Application of long-chain non-coding RNA HEAL in regulation and control of plant flowering under high temperature stress

Abstract

The invention provides an application of long-chain non-coding RNA HEAL in regulation and control of plant flowering under high temperature stress. The invention proves that the long-chain non-coding RNA HEAL can be combined with actin in vivo and in vitro, and the long-chain non-coding RNA HEAL and the actin can be co-localized. A microfilament marker is utilized to observe that overexpression of HEAL can improve the stability of actin in pollen after high temperature stress. HEAL is separated and cloned, a recombinant overexpression vector is obtained by utilizing a plant genetic engineering technology and is transformed into a plant to obtain wheat and corn positive seedlings for overexpression of HEAL, a homozygous strain is obtained through subsequent screening and identification, and the homozygous strain is compared with a wild type plant growing under the same condition, so that the wheat and corn positive seedlings with overexpression of HEAL are obtained. The wheat and corn overexpressed with HEAL retain more active pollen after high temperature stress, and the HEAL is further proved to be capable of improving the high temperature tolerance of the plant. Therefore, the HEAL gene can be applied to high-temperature stress resistance of crops, and has very high agricultural application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to long-chain non-coding RNA Application of HEAL in regulating plant flowering under high temperature stress. Background Art

[0002] Plants are affected by various biological and non-biological factors during their growth and development. With the deepening of the world's industrialization, the earth's environment is becoming increasingly severe, and the survival of plants is facing severe challenges. Among them, high temperature is one of the most important factors threatening the growth and development of crops. In recent years, extreme high temperature weather has occurred frequently, causing great losses to crop production. Therefore, finding the mechanism for plants to resist high temperatures has become an important issue that needs to be solved urgently.

[0003] Crop yield losses caused by high temperatures are partly attributed to the disruption of the flowering process. Plants are sensitive to high temperature stress during pollen germination, pollen tube growth and fertilization. In corn, the interval between flowering and silking is prolonged, the opening angle of the spikelet is reduced, the failure of anther rupture leads to a decrease in the number of pollen shed, and high temperatures one hour after pollination can cause abnormal growth of corn silk pollen tubes, leading to pollination failure. In wheat, there are two periods (8-6 days and 2-0 days before flowering) that are more sensitive to short-term (2-5 days) high temperature stress. Most studies have shown that loss of pollen viability is the main factor limiting wheat spikelet fertility under high temperature stress.

[0004] Among various non-coding RNAs (ncRNAs), long non-coding RNAs (lncRNAs) have become important regulators of temperature adaptation in many crops. At present, there have been many research advances in plant resistance to low temperature, however, there are few studies on lncRNAs in plant response to high temperature stress.

[0005] Microfilaments, or filamentous actin (F-actin), are double-stranded helical structures formed by the polymerization of globular actin (G-actin) subunits. Plant actin is one of the important components of the plant cytoskeleton, and it is of great significance in maintaining the normal growth, differentiation, and reproduction of plant cells. Actin is widely present in various tissue cells of higher plants, such as pollen, stem phloem, leaf epidermal cells, leaf sheath cells, root hairs, tendrils, etc. It is of great significance to study the mechanism of action of actin under high temperature. Summary of the invention

[0006] In view of the current status of the above-mentioned prior art, the purpose of the present invention is to provide an application of long non-coding RNA HEAL in regulating plant flowering under high temperature stress.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The present invention provides application of long-chain non-coding RNA HEAL in regulating flowering of plants under high temperature stress, and the sequence of the long-chain non-coding RNA HEAL is shown as SEQ ID NO.2.

[0008] Furthermore, the long non-coding RNA HEAL can enhance the vitality of plant pollen under high temperature stress.

[0009] Furthermore, the long non-coding RNA HEAL improves the high temperature stability of actin in pollen by binding to actin.

[0010] Furthermore, the long non-coding RNA HEAL can promote pollen germination and pollen tube growth under high temperature stress.

[0011] Furthermore, the application steps are: using upstream primers and downstream primers to perform PCR amplification, cloning the HEAL gene shown in SEQ ID NO.2, constructing a recombinant overexpression vector, and transferring it into plants through Agrobacterium transformation to obtain a HEAL overexpression plant strain.

[0012] Furthermore, the sequences of the upstream primer and the downstream primer are shown as SEQ ID NO.3 and SEQ ID NO.4.

[0013] Furthermore, the vector used for cloning is pMD19-T The expression vector used was pCAMBIA3300 .

[0014] Furthermore, the reaction system of the PCR amplification is: 25 μL of buffer, 1 μL of enzyme, 1 μL of dNTP, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of DNA template, and water is added to 50 μL; The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds, repeated for 36 cycles; and post-extension at 72°C for 5 minutes.

