Wheat heading period regulation gene, method and application

Abstract

The invention discloses a wheat heading period regulation gene and method and application, and the method comprises the following steps: picking three-leaf-stage leaves of a wheat variety 'Sumai No.3', and extracting total RNA (Ribonucleic Acid); designing a specific primer of the gene; the TaWRKY75-2D gene fragment is amplified, and the The preparation method comprises the following steps: carrying out linearization on a pcb301-bsmv-gamma-smr carrier; the preparation method comprises the following steps: constructing a pCB301-BSMV gamma-TaWRKY75-2D vector; and mixing the BSMV-alpha, the BSMV-beta and the pCB301-BSMV gamma-TaWRKY75-2D agrobacterium tumefaciens resuspension, and carrying out inoculation on the obtained mixture. By creating a TaWRKY75-2D overexpression transgenic line and a gene knockout mutant, it is found that overexpression of the gene can cause advanced heading of transgenic wheat, and heading delay is caused by the mutant; and molecular biological analysis further finds that the gene can regulate and control the heading stage of wheat by combining with a circadian rhythm pathway gene LHY.

Description

Technical Field

[0001] The present invention relates to the field of wheat heading period regulating genes, and in particular to a wheat heading period regulating gene, a method and an application thereof. Background Art

[0002] Common wheat is one of the most widely grown cereal crops, providing about 20% of the calories and protein consumed by humans worldwide. Ensuring and improving my country's wheat yield and quality is of great significance to my country's food security. Heading period is one of the most important agronomic traits of wheat, and can reflect the ecological adaptability of wheat to a certain extent. In modern wheat breeding programs, heading period is one of the most important wheat breeding target traits considered by breeders. It is not only closely related to the growth period, but also has an important influence on multiple agronomic traits such as wheat yield, disease resistance and stress resistance. Therefore, the study and analysis of the genetic regulation mechanism of wheat heading period is of great significance to sustainable agricultural production.

[0003] Vernalization is the main way to regulate wheat heading and flowering. At present, some genes involved in the regulation of flower initiation in response to vernalization have been cloned in wheat, such as CONSTANS (CO), FLOWERING LOCUS T (FT), PHOTOPERIOD1 (PPD1), VERNALIZATION1 (VRN1), VERNALIZATION3 (VRN3). In addition, a large number of studies have reported that wheat heading period is affected by many gene loci. It is pointed out that the QTLs related to wheat heading period genes are mainly distributed on chromosomes 2B, 3A, 4A, 4B, 6B, 7A, 7B and 7D. Domestic researchers used the genetic population constructed by Chinese wheat varieties to locate QTLs related to wheat heading period and precocity on chromosome 7D, which can explain 6.72% to 12.68% of the phenotypic variation. Many previous studies have located related QTLs at this site, which infers that there are key genes affecting wheat heading period on chromosome 7D.

[0004] Plant growth and development, especially the induction of heading and flowering, are regulated by both endogenous genes and environmental factors. After further studies on the segments located on different chromosomes of wheat, many genes related to heading time were cloned. For example, it has been reported that the florigen gene FT promotes heading and flowering in plants and regulates the structure of plants by switching the meristem from vegetative growth to reproductive growth. The key genes involved in vernalization in Arabidopsis are FRIGIDA (FRI) and Flowering Locus C (FLC) (MADS-box transcription factors). FRI encodes a scaffolding protein that promotes the transcription of FLC by forming a transcriptional activation complex composed of FRI, FRIGIDA Like1 (FRL1), FRIGIDA Essential1 (FES1), SUF4 repressor, and FLC expression factor. High-level expression of FLC inhibits the transcription of FT and SUPPRESSOROF OVER-EXPRESSION OF CO (SOC1), resulting in delayed flowering in Arabidopsis. In wheat, the Vrn-1 gene directly affects the heading and flowering periods of wheat, and is located on chromosomes 5AL, 5BL, and 5DL, respectively. This gene is the first vernalization gene in common wheat obtained through map-based cloning technology, including three loci: Vrn-A1, Vrn-B1, and Vrn-D1. Subsequently, Vrn-2 and Vrn-3 (Vrn-D3) were also cloned in wheat and barley, respectively.

[0005] WRKY, as a large class of transcription factors in plants, plays an important role in the growth and development of plants. A large number of studies have shown that WRKY genes regulate the flowering process of plants, such as rice OsWRKY11 regulating the heading period of rice. However, there are relatively few studies on wheat WRKY regulating the heading period, and its molecular mechanism is still unclear.

[0006] Although genes regulating wheat heading period have been reported, no research has been reported on the regulation of wheat heading period by WRKY transcription factors. The molecular mechanism of plant heading period corresponding to the circadian rhythm pathway is still unclear. It is urgent to discover the important WRKY transcription factors regulating wheat heading period and clarify their molecular mechanisms.

