown in table 3, the connection product is transformed into competent cells of escherichia coli, white monoclonal is selected by blue and white spot screening and is put in 1mL LB culture medium, PCR colony verification is carried out after bacterial liquid is turbid, 200 mu L of correct bacterial strain liquid is inoculated in 50mL LB culture medium for amplification, pGM-T-BnBADH plasmid is extracted by an alkaline lysis method, after verification, the BADH gene fragment is cut off from pGM-T-BnBADH plasmid by a double enzyme digestion method and then connected with virus gene expression vector pTRV2, and the reaction temperature is 16 ℃ in the PCR instrument for 16 hours. The ligation product was transferred into competent cells of E.coli, and plasmids were extracted and verified by double digestion (XbaI and KpnI) to obtain the viral gene silencing expression vector plasmid pTRV2-BnBADH.

3. Agrobacterium transformation

Agrobacterium GV3101 was inoculated into 10mL of LB medium and activated by overnight culture in a shaker at 28 ℃. Extracting 500 μl of bacterial liquid from activated Agrobacterium in 50mL LB medium containing antibiotic, wherein gentamicin 40 μg/mL and rifampicin 20 μg/mL, shake culturing at 28deg.C to OD ₆₀₀ About 0.8 or so. Centrifuging the bacterial liquid at 4000rpm for 10min, removing supernatant, adding 60mM CaCl ₂ The solution was mixed with 10mL and centrifuged for 10min to remove the supernatant. 1mL of 60mM CaCl was added ₂ The solution was mixed with 100. Mu.L of dimethyl sulfoxide to prepare competent Agrobacterium and split into 100. Mu.L/tube.

Three tubes of competent Agrobacterium were taken and added with 2. Mu.L of pTRV1 and pTRV2 empty viral gene silencing expression vector plasmid and pTRV2-BnBADH expression vector plasmid, respectively, and frozen in liquid nitrogen for 5min, and after standing in a 37℃incubator for 5min, 1mL of LB was added for shaking culture based on 28℃for 2h. After centrifugation at 4000rpm for 2min, about 150. Mu.L of supernatant was removed, and the supernatant was plated on LB solid medium containing 50. Mu.g/mL kanamycin and cultured at 28℃for 2d. Single colonies were picked in LB medium containing 50. Mu.g/mL kanamycin, 40. Mu.g/mL gentamicin, and 20. Mu.g/mL rifampicin. And (5) performing PCR colony verification after the bacterial liquid is turbid, and verifying that the correct bacterial liquid is reserved for later use.

4. Preparation of conversion solution

Culturing the bacterial cells: taking 500 mu L of the remaining bacterial liquid, respectively adding into 50mL of LB medium containing 50 mu g/mL of kanamycin, 40 mu g/mL of gentamicin and 20 mu g/mL of rifampicin, adding 5 mu L of 200mmol acetosyringone and 0.1g of MES, culturing at 28 ℃ overnight, and measuring OD ₆₀₀ To between 0.6 and 0.8.

Preparation of an infection liquid: 0.205g of MgCl was added sequentially to 100mL of deionized water ₂ 0.4275g MES, 100. Mu.L of 200mmol acetosyringone, pH adjusted to between 5.7 and 5.8, and 20. Mu.L Silwet L-77 silicone surfactant. Sealing with fresh-keeping film and stirring uniformly.

Will be cultured to OD ₆₀₀ Taking out bacterial liquid with the concentration of 0.6-0.8, uniformly mixing pTRV1 with pTRV2 and bacterial liquid 1:1 containing pTRV2-BnBADH expression vector respectively, and centrifuging at 4000rpm and 4 ℃ for 5min. The supernatant was removed, 10mL of the invader solution was added to the precipitated cells and the cells were shake-cultured at 28℃for 3 hours.

5. Leaf infection

Selecting plant leaves with good growth and vigorous growth in transplanted rape seedlings, and wiping the leaves clean by deionized water. The transformation solution after 3 hours of culture was injected between veins from the back of the leaf by a syringe without needle, during which time the leaf was not crushed. The left and right parts of the same leaf blade are respectively injected with agrobacterium transformation solution containing pTRV1 and pTRV2 empty virus gene silencing expression vectors and agrobacterium transformation solution containing pTRV1 and pTRV2-BnBADH expression vectors, and marked (shown in figure 1).

6. Rape culture

The infected rape plants are cultivated for 7-14d at 25 ℃ for 15 hours under light and 9 hours under dark.

7. Target gene expression detection

Extracting RNA of rape leaves: the left and right parts of rape treated leaves cultured for 7-14d are respectively placed in 2mL of EP tube, 1mL of Trizol, zirconia and 20 mu L of 3M sodium acetate are added, and the mixture is crushed in a tissue crusher at 50HZ for 180 s. Crushing, centrifuging at 13000rpm at 4deg.C for 30min, collecting supernatant, adding chloroform with equal volume, shaking for 5min, and centrifuging for 20min. The supernatant was removed by repeated addition of chloroform and centrifugation twice to remove protein. Taking the supernatant to a new tube, adding 1:0.7 volume of isopropanol is swayed up and down, gently flicked, and centrifuged for 30min. Removing supernatant, adding 75% ethanol 1mL, bouncing the precipitate, and centrifuging for 15min. Removing supernatant, repeatedly adding 1mL of 75% ethanol, bouncing the precipitate, and centrifuging for 15min. Removing the supernatant, spin-drying for 30s, and adding 30-50 mu L of DEPC water.

