Application of switchgrass PvCOL16 gene in regulation and control of plant flowering period

Abstract

The invention discloses an application of a switchgrass PvCOL16 gene in regulating and controlling the flowering period of plants. According to the invention, a recombinant over-expression vector is obtained by using an existing plant genetic engineering technology, then rice callus is transformed by using an agrobacterium infection transformation method, and a homozygous PvCOL16 transgenic rice plant is obtained through tissue culture and identification screening. Compared with a wild type plant growing under the same condition, a rice plant with the overexpressed PvCOL16 gene shows a late flowering phenotype, delays flowering of rice, reduces the plant height and participates in regulation and control of expression of genes related to flowering, so that the PvCOL16 gene can be applied to cultivation of late flowering or dwarf varieties and has very high agricultural utilization value.

Description

Technical Field

[0001] The present application relates to the technical field of plant genetic engineering, and specifically to the application of switchgrass PvCOL16 gene in regulating the flowering period of plants. Background Art

[0002] Flowering is a complex transition from vegetative to reproductive growth in plants, triggered by endogenous and environmental signals such as light and temperature. Among environmental signals, photoperiod is one of the most important influencing factors of flowering and is sensed through plant leaves. Based on their response to photoperiod, plants are generally divided into three categories: long-day (LD), short-day (SD) plants, and day-neutral plants. Control of flowering time is crucial for reproductive success and is also important for regional adaptability and potential yield capacity of crops.

[0003] In the long-day plant Arabidopsis thaliana, a large number of genetic and molecular studies have been conducted on photoperiod control of flowering, and the GIGANTEA (GI-CONSTANS (CO)-FLOWERINGLOCUS (FT)) pathway has been established. GI integrates cellular signals from photosensory transduction and the circadian clock, and then activates CO, a plant-specific CCT (CO, CO-like and TOC1) domain transcription activator. CO promotes flowering by directly activating FT, which encodes a small mobile protein synthesized in the leaf phloem. This protein is then transported to the shoot apical meristem, where it induces the expression of floral meristem identity genes (such as AP1 and LEAFY) to initiate the transition to reproductive growth.

[0004] Previous studies have shown that the CONSTANS-like (COL) gene family plays a key role in regulating flowering time in Arabidopsis and other plants. For example, Arabidopsis AtCOL3 acts as a flowering repressor under SDs and LDs, and also acts as a positive regulator of red light signaling and root growth. AtCOL9 weakens flowering under LDs and acts as a flowering activator under SDs. The rice genome contains 16 COL members. Although some of them have been shown to be regulators of photoperiodic flowering, multiple genes in the rice genome have been shown to be involved in the plant flowering regulation process, including Hd1, Ehd1, Hd3a, Ghd7, etc.

[0005] The study of the regulation mechanism of plant flowering time plays an important role in human research on plant evolution and reproduction. Changes in heading period and inflorescence development can directly affect plant yields. The early or late flowering time will also reflect different values ​​according to actual production and market demand. Moderate late flowering is also an agronomic trait of great significance to agricultural production.

[0006] However, there is still no known research on flowering genes in switchgrass (Panicumvirgatum L.) in the prior art. Summary of the invention

[0007] The present application provides the application of switchgrass PvCOL16 gene in regulating the flowering period of plants. The present invention connects the full-length CDS of PvCOL16 to an expression vector with a UBI promoter, and uses Agrobacterium to infect rice callus to verify that overexpression of the gene can cause plants to have late flowering traits and reduce plant height.

[0008] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0009] The present invention provides application of switchgrass PvCOL16 in regulating the flowering period of plants. The nucleotide sequence of the switchgrass PvCOL16 is shown in SEQ ID NO.1.

[0010] Furthermore, the strains overexpressing switchgrass PvCOL16 had late flowering traits compared with the wild-type strains.

[0011] Furthermore, during the vegetative and reproductive growth stages of the plant, the plant height of the strain overexpressing switchgrass PvCOL16 was reduced compared with the wild-type strain.

[0012] Furthermore, the flowering-related genes whose expression levels change after the overexpression of the switchgrass PvCOL16 gene include Ehd1, Ehd2, Ehd4, Hd3a, Ghd7, Ghd8, Hd16, and RCN1.

