Orobanche coerulescens gene related to parasitism and application thereof
By silencing the OcPa1 gene of sunflower Orobancha through virus-mediated gene silencing technology, the problem of prevention and control of sunflower Orobancha parasitism was solved, and the resistance breeding process of sunflower was accelerated and the growth was improved.
Patent Information
- Application Number
- CN202511087202.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-04
- Publication Date
- 2025-10-24
AI Technical Summary
Existing technologies are difficult to effectively control the parasitism of sunflower broomrape, especially the difficulty in breeding resistance caused by the long-term survival and rapid evolution of its seed bank, and the effectiveness of chemical and biological control is limited.
Virus-mediated gene silencing (VIGS) technology was used to silence the parasitism-related gene OcPa1 of Orobancha. Recombinant vectors and engineered bacteria were used to achieve gene silencing in sunflower, thereby reducing the parasitic degree of Orobancha.
The effective inhibition of the parasitism of Orobancha on sunflower revealed the key role of the OcPa1 gene in the parasitic process, improved the growth state of sunflower plants and reduced the number of parasites, providing a theoretical basis for resistance breeding.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of plant genetic engineering, in particular, to a broomrape gene associated with parasitism and its application. BACKGROUND
[0002] Sunflower is one of the important economic crops in China. Due to its tolerance to poor soil, salt-alkali, drought resistance, strong adaptability, simple management, high efficiency, the planting area is increasing. However, due to the large-scale and disorderly introduction of sunflower in recent years, and the lag of plant quarantine work, the occurrence area of broomrape in northern China is expanding, causing huge economic losses. At present, chemical control and biological control are the main methods to control sunflower broomrape in the field. However, as a root parasitic weed, it has a great impact on the host crop before it emerges, and once mature, each sunflower broomrape can produce hundreds of thousands of seeds, which can survive in the soil for decades, forming a seed bank, ensuring the adaptation of the root parasitic weed to environmental changes, and it is almost impossible to completely eliminate the seed bank, so it is difficult to effectively control it.
[0003] Resistance breeding is considered to be the most effective method to control broomrape. However, the evolution of broomrape is quite fast, and under the selection pressure of sunflower resistance varieties, new toxic broomrape physiological races often appear in 2-3 years, thus overcoming most of the resistance of the host sunflower. Despite this, resistance breeding is still an economic, environmentally friendly and effective method to control broomrape. Resistance breeding is a slow process. Identifying genes associated with broomrape parasitism will help understand the parasitic mechanism of broomrape and provide a theoretical direction for sunflower resistance breeding, thereby accelerating the breeding process.
[0004] The application of virus-induced gene silencing (VIGS) assay in plants has become an important tool for plant gene function research and plant breeding. It introduces small interfering RNA (siRNA) or short hairpin RNA (shRNA) through a viral vector to achieve the silencing of specific genes in plant cells, and then study the function of the genes or improve plant traits. The prior art document (Host sunflower-induced silencing of parasitism-related genes confers resistance to invading Orobanche cumana. (2021). Plant Physiology, 185(2), 424-440) discloses the use of VIGS technology to silence the genes of orobanche across species. In the study, tobacco rattle virus (TRV) was spread in sunflower by seed soaking inoculation method, and the target genes OcCKX5 and OcWRI1 were silenced across species after orobanche parasitism, thereby effectively reducing the parasitism degree of orobanche, and confirming the feasibility of the technology. SUMMARY
[0005] On the basis of the prior art, the inventors of the present application have newly discovered an orobanche gene OcPa1, and found through experimental verification that the gene is related to the parasitism of orobanche on sunflower.
[0006] In this case, the present application includes but is not limited to the following:
[0007] In one aspect, the present application provides a parasitism-related orobanche gene, characterized in that the gene encodes an amino acid sequence as shown in SEQ ID NO: 1.
[0008] In one aspect, the nucleotide sequence of the gene according to the present application is as shown in SEQ ID NO: 2.
[0009] In another aspect, the present application provides a recombinant vector for reducing the expression or silencing of the orobanche gene according to the present application.
[0010] In one aspect, the recombinant vector according to the present application is a virus-mediated gene silencing recombinant vector.
[0011] In one aspect, the recombinant vector according to the present application comprises a target gene as shown in SEQ ID NO: 3.
[0012] In one aspect, the recombinant vector according to the present application is a pTRV2 vector containing a target gene as shown in SEQ ID NO: 3.
[0013] In one aspect, the recombinant vector according to the present application is selected from a recombinant pTRV2 vector.
[0014] In one aspect, the recombinant pTRV2 vector according to the present application requires the presence of a helper vector to function.
[0015] In one aspect, the helper vector according to the present application is selected from a pTRV1 vector.
