Application of HaSnRK2.5 gene in improving parasitic resistance of sunflower to Orobanche coerulescens

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a HaSnRK2.5 gene in improving parasitic resistance of sunflowers to orobanche coerulescens. A complete HaSnRK2.5 gene sequence is obtained from detected transcriptome data of a sunflower, the HaSnRK2.5 gene is transferred into a body plant sunflower, a sunflower plant with the overexpressed HaSnRK2.5 gene is obtained, and through an Orobanche coerulescens parasitic experiment of the obtained transgenic plant, the Orobanche coerulescens can be used for screening the Orobanche coerulescens. It is found that the Orobanche coerulescens parasitism resistance of a plant with the overexpressed HaSnRK2.5 gene is remarkably improved compared with that of a wild sunflower, and the discovery has important value in sunflower molecular breeding and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the application of the HaSnRK2.5 gene in improving the resistance of sunflowers to Orobanche cumana parasitism. Background Art

[0002] Sunflowers (Helianthus annuus L.) can be divided into three categories: oil sunflowers, edible sunflowers, and ornamental sunflowers. Sunflowers can be divided into three categories: oil sunflowers, edible sunflowers, and ornamental sunflowers. Among them, oil sunflowers are one of the important oil crops in the world, edible sunflowers are the main leisure snacks for people, and ornamental sunflowers are used for environmental beautification.

[0003] Sunflower broomrape (Orobanche cumana), also known as poisonous root grass, is an annual holoparasitic herbaceous plant of the genus Orobanche in the family Orobanchaceae. Its plant height ranges from 15 to 54 cm. Sunflower broomrape is a holoparasitic plant that specifically parasitizes the roots of sunflowers through haustoria. Once parasitism is successful, sunflower broomrape absorbs the nutrients and water of sunflowers through the haustoria connected to the roots, resulting in slow growth, yellowing of leaves, lodging, reduced yield, and even death of sunflowers, thus causing serious impacts on the sunflower planting industry.

[0004] After sunflower broomrape parasitism, it will significantly reduce the leaf area index of sunflower plants during the filling period, reduce the population photosynthetic rate of sunflowers, and thus affect the synthesis and accumulation of dry matter. At the same time, it will also damage the root morphology of sunflowers and affect their normal growth and development (CUI C, WANG J, WANG H W, et al. Effect of parasitism severity by sunflower broomrape on yield formation and physiological characteristics of sunflower. [J]. Chinese Journal of Oil Crop Sciences, 2016, 38(4): 518 - 523.).

[0005] Currently, the control of sunflower broomrape mainly includes deep plowing, crop rotation, and application of pesticides. Cultivating broomrape-resistant varieties is also an important way to control broomrape. However, broomrape has the ability to evolve rapidly. Once a resistant sunflower variety effective against existing broomrape races is discovered, after a period of time, broomrape will also evolve into more virulent physiological races. Therefore, cultivating broomrape-resistant varieties is a long-term and continuous task.

[0006] The ABA pathway has been relatively well studied, including its role in plant growth and stress responses. However, there is little research on it in sunflowers, especially its role in sunflower resistance to Orobanche cumana. Therefore, screening for key genes in the ABA pathway of sunflower in response to Orobanche cumana stress and analyzing its molecular mechanism can provide an important basis for molecular breeding and application of sunflower resistance. Summary of the Invention

[0007] In response to the need in the prior art to explore new varieties and genes of sunflower resistant to Orobanche cumana, the present invention provides the application of the HaSnRK2.5 gene in enhancing the resistance of sunflower to Orobanche cumana parasitism. The specific technical solutions are as follows:

[0008] First, the present invention provides the application of the HaSnRK2.5 gene in enhancing the resistance of sunflower to Orobanche cumana parasitism. The nucleotide sequence of the HaSnRK2.5 gene is shown in SEQ ID NO.1.

[0009] Furthermore, the ways of the application include:

[0010] By overexpressing the HaSnRK2.5 gene, the ability of sunflower to resist Orobanche cumana stress is enhanced.