[0013] Furthermore, the expression levels of genes Ghd7, Ghd8, Hd16, and RCN1 in the strains overexpressing switchgrass PvCOL16 were significantly upregulated and higher than those in wild-type plants; the expression levels of genes Ehd1, Ehd2, Ehd4, and Hd3a were significantly decreased and lower than those in wild-type plants.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention separates and clones the PvCOL16 gene from switchgrass, uses the existing plant genetic engineering technology to obtain a recombinant overexpression vector, and then uses the Agrobacterium infection transformation method to transform rice callus tissue, and obtains a homozygous rice strain that overexpresses the PvCOL16 gene through tissue culture and identification screening. Compared with wild-type plants grown under the same conditions, the rice plants overexpressing the PvCOL16 gene show a phenotype of 4-6 weeks late flowering, delaying rice flowering, and significantly reducing plant height, and participate in regulating the expression of flowering-related genes, so the PvCOL16 gene can be used to cultivate varieties that bloom late or reduce plant height, and has high agricultural utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0017] Figure 1 :Evolutionary tree constructed based on the amino acids encoded by the switchgrass PvCOL16 gene and proteins in Arabidopsis thaliana (AtCOL16), rice (OsCOL16), soybean (GmCOL16), wheat (TaCOL16), sorghum (SbCOL16) and Brachypodium distachyon (BdCOL16).

[0018] Figure 2 : Electrophoretic bands of PCR amplified switchgrass PvCOL16 gene detection.

[0019] Figure 3 : Schematic diagram of the constructed overexpression vector and restriction enzyme cutting sites.

[0020] Figure 4 : Leaf DNA identification results of T0 generation transgenic lines; wherein, M is a marker, + is a positive control of the plasmid vector connected to switchgrass PvCOL16, and - is a negative control of the wild-type rice genome.

[0021] Figure 5 : Statistics of plant height and biomass of transgenic lines at seedling stage. a and b are comparison and statistical analysis of plant height, c is statistical analysis of fresh weight, and d is statistical analysis of dry weight.

[0022] Figure 6 :Statistical results of flowering time and biomass. a, e, f are the comparison and statistical analysis of plant height and heading time of transgenic lines, b, i are the length of stem nodes and statistical analysis, c, d, j are the number of grains per ear and statistical analysis, g, h are the comparison and statistical analysis of plant bright red and dry weight.

[0023] Figure 7:Quantitative expression analysis of flowering-related genes in transgenic rice plants overexpressing PvCOL16. 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: Sequence analysis, cloning and vector construction of switchgrass PvCOL16 gene

[0026] 1. Acquisition of switchgrass PvCOL16 gene

[0027] The gene Pavir.9NG134200.1 was found using the public genome database website Phytozome and named PvCOL16. The genome sequence of PvCOL16 is shown in SEQ ID NO.1, with a full length of 7819 bp; its transcript sequence is shown in SEQ ID NO.2, with a full length of 2835 bp; its CDS sequence is shown in SEQ ID NO.3, with a length of 1212 bp, and encodes 403 amino acids, and the encoded amino acid sequence is shown in SEQ ID NO.4.

[0028] The amino acid sequence of switchgrass PvCOL16 was compared with the COL16 proteins in Arabidopsis, rice, soybean, wheat, wheat, sorghum, and Brachypodium distachyon to construct an evolutionary tree, such as Figure 1 As shown, the switchgrass PvCOL16 protein is closely homologous to the sorghum SbCOL16 protein.

[0029] 2. Cloning and vector construction of switchgrass PvCOL16 gene

[0030] 1. RNA extraction: Total RNA from switchgrass was extracted using TRIzon;

[0031] (1) Grind fresh plant tissue thoroughly in liquid nitrogen or chop the plant tissue into small pieces and grind it quickly in TRIzon. Add 1 ml of TRIzon for every 30-50 mg of tissue and mix well.

[0032] (2) Add chloroform to the above solution, 0.2 ml of chloroform for every 1 ml of TRIzon, cap the tube, shake vigorously for 15 seconds, and leave at room temperature for 2-3 minutes.