[0016] In one aspect, the vectors pTRV1 and pTRV2 used according to the present application are two binary vectors from tobacco rattle virus, and after transforming plants with them simultaneously, virus-mediated gene silencing (VIGS) can be achieved, which is a conventional method of gene silencing. pTRV1 and the silencing vector pTRV2 co-infect plants to form a complete TRV virus system, in which the helper vector is pTRV1, and the replicase produced by pTRV1 replicates the RNA of pTRV2; the movement protein of pTRV1 helps the virus to transport long distances in the vascular bundle, so that the silencing effect spreads throughout the whole plant. The role of pTRV1 is mainly reflected in virus replication, systemic movement, and cooperation with the silencing vector to achieve whole-plant gene silencing. In the experimental process, pTRV1 does not need to be modified, and it can be directly transformed into Agrobacterium.
[0017] In another aspect, the present application provides a recombinant engineering bacteria, characterized in that the recombinant engineering bacteria comprises the recombinant vector according to the present application.
[0018] In one aspect, the recombinant engineering bacteria according to the present application is selected from a recombinant Agrobacterium. Preferably, the Agrobacterium is Agrobacterium tumefaciens GV3101.
[0019] In another aspect, the present application provides the use of the orobanche gene according to the present application, the recombinant vector according to the present application, or the recombinant engineering bacteria according to the present application in reducing the parasitism of orobanche on plants and / or plant resistance breeding.
[0020] In one aspect, the plant according to the present application is selected from a chenopodiaceae plant, a leguminous plant, a solanaceae plant, a cucurbitaceae plant, a cruciferous plant, a cannabis plant, a linaceae plant, an umbelliferae plant, or a gramineae plant, preferably a chenopodiaceae plant, more preferably a sunflower.
[0021] In another aspect, the present application provides a method for reducing the parasitism of orobanche on plants and / or plant resistance breeding, characterized in that the method comprises: introducing the recombinant vector or the recombinant engineering bacteria according to the present application into the plant.
[0022] In one aspect, the plant resistance according to the present application refers to the resistance of a plant to the parasitism of orobanche.
[0023] In another aspect, the present application provides a method for obtaining a plant with improved traits, characterized in that it comprises the following treatment steps:
[0024] (1) using the recombinant vector or the recombinant engineering bacteria according to the present application to infect the plant callus or seeds; and
[0025] (2) culturing the infected callus or seeds into a whole plant.
[0026] In one aspect, the improved traits according to the present application refer to the improved resistance to the orobanche parasitism.
[0027] In one aspect, the method for obtaining a plant with improved traits according to the present application comprises the following steps:
[0028] Step S1, selecting healthy sunflower seeds with a germination rate of 95% or above and performing a shelling treatment;
[0029] Step S2, placing the shelled sunflower seeds into distilled water and performing a soaking treatment at room temperature for 2 hours;
[0030] Step S3, after the soaking is completed, removing the seed coat to expose the embryo;
[0031] Step S4, using a sterile needle to make a wound on the surface of each seed and controlling the wound within the epidermis layer without penetrating the internal tissue of the embryo;
[0032] Step S5, preparing an infiltration solution for silencing the orobanche parasitism related gene OcPa1, mixing with an equal volume of pTRV1 at 1:1 before use, and measuring the absorbance value at 600 nm to ensure it is between 0.6 and 1.0;
[0033] Step S6, completely immersing the seed treated with the wound in the prepared infiltration solution, and soaking at room temperature in the dark for more than 6 hours;
[0034] Step S7, taking out the soaked seeds and washing them with distilled water at least three times, each time using clean distilled water;
[0035] Step S8, transferring the washed seeds to MS liquid medium and culturing them at room temperature in the dark for 1 day;
[0036] Step S9, moving the cultured seeds into the soil mixed with orobanche in advance.
[0037] In one embodiment of the present application, the amino acid sequence of the orobanche parasitism related gene OcPa1 according to the present application is shown in SEQ ID NO: 1.
[0038] SEQ ID NO: 1:
[0039] In one embodiment of the present application, the nucleotide sequence of the parasitism-related Orobanche gene OcPai described in the present application is shown as SEQ ID NO: 2, and is specifically as follows:
[0040] SEQ ID NO: 2:
[0041]
[0042] In one embodiment of the present application, the target sequence for silencing the parasitism-related Orobanche gene OcPai described in the present application is shown as SEQ ID NO: 3, and is specifically as follows:
[0043] SEQ ID NO: 3:
[0044] Compared with the prior art, the parasitism-related Orobanche gene and the application thereof have at least the following beneficial effects:
[0045] The present application specifically silences the parasitism-related Orobanche gene OcPai by using virus-mediated gene silencing technology (VIGS), effectively inhibits the parasitism of Orobanche on plants (for example, sunflower), reveals the key role of the OcPai gene in the parasitism process of Orobanche, and proves the importance of the gene in the plant parasitism network. BRIEF DESCRIPTION OF DRAWINGS
[0046] Figure 1 A VIGS virus vector construction map of the present application.