[0011] Second, the present invention provides the application of the HaSnRK2.5 protein in enhancing the resistance of sunflower to Orobanche cumana parasitism. The amino acid sequence of the HaSnRK2.5 protein is shown in SEQ ID NO.2.

[0012] Third, the present invention provides the application of a recombinant vector in enhancing the resistance of sunflower to Orobanche cumana parasitism. The recombinant vector contains the HaSnRK2.5 gene, and the nucleotide sequence of the HaSnRK2.5 gene is shown in SEQ ID NO.1.

[0013] Fourth, the present invention provides the application of a genetically engineered bacterium in enhancing the resistance of sunflower to Orobanche cumana parasitism. The genetically engineered bacterium contains the HaSnRK2.5 gene, and the nucleotide sequence of the HaSnRK2.5 gene is shown in SEQ ID NO.1.

[0014] Fifth, the present invention provides a method for constructing a sunflower plant with enhanced resistance to Orobanche cumana parasitism, including the following steps:

[0015] (1) Obtain the HaSnRK2.5 gene by gene cloning method;

[0016] (2) Design primer sequences using the HaSnRK2.5 gene as a template and construct an overexpression vector of the HaSnRK2.5 gene;

[0017] (3) Transfer the vector into Agrobacterium competent cells to obtain recombinant Agrobacterium overexpressing the HaSnRK2.5 gene;

[0018] (4) Infect sunflower seeds with the recombinant Agrobacterium to obtain sunflower plants with enhanced resistance to Orobanche cumana Wallr.

[0019] Furthermore, the variety of the sunflower is Tonghui 15 and / or TY0409.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention obtains the complete HaSnRK2.5 gene sequence from the measured transcriptome data of sunflowers, transfers the HaSnRK2.5 gene into the host plant sunflower, and obtains sunflower plants overexpressing the HaSnRK2.5 gene. Through the Orobanche cumana Wallr. parasitism experiment on the obtained transgenic plants, it is found that the plants overexpressing the HaSnRK2.5 gene have significantly improved resistance to Orobanche cumana Wallr. parasitism compared with wild-type sunflowers. This discovery has important value for the molecular breeding and application of sunflowers. Description of the Drawings

[0022] Figure 1 It is the PCR electrophoresis map for the cloning of the HaSnRK2 gene.

[0023] Figure 2 It is the schematic diagram of the expression of the HaSnRk2 gene after Orobanche cumana Wallr. infection in sunflowers; among them, the HaSnRk2 gene is the HaSnRk2.5 gene and the HaSnRk2.14 gene; A is the schematic diagram of the relative expression of the HaSnRk2.5 gene and the HaSnRk2.14 gene after Orobanche cumana Wallr. infects Tonghui 15 plants, TH represents the normally growing Tonghui 15 plants, and THO represents the Tonghui 15 plants after Orobanche cumana Wallr. infection; B is the schematic diagram of the relative expression of the HaSnRk2.5 gene and the HaSnRk2.14 gene after Orobanche cumana Wallr. infects TY0409 plants, TY represents the normally growing TY0409 plants, and TYO represents the TY0409 plants after Orobanche cumana Wallr. infection.

[0024] Figure 3 It is the subcellular localization map of the HaSnRK2.5 gene transferred into tobacco.

[0025] Figure 4 It is the co-culture result of the transient overexpression plants of the HaSnRK2 gene and the control group plants with Orobanche cumana Wallr. in the Orobanche cumana Wallr.-sensitive variety TY0409 of sunflowers; among them, the circled position is the Orobanche cumana Wallr. parasitism position, A is the control group plants, B is the plants overexpressing the HaSnRk2.5 gene, and C is the plants overexpressing the HaSnRk2.14 gene.

[0026] Figure 5 Statistical chart of the number of Orobanche cumana parasitizing on the root tissues of the transient overexpression plants of the HaSnRK2 gene and the control group plants in the sunflower variety TY0409 sensitive to Orobanche cumana and Orobanche cumana after co-culturing for 20 days.