[0015] Furthermore, the recombinant expression vector containing the HEAL gene was introduced into wheat and corn, respectively, to obtain transgenic wheat and corn lines overexpressing HEAL. Under high temperature stress, the pollen grain activity of the transgenic wheat lines overexpressing HEAL was higher than that of the wild type, and the pollen tube germination rate was also higher than that of the wild type; the pollen grain activity of the transgenic corn lines overexpressing HEAL was higher than that of the wild type.

[0016] The present invention also provides the use of long-chain non-coding RNA HEAL in improving the yield of corn and wheat. The sequence of the long-chain non-coding RNA HEAL is shown in SEQ ID NO.2.

[0017] Compared with the prior art, the advantages and beneficial technical effects of the present invention are: the long non-coding RNA HEAL provided by the present invention has an important function in Arabidopsis thaliana to resist high temperature stress, and the present invention proves that the binding protein of HEAL is actin by RNA Pull-down technology and mass spectrometry analysis technology. The RNA FISH technology proves that HEAL and F-actin are co-localized in vivo.

[0018] The present invention uses microfilament markers to prove that overexpression of HEAL can improve the stability of actin in pollen after high temperature stress. The present invention also separates and clones HEAL, uses existing plant genetic engineering technology to obtain a recombinant overexpression vector, transforms it into a plant, obtains wheat and corn positive seedlings overexpressing HEAL, and obtains homozygous strains after subsequent screening and identification. Comparing it with wild-type plants grown under the same conditions, the pollen grain activity of wheat overexpressing the HEAL gene after high temperature treatment is higher than that of the wild type, and the germination rate of pollen tubes is also higher than that of the wild type; the pollen grain activity of corn overexpressing the HEAL gene after high temperature treatment is higher than that of the wild type. It is further proved that HEAL can improve the high temperature tolerance of plant pollen. Therefore, the HEAL gene can be used in crops to resist high temperature stress, can increase plant yield, and has a high agricultural application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :The HEAL binding protein is actin. A is the RNA Pull-down result diagram, the arrows point to the differential bands, and B is the RNA FISH result diagram.

[0020] Figure 2 : Schematic diagram of the constructed overexpression vector and restriction enzyme cutting sites.

[0021] Figure 3 : Changes of F-actin before and after high temperature. A shows the changes of Arabidopsis pollen F-actin in wild type, HEAL overexpression and mutant, and B shows the changes of in vitro polymerized F-actin with or without HEAL.

[0022] Figure 4 : Pollen viability of wheat overexpressing HEAL before and after high temperature treatment.

[0023] Figure 5 : Pollen viability of corn overexpressing HEAL before and after high temperature treatment. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. The experimental methods in the following embodiments without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer; the reagents and materials used are all commercially available products unless otherwise specified.

[0025] Example 1: HEAL directly binds to actin The present invention firstly proves that the binding protein of HEAL is actin ( Figure 1 A).

[0026] HEAL colocalized with F-actin by RNA FISH. A Cy5-labeled HEAL probe was synthesized at Shanghai Shenggong Biotechnology Co., Ltd. The probe sequence used to design HEAL is shown in SEQ ID NO.1. UBQpro:GFP-ABD2-GFP The transgenic Arabidopsis seedlings were fixed in 4% paraformaldehyde for 2 hours, then transferred to 0.5% TritonX-100 for 30 minutes, and then transferred to 2×SSC containing 2 pmol of probe and 50% formamide for overnight hybridization at 37°C. The seedlings were taken out and photographed using a laser confocal microscope. Under a 40× oil lens, the GFP and Cy5 fluorescence overlaps can be observed, as shown in Figure 2. Figure 1 As shown in B, it is confirmed that the two can indeed co-localize.

[0027] Example 2: Construction of HEAL recombinant expression vector The HEAL genome sequence was obtained using the Arabidopsis genome database website TAIR, and its sequence is shown in SEQ ID NO.2, with a total length of 448 bp.

[0028] The primer pair for amplifying the HEAL gene was designed based on the HEAL gene sequence, and its sequence is (5'-3'): Upstream primer: ggatccGGTGTCAACGTCTCCACTGG (SEQ ID NO. 3); Downstream primer: gagctcCGGGAATGAACAACGAGGCG (SEQ ID NO. 4); 2×Phanta Max Master was used for PCR amplification. The reaction system was: 25 μL Buffer, 1 μL enzyme, 1 μL dNTP, 2 μL upstream primer, 2 μL downstream primer, 2 μL DNA template, and water was added to 50 μL.

[0029] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds, repeated for 36 cycles; and post-extension at 72°C for 5 minutes.

[0030] After the PCR reaction is completed, agarose gel electrophoresis is performed. After the target band is detected, the gel is cut and the gel is recovered. The gel recovery method is performed according to the gel recovery kit of OMEGA Company.