[0047] Figure 2 A comparison chart of the inhibitory effect of OcPai gene silencing on the infestation of sunflower by Orobanche.
[0048] Figure 3 A chart of the influence of OcPai gene silencing on the phenotype of sunflower leaves.
[0049] Figure 4 A comparison chart of the influence of OcPai gene silencing on the overall growth of sunflower plants.
[0050] Figure 5 A comparison chart of sunflower root tissue cross sections.
[0051] Figure 6 A statistical analysis chart of the influence of OcPai gene silencing on the number of Orobanche parasitism. DETAILED DESCRIPTION
[0052] The technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present application. The described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.
[0053] Example 1 Screening of sunn hemp gene
[0054] The whole growth cycle of sunn hemp is subjected to transcriptome sequencing, and time series analysis is performed on all expressed genes. After the genes expressed higher in the sunn hemp infection stage than in other stages are translated into protein sequences, secretion group prediction is performed, and candidate effector proteins containing signal peptides, not containing transmembrane domains, and C-terminal not containing 'KDEL' or 'HDEL' motif are screened. After functional verification of the candidate effector genes with high expression, it is found that OcPai can promote the parasitism of sunn hemp.
[0055] Example 2 Construction of virus-mediated gene silencing vector
[0056] The virus-mediated gene silencing vector for silencing the sunn hemp gene OcPai related to parasitism in the embodiments of the present application comprises a target sequence as shown in SEQ ID NO: 3, wherein the backbone vector of the virus-mediated gene silencing vector is preferably a pTRV2 vector (in particular, pTRV2-GFP (purchased from HonorGene, item number HG-VRH1165)), and the target sequence is inserted between the BamH I and Kpn I enzyme cutting sites of the pTRV2 vector, for example, by means of homologous recombination, to construct a vector containing the target sequence (for example, as shown in SEQ ID NO: 4), wherein the primers used in the homologous recombination process are preferably as shown in Table 1. Figure 1
[0057] Table 1 Primers for constructing pTRV2 vector
[0058]
[0059] Example 3 Construction of agrobacterium containing virus-mediated gene silencing vector for infecting plants
[0060] In the present embodiment, the virus-mediated gene silencing vector obtained in Example 2 is introduced into agrobacterium to obtain agrobacterium for silencing the sunn hemp gene OcPai related to parasitism; wherein the agrobacterium is preferably Agrobacterium tumefaciens, and more preferably Agrobacterium tumefaciens GV3101.
[0061] Example 4 Using agrobacterium containing virus-mediated gene silencing vector to infect sunn hemp
[0062] In the embodiments of the present application, the infection solution for silencing the parasitism-related orobanche gene OcPai includes Agrobacterium obtained in Embodiment 3; wherein the OD600 value of the Agrobacterium is preferably 0.6 to 1.0, and further preferably 0.8; the infection solution further includes Agrobacterium containing a helper vector and an infection medium; the Agrobacterium containing the helper vector is preferably Agrobacterium tumefaciens, and more preferably Agrobacterium tumefaciens GV3101; the helper vector is preferably a pTRV1 vector (for example, purchased from HonorGene, item number HG-VRW0365); and the infection medium is a MS liquid medium (Phytotech, M519) as a base medium, and further preferably includes 10 mM magnesium chloride, 10 mM 2-morpholinoethanesulfonic acid and 200 μM acetyl-syringone.
[0063] The method for silencing the parasitism-related orobanche gene OcPai in sunflower using Agrobacterium containing a virus-mediated gene silencing vector in the embodiments of the present application includes the following steps:
[0064] Step S1, selecting sunflower seeds with a germination rate of 95% or more and healthy and full, and performing a shelling treatment;
[0065] Step S2, placing the shelled sunflower seeds in distilled water, and performing an immersion treatment at room temperature for 2 hours;
[0066] Step S3, after the immersion is completed, removing the seed coat to expose the embryo;
[0067] Step S4, using a sterile needle to make a wound on the surface of each seed, and controlling the wound to the epidermis layer of the seed without penetrating the internal tissue of the embryo;
[0068] Step S5, preparing an infection solution for silencing the parasitism-related orobanche gene OcPai, uniformly mixing the infection solution with an equal volume of a pTRV1 vector at a ratio of 1:1 before use, and measuring the absorbance value at a wavelength of 600 nm to ensure that the value is between 0.6 and 1.0;
[0069] Step S6, completely immersing the seed treated by the wound in the prepared infection solution, and performing an immersion for more than 6 hours under room temperature and dark conditions, and stirring the infection solution gently in a timely manner to ensure that the infection solution is in sufficient contact with the surface of the seed;
[0070] Step S7, taking out the immersed seed, and washing the seed with distilled water at least three times, and changing the clean distilled water each time to remove the excess infection solution attached to the surface of the seed to avoid affecting the subsequent culture;
[0071] Step S8, transferring the washed seed to a MS liquid medium, and culturing the seed under room temperature and dark conditions for 1 day;
[0072] Step S9, the seeds after culture are moved into the soil mixed with Orobanche in advance, and are lightly covered with fine soil to ensure full contact of the seeds with the soil, and are regularly sprayed with an appropriate amount of water to maintain soil moisture to promote seed germination and subsequent growth.