[0027] Figure 6 Result chart of the expression level of the HaSnRK2 gene in sunflower plants after transient overexpression of the SnRK2 gene in the sunflower variety TY0409 sensitive to Orobanche cumana. Detailed implementation manners

[0028] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and only a part of the embodiments of the present invention, rather than all of the embodiments.

[0029] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels. The experimental methods without detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

[0031] In the following embodiments, the sunflower varieties used are: Tonghui 15, TY0409; the types of Orobanche cumana are: a mixed seed sample of Orobanche cumana physiological races above grade E. At the early stage of Orobanche cumana parasitizing sunflowers, root nodules are formed on the sunflower roots, which are white in color at the early stage and gradually turn yellow at the later stage.

[0032] The nucleotide sequence of the HaSnRK2 gene is shown in SEQ ID NO.1 (this gene is renamed HaSnRk2.5 among the members of the HaSnRk2 family), and the amino acid sequence of the HaSnRK2.5 protein is shown in SEQ ID NO.2; the nucleotide sequence of the HaSnRK2.14 gene is shown in SEQ ID NO.3.

[0033] SEQ ID NO.1:

[0034]

[0035] SEQ ID NO.2:

[0036] MERYEVVKELGSGNFGVAKLVRDKGTRELFAVKFIERGQKIDEHVEREILNHRSLRHPNIIRFKEVLLTPTHLAIVMEYAAGGELFERICHAGKFSENEARFFFQQLISGVSYCHSMEICHRDLKLENTLLDGSTAPRVKICDFGYSKSSVLHSQPKSAVGTPAYIAPEVLSRKEYDGKLADVWSCGVTLYVMLVGTYPFGHPDDPNNFRTTISRILSVQYAIPDNVEVSLECRHLLYRIFVANPEKRISIQEIQIHPWFLKNLPTDLVTGASSHNTNLNDTNQNVDEILSIIQEARTHPGMLNDGSLQLLGDSMDFDDLDDSDIEDIDISDDYDSSF

[0037] SEQ ID NO.3:

[0038]

[0039] Example 1 Obtaining the full-length cDNA sequence of the sunflower HaSnRK2 gene

[0040] Take 0.1 g of fresh sunflower root tissue samples, add an appropriate amount of grinding beads to a 2 mL centrifuge tube, and quickly grind them into powder in a grinding machine pre-cooled with liquid nitrogen in advance. Use Universal Plant Total RNA Isolation Kit to extract total RNA, and detect its integrity and concentration by 1% agarose gel electrophoresis and NanoDrop ultra-micro spectrophotometer. Then use HiScript III 1st Strand cDNA Synthesis Kit(+gDNAwiper) to reverse the extracted RNA into cDNA.

[0041] Use Max DNAPolymerase to perform PCR amplification using cDNA as a template (as Figure 1 shown), and the amplification primers are:

[0042] HaSnRK2-F: TCAACACGGGGGACGatggagagatatgaagtggttaaagaattggg,

[0043] HaSnRK2-R: CCATGTCGACTCTAGaaatgaagaatcataatcatcacttatatctatatcttcaatgtctg.

[0044] 50 μL PCR amplification system: 25 μL PrimeSTAR Max Premix(2×), 1 μL Forward primer, 1 μL Reverse primer, 1000 ng Template cDNA, supplemented with RNase Free ddH2O to 50 μL. PCR instrument program settings: 98 °C for 10 s, 60 °C for 30 s, 72 °C for 90 s, 32 cycles, 72 °C for 5 min, and stored at 12 °C.

[0045] Observe whether the size of the target band is correct by 1% agarose gel electrophoresis, use Quick GelExtraction Kit to perform gel cutting and recovery, sequence the recovered product, and obtain the nucleotide sequence of the sunflower HaSnRK2.5 gene.

[0046] By identifying and analyzing the HaSnRK2 gene family to which the HaSnRK2.5 gene belongs, it was found that the HaSnRK2.14 gene and the HaSnRK2.5 gene in sunflower belong to the same family, and their protein sequences are similar, both being serine / threonine protein kinases. Therefore, these two genes were used as candidate genes for subsequent detection.