[0031] 3. Construction of expression vector: First, add the gel recovery product to the following reaction system A: 25.5μL of gel recovery product, 3μL of 10×PCR buffer, 1.2μL of dNTP, and 0.3μL of rTaq enzyme. In a PCR instrument, react at 72℃ for 5 min.

[0032] Connect the A-added product to the cloning vector pMD19-T : Add 4.5 μL of product A, 5 μL of Solution I, and 0.5 μL of pMD19-Tsimple vector. Ligate overnight at 16°C.

[0033] The ligation product was transformed into the competent E. coli DH5α, and then spread on the LB plate containing ampicillin to grow overnight. Pick a single white colony in 100μL LB liquid culture medium containing ampicillin, culture at 37℃ for 3-4 hours, and perform PCR verification of the bacterial solution. Select the bacterial solution with the correct size of the amplified target band and transfer it to the shaking bacterial tube. Add 3-5 mL of LB liquid culture medium containing ampicillin to the shaking bacterial tube, and culture it overnight at 37℃ constant temperature shaker and 220 rpm. Use Shanghai Jierui high-purity plasmid mini extraction kit to extract plasmid DNA, and send it to Shanghai Shenggong Biotechnology Co., Ltd. for DNA sequencing. The results confirmed that the HEAL gene sequence was amplified correctly, and the subsequent expression vector construction can be carried out.

[0034] The ubiquitin promoter was digested with BamH1 and Sac1 pCAMBIA3300 After the empty vector and the cloning vector plasmid containing the target gene were digested at 37°C for two hours, agarose gel electrophoresis was performed, and the correct band was cut out for gel recovery. The gel recovery products were ligated using T4 DNA ligase, and the ligation products were transformed into the Escherichia coli TOP10 competent strain, and then spread on LB plates containing kanamycin and grown overnight.

[0035] Pick a single white colony and place it in 100 μL LB liquid culture medium containing kanamycin. After culturing at 37°C for 3-4 hours, perform PCR verification on the bacterial solution. Select the bacterial solution with the correct and bright amplified target band size and transfer it to a shaking tube. Add 3-5 mL LB liquid culture medium containing kanamycin. Incubate overnight at 37°C in a constant temperature shaker at 220 rpm. After extracting the plasmid, perform double enzyme digestion identification. The consistency with the target gene band size proves that it is the correct overexpression vector. Save the strain and plasmid for subsequent experiments. So far, the overexpression vector has been obtained. UBI:: HEAL Figure 2 ).

[0036] Take 2 μL of the recombinant overexpression vector and transform Agrobacterium EHA105 by freeze-thaw method. The obtained bacterial solution is spread on LB solid culture medium. After inverted culture for 36-48 hours, single colonies are picked for PCR and enzyme digestion double identification. The recombinant Agrobacterium single colonies with positive identification results are saved for subsequent experiments.

[0037] Example 3: HEAL enhances the stability of actin at high temperatures 1. HEAL enhances the stability of actin in Arabidopsis pollen under high temperature The above recombinant expression vector was infected into Arabidopsis thaliana by Agrobacterium transformation, and a homozygous HEAL overexpression strain was obtained by resistance and PCR identification.

[0038] Microfilament marker UBQpro:GFP-ABD2-GFP The homozygous strain was hybridized with the HEAL overexpression strain and the original mutant strain, and the homozygous strain was obtained after resistance screening and PCR identification for observing changes in microfilaments. After Arabidopsis thaliana in full flowering period was subjected to high temperature treatment at 42°C for 0.5 hours, pollen preparation was taken, and the state of microfilaments in pollen was observed under a laser confocal microscope using a 63× oil lens, and photographs were taken. The fluorescence was then quantified using imageJ software. The results showed that compared with the wild type, the fluorescence of microfilaments in the pollen of the overexpression strain was stronger, while that of the mutant was weaker, indicating that HEAL can enhance the stability of microfilaments in pollen under high temperature ( Figure 3 A).

[0039] 2. HEAL enhances the stability of polymerized F-actin in vitro The present invention uses an in vitro transcription kit to perform in vitro transcription to obtain HEAL.

[0040] The present invention requires extracting G-actin protein from rabbit muscle and polymerizing it in vitro to form F-actin: g Centrifuge for 30 min, collect the supernatant, assemble microfilaments in 1× KMEI buffer to a final actin concentration of 4 μM, and polymerize at room temperature for 30 min.

