Application of HaSnRK2.5 gene in improving the resistance of sunflower to the broomrape parasitism

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of HaSnRK2.5 gene in improving resistance of sunflower to orobanche parasitism. The complete HaSnRK2.5 gene sequence is obtained from the measured transcriptome data of sunflower, the HaSnRK2.5 gene is transferred into the sunflower, and the sunflower plant overexpressing the HaSnRK2.5 gene is obtained. Through the orobanche parasitism experiment on the obtained transgenic plant, it is found that the resistance of the plant overexpressing the HaSnRK2.5 gene to orobanche parasitism is significantly improved compared with the wild type sunflower. The finding has important value for molecular breeding and application of sunflower.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the HaSnRK2.5 gene in improving the resistance of sunflower to broomrape parasite. Background Technology

[0002] Sunflowers (Helianthus annuus L.) can be divided into three categories: oilseed sunflowers, edible sunflowers, and ornamental sunflowers. Oilseed sunflowers are one of the world's most important oil crops, edible sunflowers are a popular snack, and ornamental sunflowers are used for landscaping.

[0003] Sunflower broomrape (Orobanche cumana), also known as poisonous root grass, is an annual holoparasitic herb belonging to the genus Orobanche in the family Orobanchaceae. It grows to a height of 15–54 cm. As a holoparasitic plant, sunflower broomrape specifically parasitizes the roots of sunflowers through haustoria. Once successfully parasitized, it absorbs nutrients and water from the sunflower through these haustoria connected to the roots, causing slow growth, yellowing leaves, lodging, reduced yield, and even death, thus severely impacting sunflower cultivation.

[0004] After being parasitized by sunflower broomrape, the leaf area index of sunflower plants during the grain-filling stage is significantly reduced, the photosynthetic rate of the sunflower population is decreased, and thus the synthesis and accumulation of dry matter are affected. At the same time, it also damages the root morphology of sunflower and affects its normal growth and development (CUI C, WANG J, WANG HW, et al. Effect of parasitism severity by sunflowerbroomrape on yield formation and physiological characteristics of sunflower.[J].Chinese Journal of Oi Crop Sciences,2016,38(4):518-523.).

[0005] Currently, the control of broomrape in sunflowers is mainly carried out through deep plowing, crop rotation, and pesticide application. Breeding broomrape-resistant varieties is also an important way to control broomrape. However, broomrape has the ability to evolve rapidly. Once a resistant sunflower variety that is effective against existing broomrape races is discovered, broomrape will evolve into more toxic physiological races after a period of time. Therefore, breeding broomrape-resistant varieties is a long-term and continuous task.

[0006] The ABA pathway has been thoroughly studied, including its role in plant growth and abiotic stress. However, research on its application in sunflower is limited, especially regarding its role in sunflower resistance to scours. Therefore, screening key genes in the ABA pathway of sunflower's scours stress response and elucidating its molecular mechanisms can provide an important foundation for molecular breeding and application of sunflower resistance. Summary of the Invention

[0007] To address the need in existing technologies to discover new sunflower varieties and genes resistant to broomrape, this invention provides the application of the HaSnRK2.5 gene in improving sunflower resistance to broomrape parasitism. The specific technical solution is as follows:

[0008] In a first aspect, the present invention provides the application of the HaSnRK2.5 gene in improving the resistance of sunflower to broomrape parasite, the nucleotide sequence of the HaSnRK2.5 gene being shown in SEQ ID NO.1.

[0009] Furthermore, the application methods include:

[0010] Overexpression of the HaSnRK2.5 gene enhances the sunflower's resistance to broomrape stress.

[0011] Secondly, the present invention provides the application of HaSnRK2.5 protein in improving the resistance of sunflower to broomrape parasite, the amino acid sequence of HaSnRK2.5 protein being shown in SEQ ID NO.2.