[0047] Example 2 Functional verification of the HaSnRK2.5 gene and the HaSnRK2.14 gene in sunflower

[0048] The sunflower varieties used in this example were Tonghui 15 and TY0409.

[0049] (1) Remove the husks of sunflower seeds, pour distilled water into a petri dish to soak the dehusked seeds, use forceps to peel off the transparent seed coats on the surfaces of the seeds, and then gently scratch the sunflower seeds with the peeled seed coats with forceps, taking care not to scratch important parts such as the germ of the seeds. Place the sunflower seeds in MS liquid medium, and transfer them to MS solid medium after germination. After 24 - 48 hours, transfer the sunflower seeds to a square dish and let them grow normally in the greenhouse for two weeks.

[0050] The sunflower seedlings that had grown normally for two weeks were co - cultured with the sunflower broomrape seeds that had been pre - germinated with the germination stimulant GR24. After 10 - 14 days, it was observed that the sunflower seedlings were parasitized by the sunflower broomrape. RNA was extracted from the sunflower parasitized by the sunflower broomrape, and RNA from the normally growing sunflower was extracted as a control. After reverse transcription into cDNA, fluorescence quantitative PCR was performed to verify the gene expression.

[0051] The results showed that after the sunflower seedlings were parasitized by the sunflower broomrape, in the resistant variety Tonghui 15, the expression level of the HaSnRK2.5 gene increased significantly, while in the sensitive variety, the expression level of the HaSnRK2.5 gene decreased; compared with the control plants, the expression levels of the HaSnRK2.14 gene did not change significantly in both sunflower varieties (as Figure 2 shown).

[0052] (2) Use the restriction endonucleases Sac I and XbaI from NEB to linearize the pCAMBIA1300 - eGFP vector. The linearization system is 5 μL of rCutSmart buffer, 1 μg of the vector, 1 μL each of Sac I and XbaI, and ddH 2 O is added to make up to 50 μL. Incubate at 37 °C for 2 hours, and after agarose gel electrophoresis, purify and recover. The gel recovery product, the cloned HaSnRK2.5 gene product, and the HaSnRK2.14 gene product are passed through -Perform homologous recombination using the Uni Seamless Cloning and Assembly Kit. The recombination system consists of 5 μL of 2×Assembly Mix, 0.03 pmol of linearized vector, 0.06 pmol of gene fragment, and ddH 2 O is added to make up to 10 μL. React at 50 °C for 15 min. The product can be stored in a -20 °C refrigerator.

[0053] Transform the homologous recombination product into competent Escherichia coli DH5α cells. Take one-tenth of the volume of the competent cells and add the homologous recombination product to it. Gently flick the centrifuge tube wall to mix well, then place it on ice for 30 min. After that, heat shock at 42 °C for 45 s, immediately place it in ice for 2 min, add antibiotic-free LB medium, resuscitate on a shaker for 1 h, centrifuge at 5000 rpm for 1 min, and leave 30 - 50 μL of the resuspended cells to coat on an LB plate containing 50 ng / μL kanamycin. Incubate overnight in a 37 °C incubator. Select single colonies for labeling, perform colony PCR, and send the samples with correct bands verified by agarose gel electrophoresis for sequencing. Shake the bacteria with correct sequencing results, extract the plasmid, and store it at -20 °C for the next experiment.

[0054] Transform the extracted plasmid into competent Agrobacterium tumefaciens GV3101 cells. Resuspend the collected bacterial solution and coat it on an LB plate containing kanamycin. Incubate at 28 °C in an inverted position for 2 - 3 days. Pick single colonies from the LB liquid medium containing kanamycin and rifampicin and shake the bacteria for PCR verification to screen for positive clones. Shake the bacteria and perform tobacco injection, and observe the fluorescence results using a laser confocal microscope. The results show that the HaSnRK2.5 gene exists in various parts of the cell (as Figure 3 shown), and the expression of the HaSnRK2.5 gene exists in the cytoplasm, nucleus, cytoskeleton, chloroplasts, mitochondria, Golgi apparatus, etc. of the cell.