[0033] (3) Centrifuge at 12,000 rpm at 4°C for 15 min and transfer the upper aqueous phase to a new RNase-free centrifuge tube.

[0034] (4) Add an equal volume of isopropanol to the aqueous solution, mix thoroughly by inversion, and place at room temperature for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes and discard the supernatant.

[0035] (5) Add 75% ethanol (prepared with RNase-free water) to wash the precipitate. For every 1 ml of TRIzon used, use 1 ml of 75% ethanol to wash the precipitate.

[0036] (6) Centrifuge at 12,000 rpm at 4°C for 3 min. Carefully aspirate the supernatant, taking care not to aspirate the RNA precipitate.

[0037] (7) Leave at room temperature for 2-3 minutes and air dry. Add 30-100 μL of RNase-free water to fully dissolve the RNA. The obtained RNA is stored at -80°C to prevent degradation.

[0038] 2. Synthesis of the first strand of reverse transcribed cDNA: After measuring the concentration of the above RNA, use PrimeScript TM cDNA was obtained by reverse transcription using 1stStrand cDNA Synthesis Kit according to the instruction manual.

[0039] 3. Cloning of switchgrass PvCOL16 gene and construction of expression vector

[0040] According to the seamless cloning instructions, the KpnI and XbaI sites were selected to linearize the 1302 vector, and the PvCOL16 gene primer pair was designed based on the CDS sequence of the switchgrass PvCOL16 gene. After the fragment was amplified, a recombination reaction was performed. The primer pair sequence with the homologous sequence was (5'-3'):

[0041] Upstream primer - gattatttttgcagggtaccCGGGAGAAGACGAAGTGTTC

[0042] Downstream primer - aatgtttgaacgatctctagaGCAATGCACGACGAGTAGT

[0043] Toyobo KOD fx high-fidelity enzyme was used for PCR amplification. The reaction system was 2x PCR buffer for KODFX 25μL, 2mM dNTPs 10μL, 2μL of upstream and downstream primers at 10pmol / L each, 3μL of template DNA, 1μL of KOD FX, and 7μL of ddH2O.

[0044] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 56°C for 30 seconds, extension at 68°C for 90 seconds, 32 cycles; extension at 68°C for 5 minutes; and insulation at 4°C.

[0045] After PCR, agarose gel electrophoresis was performed, the gel was cut to obtain the target band, and a DNA purification and recovery kit was used according to the instructions to obtain the purified DNA fragment.

[0046] The recombinant product was transformed into E. coli DH5α strain, plated on LB plates with kanamycin resistance and grown overnight.

[0047] A single white colony was picked and grown in LB liquid culture medium for 8 hours, and PCR verification was performed on the bacterial solution. The strains with correct bands were sent to the company for sequencing. The strains and plasmids with correct sequencing were preserved for subsequent experiments.

[0048] Example 2: Screening of rice overexpressing PvCOL16 positive seedlings

[0049] 1. Seed disinfection and callus induction

[0050] (1) Prepare the induction medium and dispense it into 100 ml Erlenmeyer bottles. After sealing, sterilize at 121°C for 15 min and cool for later use. After shelling, wash the rice seeds with 75% ethanol for 1 min, sterilize with 15% NaClO for 15-20 min, and finally wash with sterile ddH2O for 4-5 times. Inoculate 8-12 sterilized rice seeds into each bottle of induction medium and culture in the dark at 30°C for 40-45 days to induce callus formation.

[0051] (2) Streak the Agrobacterium strain containing the target gene in Example 1 on a plate containing kanamycin and rifampicin. After two days, pick a single colony and culture it in a shaking incubator at 28°C.

[0052] (3) Centrifuge the cultured bacterial solution at 4000 rpm for 15 minutes, discard the supernatant, add the infection solution to resuspend the bacteria to OD = 0.3, add 100 μL AS and 2 ml 50% glucose per 100 ml infection solution, and continue culturing for 30 minutes.

[0053] (4) Introduce the Agrobacterium suspension into the flask containing the callus tissue and let it stand for 10 minutes. Pour off the bacterial solution and use absorbent paper to absorb the remaining suspension in the callus tissue. Finally, place it in a petri dish covered with filter paper and let it dry naturally for 1-2 hours.