[0012] Thirdly, the present invention provides an application of a recombinant vector in improving the resistance of sunflower to orobanche deserticola, the recombinant vector containing the HaSnRK2.5 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0013] Fourthly, the present invention provides an application of a genetically engineered bacterium in improving the resistance of sunflower to orobanche deserticola, the genetically engineered bacterium containing the HaSnRK2.5 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0014] Fifthly, the present invention provides a method for constructing sunflower plants with enhanced resistance to broomrape parasitism, comprising the following steps:

[0015] (1) The HaSnRK2.5 gene was obtained by gene cloning;

[0016] (2) Primer sequences were designed using the HaSnRK2.5 gene as a template to construct an overexpression vector for the HaSnRK2.5 gene;

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

[0018] (4) Recombinant Agrobacterium was used to infect sunflower seeds to obtain sunflower plants with enhanced resistance to broom stress.

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

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

[0021] This invention obtained the complete HaSnRK2.5 gene sequence from the sequenced transcriptome data of sunflower and transferred the HaSnRK2.5 gene into the native plant, sunflower, to obtain sunflower plants overexpressing the HaSnRK2.5 gene. Through broom parasitism experiments on the obtained transgenic plants, it was found that the plants overexpressing the HaSnRK2.5 gene showed significantly improved resistance to broom parasitism compared to wild-type sunflowers. This discovery has important value for the molecular breeding and application of sunflowers. Attached Figure Description

[0022] Figure 1 This is a PCR electrophoresis image of a HaSnRK2 gene clone.

[0023] Figure 2 This diagram illustrates the expression of the HaSnRk2 gene in sunflower broomrape after infection. The HaSnRk2 gene represents both the HaSnRk2.5 and HaSnRk2.14 genes. A shows the relative expression of the HaSnRk2.5 and HaSnRk2.14 genes in Tonghui 15 sunflower plants infected with broomrape; TH represents normally growing Tonghui 15 plants, and THO represents broomrape-infected Tonghui 15 plants. B shows the relative expression of the HaSnRk2.5 and HaSnRk2.14 genes in TY0409 sunflower plants infected with broomrape; TY represents normally growing TY0409 plants, and TYO represents broomrape-infected TY0409 plants.

[0024] Figure 3 Subcellular localization map of the HaSnRK2.5 gene after its transfer into tobacco.

[0025] Figure 4 The results of co-culturing sunflower broomrape with plants transiently overexpressing the HaSnRK2 gene and control plants in the sunflower broomrape-sensitive variety TY0409 are shown. The circled locations indicate the parasitic sites of sunflower broomrape. A is the control plant, B is the plant overexpressing the HaSnRk2.5 gene, and C is the plant overexpressing the HaSnRk2.14 gene.

[0026] Figure 5 This is a statistical chart showing the number of sunflower broomrape parasites on the root tissues of sunflower broomrape-sensitive varieties TY0409 after transient overexpression of the HaSnRK2 gene, control plants, and sunflower broomrape co-cultured for 20 days.

[0027] Figure 6 The graph shows the expression level of the HaSnRK2 gene in sunflower plants after transient overexpression of the SnRK2 gene in the broomrape-sensitive sunflower variety TY0409. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0029] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should 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 one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0031] In the following examples, the sunflower varieties used are: Tonghui 15 and TY0409; the species of sunflower broomrape is: a mixed seed sample of broomrape physiological races of grade E or above. In the early stage of sunflower broomrape parasitizing sunflower, root nodules are formed on the sunflower roots. The color is white in the early stage and gradually turns yellow in the later stage.

[0032] The nucleotide sequence of the HaSnRK2 gene is shown in SEQ ID NO.1 (this gene is renamed HaSnRk2.5 in the HaSnRk2 family), the amino acid sequence of the HaSnRK2.5 protein is shown in SEQ ID NO.2, and 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 0.1g of fresh sunflower root tissue sample, add an appropriate amount of grinding beads to a 2mL centrifuge tube, and rapidly grind it into powder in a sample grinder pre-cooled with liquid nitrogen. Total RNA was extracted using the Universal Plant Total RNA Isolation Kit, and its integrity and concentration were assessed using 1% agarose gel electrophoresis and a NanoDrop micro-spectrophotometer. The extracted RNA was then reverse-engineered into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper).

[0041] use Max DNA Polymerase uses cDNA as a template for PCR amplification (e.g., ...). Figure 1 (As shown), the amplification primers are:

[0042] HaSnRK2-F:TCAACACGGGGGGACGatggagagatatgaagtggttaaagaattggg,

[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, RNase-free ddH2O added to 50 μL. PCR instrument program settings: 98℃ for 10 s, 60℃ for 30 s, 72℃ for 90 s, 32 cycles, 72℃ for 5 min, stored at 12℃.