[0007] The present invention creates TaWRKY75-2D overexpression transgenic strains and gene knockout mutants, and finds that overexpression of the gene causes transgenic wheat to head prematurely, while the mutant causes delayed heading; further molecular biological analysis finds that the gene can regulate the heading period of wheat by binding to the circadian rhythm pathway gene LHY. Summary of the invention

[0008] In view of this, the present invention provides a wheat heading period regulating gene, method and application.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A wheat heading period regulatory gene, which is the TaWRKY75-2D gene.

[0011] A method for regulating genes in wheat heading period, comprising the following steps:

[0012] Step 1:

[0013] Leaves of the wheat variety "Sumai No. 3" at the three-leaf stage were picked and total RNA was extracted using RNAprep pure Plant Kit;

[0014] Step 2:

[0015] Obtain the full-length sequence of TaWRKY75-2D and design specific primers for the gene;

[0016] Step 3: amplify the TaWRKY75-2D gene fragment using specific primers and clone it into the wheat genetic transformation vector PC186;

[0017] Step 4:

[0018] The pcb301-bsmv-γ-smr vector was linearized with the restriction endonuclease SapI;

[0019] Step 5:

[0020] The TaWRKY75-2D gene target was designed and assembled into the pCB301-BSMVγ-SmR linearized vector using homologous recombination technology to construct the pCB301-BSMVγ-TaWRKY75-2D vector;

[0021] Step 6:

[0022] BSMV-α, BSMV-β and pCB301-BSMVγ-TaWRKY75-2D were transferred into Agrobacterium GV3101, and equal volumes of BSMV-α, BSMV-β and pCB301-BSMVγ-TaWRKY75-2D Agrobacterium resuspension were mixed and then co-infiltrated into tobacco leaves; after inoculation, the inoculated leaves were ground in inoculation buffer and inoculated into wheat at the jointing stage.

[0023] Preferably, in step 2, the full-length sequence of TaWRKY75-2D is obtained from Ensembl plant.

[0024] Preferably, in step 6, equal volumes of BSMV-α, BSMV-β and pCB301-BSMVγ-TaWRKY75-2D Agrobacterium resuspension are mixed and adjusted to A600=0.3.

[0025] Preferably, in step six, the tobacco leaves are co-infiltrated with a needle-free syringe at the 5-6 leaf stage.

[0026] Preferably, in step six, 3 to 7 days after inoculation, the inoculated leaves are ground in an inoculation buffer.

[0027] Preferably, in step six, the buffer is 10 mM sodium phosphate, pH=5.85, 0.5% sodium sulfite.

[0028] A method for regulating genes during wheat heading period is used for regulating wheat heading period.

[0029] Compared with the prior art, the present invention has achieved the following technical effects:

[0030] The present invention creates TaWRKY75-2D overexpression transgenic strains and gene knockout mutants, and finds that overexpression of the gene causes transgenic wheat to head prematurely, while the mutant causes delayed heading; further molecular biological analysis finds that the gene can regulate the heading period of wheat by binding to the circadian rhythm pathway gene LHY. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram for identifying the phenotype of wheat heading period regulated by TaWRKY75-2D, a wheat heading period regulating gene, method and application of the present invention;

[0032] Among them, A. caused the transgenic wheat to head early; B. the mutant caused the heading to be delayed;

[0033] Figure 2 This is a TaWRKY75-2D downstream regulatory factor screening diagram for a wheat heading period regulatory gene, method and application of the present invention;

[0034] Among them, A. yeast one-hybrid; B. EMSA. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The invention discloses a wheat heading period regulating gene, which is a TaWRKY75-2D gene.

[0037] The present invention also discloses a method for regulating genes in the heading period of wheat, comprising the following steps:

[0038] Step 1:

[0039] Leaves of the wheat variety "Sumai No. 3" at the three-leaf stage were picked and total RNA was extracted using RNAprep pure Plant Kit;

[0040] Step 2:

[0041] The full-length sequence of TaWRKY75-2D was obtained from Ensembl Plants, and specific primers for this gene were designed;

[0042] Step 3: amplify the TaWRKY75-2D gene fragment using specific primers and clone it into the wheat genetic transformation vector PC186;

[0043] Step 4:

[0044] The pcb301-bsmv-γ-smr vector was linearized with the restriction endonuclease SapI;

[0045] Step 5:

[0046] The TaWRKY75-2D gene target was designed and assembled into the pCB301-BSMVγ-SmR linearized vector using homologous recombination technology to construct the pCB301-BSMVγ-TaWRKY75-2D vector;

[0047] Step 6:

[0048] BSMV-α, BSMV-β and pCB301-BSMVγ-TaWRKY75-2D were transferred into Agrobacterium GV3101, and equal volumes of BSMV-α, BSMV-β and pCB301-BSMVγ-TaWRKY75-2D Agrobacterium resuspension were mixed and adjusted to A600=0.3, and tobacco leaves were infiltrated with a needleless syringe at the 5-6 leaf stage; 3-7 days after inoculation, the inoculated leaves were ground in an inoculation buffer, the buffer was 10mM sodium phosphate, pH=5.85, 0.5% sodium sulfite. Grind in the inoculation buffer, and inoculate on wheat at the jointing stage.