[0054] (5) Use tweezers to transfer the dried callus particles to the co-culture medium, seal them with sealing glue, and place them in the dark at 19°C for 3 days.

[0055] (6) The callus tissue is then transferred to a screening medium and resistant calli are obtained after 40-60 days.

[0056] (7) Pick out small pieces of light yellow, dense, dry, and vigorously growing resistant callus, transfer them to differentiation medium, and culture them in light for 30-40 days to differentiate into 3-5 cm seedlings for rooting culture, and transplant them after 15-20 days.

[0057] 2. Identification of transgenic positive seedlings

[0058] (1) The PvCOL16 overexpressing transgenic plants obtained above were used as T0 generation, and genomic DNA was extracted from leaves and PCR identification was performed. The results were ( Figure 4 ) showed that all the selected plants were positive plants.

[0059] (2) The harvested T0 generation seeds were used as the T1 generation after germination, and genomic DNA was extracted from the leaves for PCR identification. The results showed that the positive:negative ratio of T1 generation plants was between 2:1 and 6:1, and the positive strains were single copies.

[0060] (3) The harvested T1 generation positive seeds are used as T2 generation after germination, and the genomic DNA is extracted from the leaves for PCR identification; if the results show that T2 generation of a certain strain does not undergo T-DNA segregation, the strain is identified as a homozygous strain and is selected for subsequent experiments.

[0061] Example 3: Phenotypic analysis of transgenic lines overexpressing PvCOL16

[0062] The plant height, fresh weight and dry weight of rice seedlings were counted after 30 days of growth. The flowering time and plant height of T2 homozygous rice plants were counted: the flowering time was the number of days from germination to awn emergence of T2 rice seeds under light conditions, using wet filter paper, and the plant height was the height of the aboveground part, from the base to the end of the main stem.

[0063] Compared with the wild type, the plant heights of the homozygous transgenic lines L1 and L2 were shorter after 30 days of growth, which were 62.6% and 56.4% of the wild type plants, respectively ( Figure 5 a, b); the fresh weight and dry weight of L1 strain were 39.6% and 25.3% of those of wild-type plants, respectively, and the fresh weight and dry weight of L2 strain were 27.3% and 27.6% of those of wild-type plants, respectively ( Figure 5 c, d).

[0064] Heading time, agronomic traits and statistical analysis of wild-type control plants and transgenic plants Figure 6 As shown, the L1 strain was about 23 days later than the wild-type control strain, and the L2 strain was about 46 days later than the wild-type control strain ( Figure 6 a, e). The plant height of the transgenic plants was significantly lower than that of the wild-type control plants. The average plant heights of lines L1 and L2 were 72.7 cm and 57.3 cm, respectively, which were 69.9% and 55.1% of those of the WT plants ( Figure 6 a, f); The internode lengths of transgenic lines L1 and L2 were significantly lower than those of WT control plants, which were 71.1% and 62.6% of those of control plants, respectively ( Figure 6 b, i). The fresh weight and dry weight of L1 and L2 were significantly lower than those of the wild-type control strains. The fresh weight and dry weight of the L1 strain were 62.9% and 69.0% of those of the wild-type plants, respectively, and the fresh weight and dry weight of the L2 strain were 71.1% and 67.4% of those of the wild-type plants, respectively ( Figure 6 g, h). The average number of grains per ear in lines L1 and L2 was 29.8% and 42.1% of that in the control, respectively. Figure 6 c, d, j).

[0065] Example 4: Detection of the expression level of flowering-related genes in the transgenic strains overexpressing PvCOL16

[0066] According to the requirements of qRT-PCR primer design, Primer Premier 6.0 software was used to design specific primers for multiple flowering-related genes. The primer pair sequences are:

[0067] qEhd1-F 5'-cctacagtgattatggcttca-3'

[0068] qEhd1-R 5'-gtgctgccaaatgttgctc-3'

[0069] qEhd2-F 5'-CGACGACAATAGCTCGATCGC-3'

[0070] qEhd2-R 5'-GTGCATGGTCACGGAGCCTT-3'