[0073] Experimental results
[0074] The Orobanche-related gene provided by the application, after gene silencing by the VIGS method, the phenotype statistics include: as shown in Figure 2 the sunflower underground Orobanche parasitic conditions under different treatments, the number of Orobanche after pTRV2-OcPa1 treatment is significantly less than that of the control group; as shown in Figure 3 the sunflower leaf morphology under different treatments, the leaf after pTRV2-OcPa1 treatment is more expanded than the control group; as shown in Figure 4 the comparison of the growth states of the control group and the pTRV2-OcPa1 treatment group, after silencing the OcPa1 gene by VIGS, the sunflower plants are obviously higher, and the leaf growth is better, which may be related to the decrease of the degree of Orobanche parasitism, indicating that the OcPa1 gene is related to Orobanche parasitism, and silencing the OcPa1 gene can improve the overall growth state of the sunflower plants.
[0075] Figure 5 The sunflower root tissue cross section is shown, the Orobanche successfully passes through the sunflower root under the treatment of empty vector pTRV2, and establishes a connection with the vascular tissue, while under the treatment of pTRV2-OcPa1, the Orobanche is blocked in the cortex of the sunflower root, resulting in failure of its infection.
[0076] As shown in Figure 6 the statistical analysis chart of the influence of OcPa1 gene silencing on the number of Orobanche parasitism. The vertical coordinate in the figure represents the number of Orobanche parasitism, by comparing the control group (empty vector pTRV2) and the gene silencing treatment group (pTRV2-OcPa1), it can be observed that the gene silencing treatment has an influence on the number of Orobanche parasitism. In addition, Figure 6 the number of parasitism of each treatment group is also shown. Overall, the number of Orobanche parasitism in the pTRV2-OcPa1 treatment group is significantly lower than that in the empty vector pTRV2 control group, indicating that silencing the OcPa1 gene can reduce the number of Orobanche parasitism, indicating that the OcPa1 gene plays an important role in the process of Orobanche parasitism.
[0077] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.
[0078] Finally: the above only for the preferred embodiments of the present application, and not for limiting the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application, should be included in the scope of protection of the present application.
Claims
1. An orobanchae related gene, characterized in that, The gene encodes an amino acid sequence as shown in SEQ ID NO:
1.
2. The orobanche gene of claim 1, wherein, The nucleotide sequence of the gene is shown in SEQ ID NO:
2.
3. A recombinant vector for reducing expression of or silencing the Orobanche gene according to claim 1 or 2.
4. The recombinant vector of claim 3, wherein, The recombinant vector is a virus-mediated recombinant vector.
5. The recombinant vector of claim 3 or 4, wherein, The target gene comprises the sequence as shown in SEQ ID NO:
3.
6. A recombinant engineered bacterium, characterized in that, The recombinant engineering bacteria comprise the recombinant vector according to any one of claims 3-5.
7. Use of the Orobanche gene according to claim 1 or 2, the recombinant vector according to any one of claims 3-5, or the recombinant engineering bacteria according to claim 6 in reducing the parasitism of Orobanche on plants and / or breeding of plant resistance.
8. Use according to any one of claim 7, characterized in that, The plant is selected from a plant of the family Asteraceae, Leguminosae, Solanaceae, Cucurbitaceae, Cruciferae, Cannabaceae, Linaceae, Umbelliferae or Gramineae, preferably a plant of the family Asteraceae, more preferably sunflower.
9. A method for reducing the parasitism of plants by Orobanche and / or breeding for plant resistance, characterized in that, The method comprises: introducing the recombinant vector according to any one of claims 3-5 or the recombinant engineering bacteria according to claim 6 into the plant.
10. A method of obtaining a plant with improved traits, comprising, The method comprises the following treatment steps: (1) using the recombinant vector according to any one of claims 3-5 or the recombinant engineering bacteria according to claim 6 to infect plant callus or seeds; and (2) culturing the infected callus or seeds into whole plants. The method comprises the following treatment steps: (1) using the recombinant vector according to any one of claims 3-5 or the recombinant engineering bacteria according to claim 6 to infect plant callus or seeds; and (2) culturing the infected callus or seeds into whole plants.