[0045] To observe whether the target band size is correct, use 1% agarose gel electrophoresis. The Quick Gel Extraction Kit was used to cut and recover the gel, and the recovered product was sequenced to obtain the nucleotide sequence of the sunflower HaSnRK2.5 gene.

[0046] Identification and analysis of the HaSnRK2 gene family to which the HaSnRK2.5 gene belongs revealed that the HaSnRK2.14 gene and the HaSnRK2.5 gene in sunflower belong to the same family and have similar protein sequences, both being serine / threonine protein kinases. Therefore, these two genes were selected as candidate genes for further testing.

[0047] Example 2: Functional verification of sunflower HaSnRK2.5 and HaSnRK2.14 genes

[0048] The sunflower varieties used in this embodiment are Tonghui 15 and TY0409.

[0049] (1) Remove the shells from the sunflower seeds, pour distilled water into a petri dish to soak the shelled seeds, peel off the transparent seed coat with tweezers, and then gently scratch the peeled sunflower seeds with tweezers, being careful not to scratch the embryo or other important parts of the seed. Place the sunflower seeds in MS liquid medium, and after germination, transfer them to MS solid medium. After 24-48 hours, transfer the sunflower seeds to square dishes and allow them to grow normally in a greenhouse for two weeks.

[0050] Two weeks after normal growth, sunflower seedlings were co-cultured with sunflower broomrape seeds that had been pre-germinated by the germination stimulus GR24. After 10-14 days, sunflower seedlings were observed to be parasitized by sunflower broomrape. RNA was extracted from sunflowers parasitized by sunflower broomrape and RNA was extracted from normally growing sunflowers as a control. The RNA was reverse transcribed into cDNA and then used for quantitative real-time PCR to verify gene expression.

[0051] The results showed that after sunflower seedlings were parasitized by *Broomrape nigra*, the expression level of the HaSnRK2.5 gene significantly increased in the resistant variety Tonghui 15, while the expression level decreased in the susceptible variety. The expression level of the HaSnRK2.14 gene did not change significantly in either sunflower variety compared to the control (e.g., ...). Figure 2 (As shown).

[0052] (2) The pCAMBIA1300-eGFP vector was linearized using NEB restriction endonucleases Sac I and XbaI. The linearization system consisted of 5 μL rCutSmart buffer, 1 μg vector, 1 μL each of Sac I and XbaI, with ddH2O added to a final volume of 50 μL. The mixture was digested at 37°C for 2 h, and purified by agarose gel electrophoresis. The gel-recovered product was compared with the cloned HaSnRK2.5 and HaSnRK2.14 gene products. - The Uni Seamless Cloning and Assembly Kit was used for homologous recombination. The recombination system consisted of 5 μL of 2×Assembly Mix, 0.03 pmol of linearized vector, 0.06 pmol of gene fragment, and ddH2O added to a final volume of 10 μL. The reaction was carried out at 50 °C for 15 min. The product can be stored at -20 °C.

[0053] The homologous recombination product was transformed into *E. coli* DH5α competent cells. One-tenth of the homologous recombination product was added to the competent cells, and the mixture was gently agitated by tapping the centrifuge tube. The cells were then placed on ice for 30 min, followed by heat shock at 42°C for 45 s, and immediately placed on ice for 2 min. Antibiotic-free LB medium was added, and the cells were incubated on a shaker for 1 h. After centrifugation at 5000 rpm for 1 min, 30-50 μL of the resuspended cells were spread onto LB agar plates containing 50 ng / μL kanamycin and incubated overnight at 37°C. Single colonies were selected for labeling, and culture PCR was performed using the culture. The correctly sequenced bands were sent for sequencing. The correctly sequenced bacterial cultures were then shaken, plasmids were extracted, and stored at -20°C for further experiments.