Obtaining total cDNA of rape: the RNA was subjected to reverse transcription by a reverse transcription system (shown in Table 5) to obtain total cDNA of canola.

Reverse transcription PCR reaction condition 1:70 ℃ for 5min;16 ℃ for 2min; adding 5 Xbuffer and reverse transcriptase after PCR;

reverse transcription PCR reaction condition 2:42 ℃ for 60min;95 ℃ for 5min;

and (3) verification: specific primers of BnBADH gene and specific primers of BnGSci and BnMKK2 related to salt and alkali resistance are designed (shown in table 6), real-time fluorescence quantitative PCR is used, and reference gene is utilized to pass through 2 ^-△△CT And (5) calculating a method. Error was reduced for each of the 3 replicates tested. And (3) carrying out mapping analysis on the relative expression change of BnBADH genes and the relative expression amounts of BnGSci and BnMKK2 genes in each sample (shown in figures 2 and 3).

The results in FIGS. 2-3 show that the relative expression levels of the BnBADH, bnGSci, bnMKK gene were significantly changed.

TABLE 1 PCR cloning System

Reagent name	Volume of
		ddH ₂ O	13.8μL
FastPfu Buffer	2μL
		dNTP	1μL
Upstream primer	1μL
		Downstream primer	1μL
cDNA	1μL
		Fastpfu enzyme	0.2μL

TABLE 2 addition A System

Reagent name	Volume of
		Ex-Taq Buffer	2μL
dATP	1μL
		PCR products	16.9μL
Ex-Taq enzyme	0.1μL

TABLE 3 cloning vector ligation System

Reagent name	Volume of
		Addition of A product	7.5μL
pGM-T vectors	0.5μL
		10×T4Buffer	1μL
T4 ligase	1μL

TABLE 4 expression vector ligation System

Reagent name	Volume of
		Cleavage product	5μL
PTRV2 vector	3μL
		10×T4Buffer	1μL
T4 ligase	1μL

TABLE 5 reverse transcription System

Reagent name	Volume of
		RNA	11.5μL
dNTP	2μL
		Oligo dT	2μL
Reverse transcriptase	0.5μL
		5×buffer	4μL

TABLE 6 primers required in the examples

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A method for virus-induced gene silencing of canola, comprising the steps of:

(3) Culturing the bacterial liquid in the step (2) to OD ₆₀₀ Between 0.6 and 0.8, pTRV1 and the obtained bacterial liquid are mixed according to the volume ratioMixing at a ratio of 1:2-2:1, centrifuging, removing supernatant, adding invasion dye solution into the precipitate thallus, and shake culturing at 25-30deg.C for 2-5 hr;

2. The method of claim 1, wherein the PCR amplification conditions in step (1) are: 94℃for 5min,94℃for 30s,60℃for 30s,72℃for 15s and 72℃for 10min.

3. The method of claim 1, wherein the nucleotide sequence of the primer for PCR amplification in step (1) is set forth in SEQ ID NO: 1-2.

4. The method according to claim 1, wherein the specific process of cutting and recycling in the step (1) is as follows: adding 600 μL of sol Buffer into EP tube filled with gel block, standing for 3min after gel block is melted, centrifuging at 12000rpm for 1min, adding into adsorption column, centrifuging at 12000rpm for 1min, pouring centrifugate, adding 600 μL of Washing Buffer, standing for 3min, centrifuging for 1min, changing adsorption column into 1.5mL EP tube, adding 50 μL of ddH ₂ Eluting the DNA by O, and spin-drying the eluent on a vacuum spin-drying instrument for 20min to obtain the DNA.

5. The method according to claim 1, wherein the conditions for the reaction of adding a in step (1) are: 60min at 72℃and 10min at 65 ℃.

6. The method according to claim 1, wherein in step (2) the agrobacterium is in particular agrobacterium GV3101.

7. The method according to claim 1, wherein the preparation process of competent agrobacterium in step (2) is specifically: inoculating Agrobacterium into LB culture medium, culturing and activating overnight in a shaker at 28 ℃,extracting 500 μl of bacterial liquid from activated Agrobacterium in 50mL LB medium containing antibiotic, wherein gentamicin 40 μg/mL and rifampicin 20 μg/mL, shake culturing at 28deg.C to OD ₆₀₀ About 0.8, centrifuging the bacterial liquid at 4000rpm for 10min, removing the supernatant, adding 60mM CaCl ₂ 10mL of the solution was mixed and centrifuged for 10min, the supernatant was removed, and 1mL of 60mM CaCl was added ₂ The solution and 100. Mu.L of dimethyl sulfoxide are mixed uniformly to prepare competent Agrobacterium.

8. The method according to claim 1, wherein the centrifugation in step (3) is carried out at 3000-5000rpm for 3-10min at 4 ℃.

9. The method according to claim 1, wherein the preparation process of the infection liquid in the step (3) is as follows: 0.205g of MgCl was added sequentially to 100mL of deionized water ₂ 0.4275g MES, 100 mu L of 200mmol acetosyringone, regulating the pH value to 5.7-5.8, adding 20 mu L of surfactant, and stirring uniformly.

10. The method according to claim 1, wherein the conditions of the culturing in step (4) are: culturing at 25deg.C for 7-14d under light for 15 hr and dark for 9 hr.