[0071] qEhd3-F 5'-gaccacctcgtcacctacaag-3'

[0072] qEhd3-R 5'-gagtgtccctccagctaatcc-3'

[0073] qEhd4-F 5'-CAGCCAGCGGAATCATCAC-3'

[0074] qEhd4-R 5'-CCAAATCCATCAGACCTACTCCT-3'

[0075] qGhd7-F 5'-AGGTGCTACGAGAAGCAAATCC-3'

[0076] qGhd7-R 5'-GGGCCTCATCTCGGCATAG-3'

[0077] qGhd8-F 5'-TCGCCGGACTCGTTGTCCAAC-3'

[0078] qGhd8-R 5'-AGAGTAGGAAGAGCTATGGGC-3'

[0079] qHd1-F 5'-ggcgtcagtgcttacacagatt-3'

[0080] qHd1-R 5'-tccagcaggtgtcaggattct-3'

[0081] qHd3a-F 5'-gctcactatcatcatccagcatg-3'

[0082] qHd3a-R 5'-ccttgctcagctatttaattgcataa-3'

[0083] qRCN1-F5'-ACTGTCTGCTCCTCTAAACA-3'

[0084] qRCN1-R 5'-GGCTGCTGATGTAGCAGAAG-3'

[0085] qhd16-F 5'-GAAGCCAATGGACGAGACACAAGAA-3'

[0086] qhd16-R 5'-GCCGTAGCAGATTGAAGCGATGT-3'

[0087] Using TB Green TM Premix Ex Taq TM II reagents, quantitative analysis was performed by QuantStudio 5 Real-Time PCR system, three biological replicates and two technical replicates were performed for each sample, and the reaction conditions were: 95°C for 10 minutes, 95°C for 5 seconds, 60°C for 31 seconds, and 40 cycles.

[0088] The results are as follows Figure 7 As shown, compared with the wild type, the expression levels of flowering-related genes Ghd7, Ghd8, Hd16, and RCN1 in the overexpressing PvCOL16 transgenic lines were significantly upregulated and higher than those in the wild type lines, but the expression levels of Hd3a, Ehd1, Ehd2, and Ehd4 genes were significantly decreased and lower than those in the wild type plants, which can explain the cause of the late flowering phenotype of the overexpressing PvCOL16 transgenic lines.

[0089] The above evidence indicates that the PvCOL16 gene is involved in regulating the expression of some flowering-related genes, and that overexpression of the PvCOL16 gene causes late flowering traits. It also indicates that overexpression of PvCOL16 can delay the flowering time of rice and reduce the plant height.

[0090] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. Application of switchgrass PvCOL16 gene in regulating flowering period of plants, characterized in that: The nucleotide sequence of the switchgrass PvCOL16 gene is shown in SEQ ID NO.

1.

2. The use of the switchgrass PvCOL16 gene in regulating the flowering period of plants according to claim 1, characterized in that: The lines overexpressing the switchgrass PvCOL16 gene had late flowering traits compared with the wild-type lines.

3. The use of the switchgrass PvCOL16 gene in regulating the flowering period of plants according to claim 1, characterized in that: During the vegetative and reproductive growth stages of the plant, the plant height of the strains overexpressing the switchgrass PvCOL16 gene was lower than that of the wild-type strain.

4. The use of the switchgrass PvCOL16 gene in regulating the flowering period of plants according to claim 1, characterized in that: The flowering-related genes whose expression levels change after the switchgrass PvCOL16 gene is overexpressed include Ghd7, Ghd8, Hd16, RCN1, Hd3a, Ehd1, Ehd2 and Ehd4.

5. The use of the switchgrass PvCOL16 gene in regulating the flowering period of plants according to claim 1, characterized in that: In the strains overexpressing the switchgrass PvCOL16 gene, the expression levels of genes Ghd7, Ghd8, Hd16 and RCN1 were significantly upregulated and higher than those in wild-type plants; the expression levels of genes Hd3a, Ehd1, Ehd2 and Ehd4 were significantly decreased and lower than those in wild-type plants.

6. Use of the switchgrass PvCOL16 gene according to any one of claims 1 to 5 in regulating the flowering period of plants, characterized in that: The plants include rice.

Images