[0054] The extracted plasmid was transformed into *Agrobacterium tumefaciens* GV3101 competent cells. The collected bacterial suspension was resuspended and plated onto LB agar plates containing kanamycin, and incubated upside down at 28°C for 2-3 days. Single colonies were picked from LB liquid medium containing kanamycin and rifampin for PCR verification. Positive clones were screened, and the cells were shaken and injected into tobacco. Fluorescence results were observed using a laser confocal microscope. The results showed that the HaSnRK2.5 gene is present in various parts of the cell (e.g., ...). Figure 3 As shown in the figure, the HaSnRK2.5 gene is expressed in the cytoplasm, nucleus, cytoskeleton, chloroplasts, mitochondria, and Golgi apparatus of the cell.

[0055] The extracted plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells, and the resuspended plasmid was plated on LB agar plates containing kanamycin and incubated upside down at 28°C for 2-3 days. Single colonies were picked for colony PCR verification, and the selected positive clones were used to infect sunflower seeds. The sunflower broomrape sensitive variety TY0409 was selected. The seeds were dehulled, and distilled water was poured into a petri dish to soak the dehulled seeds. The transparent seed coat was peeled off with tweezers, and the peeled sunflower seeds were then gently scratched a few times with tweezers, taking care not to scratch the embryo or other important parts of the seed. The treated sunflower seeds were placed in a new petri dish, and the infection solution was poured into the dish. The dish was then incubated in the dark for 6 hours. Infected sunflower seeds were placed in MS liquid medium containing cephalosporin and termethin and cultured in the dark for 48 hours. The germinated seeds were then transferred to MS solid medium containing cephalosporin and termethin. After 24-48 hours, the sunflower seeds were transferred to 13*13cm square petri dishes and allowed to grow normally in a greenhouse for two weeks to obtain sunflower plants overexpressing the HaSnRK2.5 gene.

[0056] Sunflower plants overexpressing the HaSnRK2.14 gene were prepared using the same method.

[0057] After two weeks of normal growth, sunflower seedlings overexpressing the target gene were co-cultured with sunflower broom seeds pre-germinated by the germination stimulant GR24, and the parasitism was observed for 10-14 days.

[0058] like Figure 4 A, Figure 4 B Figure 4 C and Figure 5 As shown, during the same parasitism period, no parasitism events occurred on the roots of plants overexpressing the HaSnRK2.5 gene, while sunflower broomrape parasitism events occurred in the root tissues of plants overexpressing the HaSnRK2.14 gene and the control group.

[0059] RNA was extracted from sunflower plants overexpressing the HaSnRK2.14 and HaSnRK2.5 genes, reverse transcribed into cDNA, and then subjected to quantitative real-time PCR to verify gene expression. The results are as follows: Figure 6 As shown, these two genes were successfully overexpressed in sunflower TY0409.

Claims

1. HaSnRK2.5 Use of a gene in increasing the resistance of sunflower to the broomrape, characterized in that, HaSnRK2.5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The route of the application includes: By overexpressing HaSnRK2.5 genes to increase the ability of sunflower to withstand stress from Russian thistle.

3. Use of a HaSnRK2.5 protein to increase the resistance of sunflower to the broomrape Orobanche against, The amino acid sequence of the HaSnRK2.5 protein is shown as SEQ ID NO.

2.

4. Use of a recombinant vector for increasing the resistance of sunflower to the orobanche parasitism, characterized in that, The recombinant vector comprises a sunflower HaSnRK2.5 gene, the HaSnRK2.5 nucleotide sequence of the gene is shown as SEQ ID NO.

1.

5. The use of genetically engineered bacteria in improving the resistance of sunflower to the orobanche parasitism, characterized in that, The genetically engineered bacteria comprise HaSnRK2.5 The nucleotide sequence of the gene is shown as SEQ ID NO.

1. HaSnRK2.5 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

6. A method for constructing a sunflower plant with enhanced resistance to Orobanche, characterized by, The method comprises the following steps: (1) The gene is obtained by gene cloning method HaSnRK2.5 gene; (2) to HaSnRK2.5 The primers were designed according to the gene sequence as a template, and the overexpression vector of the gene was constructed HaSnRK2.5 The primers were designed according to the gene sequence as a template, and the overexpression vector of the gene was constructed (3) transforming the vector into Agrobacterium competent cells to obtain Agrobacterium overexpressing the gene HaSnRK2.5 recombinant Agrobacterium (4) using recombinant agrobacterium to infect sunflower seeds to obtain sunflower plants with improved resistance to stress of rhizosphere stress; The HaSnRK2.5 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

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