[0055] The extracted plasmid was transformed into competent cells of Agrobacterium tumefaciens GV3101. The resuspension was spread on an LB plate containing kanamycin and cultured upside down at 28 °C for 2 - 3 days. Single colonies were picked for verification by colony PCR, and the selected positive clones were used to infect sunflower seeds. The sunflower variety TY0409, which is sensitive to Orobanche cumana Wallr., was chosen. The seeds were shelled, and distilled water was poured into a culture dish to soak the shelled seeds. The transparent seed coat on the surface of the seeds was peeled off with forceps, and then the peeled sunflower seeds were scratched a few times with forceps, taking care not to scratch important parts such as the germ of the seeds. The treated sunflower seeds were placed in a new culture dish, and then the infection solution was poured into the culture dish. They were placed in the dark for 6 h. The infected sunflower seeds were placed in MS liquid medium containing cefotaxime and ticarcillin and cultured in the dark for 48 h. Then, the germinated seeds were transferred to MS solid medium containing cefotaxime and ticarcillin. After 24 - 48 h, the sunflower seeds were transferred to a 13 * 13 cm square culture dish and allowed to grow normally in the greenhouse for two weeks to obtain sunflower plants overexpressing the HaSnRK2.5 gene.

[0056] Sunflower plants overexpressing the HaSnRK2.14 gene were prepared in the same way.

[0057] Two weeks after the overexpressed sunflower seedlings of the target gene grew normally, they were co-cultured with Orobanche cumana Wallr. seeds pre-germinated with the germination stimulant GR24, and the parasitism situation was observed after 10 d - 14 d.

[0058] As Figure 4 A, Figure 4 B, Figure 4 C and Figure 5 shown, within the same parasitism time, no parasitism event occurred on the roots of the plants overexpressing the HaSnRK2.5 gene, while parasitism events of Orobanche cumana Wallr. occurred on the root tissues of the plants overexpressing the HaSnRK2.14 gene and the control group plants.

[0059] RNA was extracted from sunflower plants overexpressing the HaSnRK2.14 gene and the HaSnRK2.5 gene, reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed to verify the gene expression. The results were as Figure 6 shown, and it could be seen that these two genes were successfully overexpressed in sunflower TY0409.

Claims

1. Application of the HaSnRK2.5 gene in improving the resistance of sunflower to Orobanche parasitism, characterized in that: The nucleotide sequence of the HaSnRK2.5 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The application pathways include: The ability of sunflower to resist Orobancha stress was improved by overexpressing the HaSnRK2.5 gene.

3. The use of HaSnRK2.5 protein in improving the resistance of sunflower to Orobanche parasitism, characterized in that: The amino acid sequence of HaSnRK2.5 protein is shown in SEQ ID NO.

2.

4. Use of a recombinant vector in improving the resistance of sunflower to Orobanche parasitism, characterized in that: The recombinant vector comprises the sunflower HaSnRK2.5 gene, and the nucleotide sequence of the HaSnRK2.5 gene is shown in SEQ ID NO.

1.

5. Use of a genetically engineered bacterium in improving the resistance of sunflower to parasitism of Orobanche deserticola, characterized in that: The genetically engineered bacteria comprises the HaSnRK2.5 gene, and the nucleotide sequence of the HaSnRK2.5 gene is shown in SEQ ID NO.

1.

6. A method for constructing a sunflower plant with improved resistance to Orobanche parasitism, characterized in that: The following steps are involved: (1) Obtaining the HaSnRK2.5 gene by gene cloning; (2) Designing primer sequences using the HaSnRK2.5 gene as a template to construct an overexpression vector of the HaSnRK2.5 gene; (3) transferring the vector into Agrobacterium competent cells to obtain recombinant Agrobacterium overexpressing the HaSnRK2.5 gene; (4) Using recombinant Agrobacterium to infect sunflower seeds, sunflower plants with improved resistance to Orobancha stress were obtained.

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