[0049] Embodiment 1:

[0050] Yeast one-hybrid assay

[0051] The interaction between TaWRKY75-2D and LHY promoter was verified using yeast one-hybrid assay (Y1H);

[0052] The full length of TaWRKY75-2D and LHY promoters were amplified from the wheat variety "Sumai No. 3". The sequence of TaWRKY75-2D was recombined into the 42-AD vector to obtain BD-TaWRKY75-2D;

[0053] The LHY promoter (TraesCS6B02G266800) was recombined into LACZ-DFR to obtain AD-LHY; Y1H detection was performed according to the protocol of the Super Yeast Transformation System (Coolaber, SK2401);

[0054] Subsequently, the recombinant plasmids BD-TaWRKY75-2D and AD-LHY / FT were co-transformed into the yeast Y1H Gold; the transformed yeast cells were placed on synthetic defined (SD) medium without Ura and Trp (SD / -Ura / -Trp).

[0055] Embodiment 2:

[0056] Electrophoretic mobility shift assay

[0057] In EMSA, the full-length CDS of TaWRKY75 was amplified from the wheat variety “SuMai 3” and cloned into the SUMO vector to generate the SUMO-TaWRKY75-2D construct;

[0058] SUMO-tagged TaWRKY75-2D protein and empty SUMO were expressed in Escherichia coli BL21(DE3); recombinant protein expression was induced by 0.1 mM isopropyl-β- d -thiogalactopyranoside (IPTG);

[0059] The recombinant protein was purified with amylose resin (NEB); the W-box binding site on the LHY promoter was predicted and used to synthesize an oligonucleotide probe with a 5' biotin modification.

[0060] Embodiment 3:

[0061] The main results are as follows:

[0062] 1. Construct TaWRKY75-2D gene overexpression vector and gene knockout vector, and obtain TaWRKY75-2D gene overexpression and gene knockout transgenic plants by Agrobacterium-mediated genetic transformation method, and observe their heading period.

[0063] It was found that overexpression of this gene caused transgenic wheat to head prematurely. Figure 1 As shown in A, the mutant leads to delayed heading. Figure 1 As shown in B.

[0064] 2. Verification of downstream regulatory factors.

[0065] TaWRKY75-2D downstream regulatory factors were screened by transcriptomics, yeast one-hybrid, and EMSA. The results showed that TaWRKY75-2D regulates wheat heading date by binding to the circadian rhythm pathway gene LHY. Figure 2 shown.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A wheat heading period regulating gene, characterized in that: The gene is TaWRKY75-2D gene.

2. A method for regulating genes in wheat heading period, characterized in that: The following steps are involved: Step 1: Leaves of wheat variety "Sumai No. 3" at the three-leaf stage were picked and total RNA was extracted using RNAprep pure Plant Kit; Step 2: Obtain the full-length sequence of TaWRKY75-2D and design specific primers for the gene; Step 3: amplify the TaWRKY75-2D gene fragment using specific primers and clone it into the wheat genetic transformation vector PC186; Step 4: The pcb301-bsmv-γ-smr vector was linearized with the restriction endonuclease SapI; Step 5: The TaWRKY75-2D gene target was designed and assembled into the pCB301-BSMVγ-SmR linearized vector using homologous recombination technology to construct the pCB301-BSMVγ-TaWRKY75-2D vector; Step 6: BSMV-α, BSMV-β and pCB301-BSMVγ-TaWRKY75-2D were transferred into Agrobacterium GV3101, and equal volumes of BSMV-α, BSMV-β and pCB301-BSMVγ-TaWRKY75-2D Agrobacterium resuspension were mixed and then co-infiltrated into tobacco leaves; after inoculation, the inoculated leaves were ground in inoculation buffer and inoculated into wheat at the jointing stage.

3. The method for regulating genes at the heading stage of wheat according to claim 2, characterized in that: In the step 2, the full-length sequence of TaWRKY75-2D was obtained from Ensembl plant.

4. The method for regulating genes at the heading stage of wheat according to claim 2, characterized in that: In step 6, equal volumes of BSMV-α, BSMV-β and pCB301-BSMVγ were added. -TaWRKY75-2D Agrobacterium resuspension and adjust A600 to 0.

3.

5. The method for regulating genes at the heading stage of wheat according to claim 2, characterized in that: In the step six, the tobacco leaves are co-infiltrated with a needle-free syringe at the 5-6 leaf stage.

6. The method for regulating genes at heading stage of wheat according to claim 2, characterized in that: In the step six, 3 to 7 days after inoculation, the inoculated leaves are ground in an inoculation buffer.

7. The method for regulating genes at the heading stage of wheat according to claim 2, characterized in that: In the step six, the buffer is 10 mM sodium phosphate, pH=5.85, and 0.5% sodium sulfite.

8. Use of a wheat heading period regulating gene and method according to claim 1 or 2-7 in regulating wheat heading period.

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