[0041]

[0042] The present invention uses Alexa 488 phalloidin to stain microfilaments: first, use 1× KMEI buffer to polymerize microfilaments so that the final concentration of actin is 5 μM. Alexa 488 phalloidin is added in a dark place to make the final concentration of actin 100nM. After incubation at room temperature for 5 minutes, 1× KMEI buffer is added to dilute the final concentration of actin to 0.05 μM, and 10 μL of the mixture is drawn onto a glass slide pretreated with 0.01% poly-lysine using a gun tip with the tip cut off, and a coverslip is covered. The state of F-actin before and after high temperature treatment and whether HEAL is applied is checked and photographed under the 63× oil lens of the LSM880 laser confocal fluorescence microscope. The results show that after applying HEAL, the F-actin after high temperature treatment is less broken or depolymerized than the F-actin without HEAL, indicating that HEAL enhances the stability of actin in vitro, and further indicating that the effect of HEAL on actin is conservative ( Figure 3 B).

[0043] Example 4: HEAL enhances the viability of wheat pollen after high temperature treatment Agrobacterium containing the recombinant vector in Example 2 was transferred into wheat to construct wheat overexpressing HEAL. The wheat used in the present invention was the spring variety fielder. After obtaining positive seedlings, the seedlings with HEAL bands after PCR identification were transgenic plants. The transgenic plants were subsequently screened and identified by PCR to obtain homozygous strains for subsequent experiments.

[0044] When the wheat flowering period comes, at 9:00 in the morning when the wheat powder is scattered, the wheat is heat treated at 42°C for 1 hour. After heat treatment, the pollen is taken for FDA staining. First, prepare the solution: dissolve FDA powder in acetone to make a solution with a concentration of 5 mg / ml, and store it in a refrigerator at 4°C. The final staining concentration is 100μg / ml. After staining in the dark for 5 minutes, wash it twice with deionized water and prepare the slices. Observe through the 10x lens of the fluorescence microscope AXIO. The pollen with green fluorescence is viable, while the pollen with weak fluorescence or no fluorescence is weak or has lost vitality. It was observed and statistically found that after high temperature treatment, the vigor of wheat pollen overexpressing HEAL was higher than that of the wild type, indicating that HEAL can enhance the vigor of wheat pollen under high temperature ( Figure 4 ).

[0045] Example 5: HEAL enhances the viability of corn pollen after high temperature treatment Agrobacterium containing the recombinant vector in Example 2 was transferred into corn to construct transgenic corn. The corn variety used in the present invention was B104. After obtaining positive seedlings, seedlings with HEAL bands after PCR identification were transgenic plants. The transgenic plants were subsequently screened and identified by PCR to obtain homozygous strains for subsequent experiments.

[0046] When the corn was scattering pollen, the corn pollen was treated at 42℃ for 1 hour, then FDA stained and observed. The 5x lens of the fluorescence microscope AXIO was used for observation and photography. The experimental results showed that after high temperature treatment, the vigor of corn pollen overexpressing HEAL was higher than that of wild type, indicating that HEAL can enhance the vigor of corn pollen under high temperature ( Figure 5 ).

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. Application of long non-coding RNA HEAL in regulating plant flowering under high temperature stress, characterized in that: The sequence of the long non-coding RNA HEAL is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The long non-coding RNA HEAL can enhance the vitality of plant pollen under high temperature stress.

3. The use according to claim 2, characterized in that: The long non-coding RNA HEAL improves the high temperature stability of actin in pollen by binding to actin.

4. The use according to claim 2, characterized in that: The long non-coding RNA HEAL can promote pollen germination and pollen tube growth under high temperature stress.

5. The use according to claim 1, characterized in that: The application steps are: using upstream primers and downstream primers to perform PCR amplification, cloning and obtaining the HEAL gene shown in SEQ ID NO.2, constructing a recombinant overexpression vector, and transferring it into plants through Agrobacterium transformation to obtain a HEAL overexpression plant strain.

6. The use according to claim 5, characterized in that: The sequences of the upstream primer and the downstream primer are shown as SEQ ID NO.3 and SEQ ID NO.

4.

7. The use according to claim 5, characterized in that: The vector used for cloning was pMD19-T The expression vector used was pCAMBIA3300 .

8. The use according to claim 5, characterized in that: The reaction system of the PCR amplification is: 25 μL of buffer, 1 μL of enzyme, 1 μL of dNTP, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of DNA template, and water is added to 50 μL; The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds, repeated for 36 cycles; and post-extension at 72°C for 5 minutes.

9. The use according to claim 5, characterized in that: The recombinant expression vector containing the HEAL gene was transferred into wheat and corn, respectively, to obtain transgenic wheat and corn lines overexpressing HEAL. Under high temperature stress, the pollen grain activity of the transgenic wheat lines overexpressing HEAL was higher than that of the wild type, and the pollen tube germination rate was also higher than that of the wild type; the pollen grain activity of the transgenic corn lines overexpressing HEAL was higher than that of the wild type.

10. Application of long non-coding RNA HEAL in increasing corn and wheat yields, characterized in that: The sequence of the long non-coding RNA HEAL is shown in SEQ ID NO.2.

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