Application of switchgrass pvcol16 gene in regulating flowering time of plants

Abstract

The application discloses application of a switchgrass PvCOL16 gene in regulation of flowering period of plants. The application obtains a recombinant overexpression vector by using existing plant genetic engineering technology, and then converts rice callus by using an agrobacterium infection conversion method; after tissue culture and identification and screening, a homozygous PvCOL16 transgenic rice plant is obtained. Compared with wild type plants grown under the same conditions, the rice plant overexpressing the PvCOL16 gene shows a late flowering phenotype, delays the flowering of the rice, reduces the plant height, and participates in regulation 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 high agricultural utilization value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to application of a switchgrass PvCOL16 gene in regulating flowering time of plants. BACKGROUND

[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 factors affecting flowering and is perceived by the leaves of plants. Plants are generally classified into three categories according to their response to photoperiod: long-day (LD), short-day (SD) and day-neutral plants. Control of flowering time is crucial for reproductive success and is also important for the regional adaptability and potential yield capacity of crops.

[0003] In Arabidopsis, a long-day model plant, much genetic and molecular research has been conducted on the photoperiodic control of flowering, and a GI-CONSTANS (CO)-FLOWERING LOCUS (FT) pathway has been established. GI integrates cellular signals from light sensing transduction and the biological clock, and then activates CO, a plant-specific CCT (CO, CO-like and TOC1) domain transcriptional 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 the flowering time of 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 attenuates flowering under LDs, but acts as a flowering activator under SDs. The rice genome contains 16 COL members. Although some of them have been proven to be modulators of photoperiodic flowering, multiple genes in the rice genome have been proven to be involved in the process of plant flowering regulation, including Hd1, Ehd1, Hd3a, Ghd7, etc.

[0005] Research on the mechanism of plant flowering time regulation is of great importance for human research on plant evolution and reproduction. Changes in heading date and inflorescence development can directly affect the yield of plants, and the early or late flowering time can also reflect different values according to actual production and market demand, and moderate late flowering is also an important agronomic trait for agricultural production.

[0006] However, the research on the flowering gene in Panicum virgatum L. in the prior art is still unknown. SUMMARY

[0007] The application provides an application of a Panicum virgatum PvCOL16 gene in regulating a flowering period of a plant.

[0008] To achieve the above-mentioned application purposes, the application adopts the following technical solutions:

[0009] The application provides an application of a Panicum virgatum PvCOL16 gene in regulating a flowering period of a plant, and the nucleotide sequence of the Panicum virgatum PvCOL16 is shown as SEQ ID NO. 1.

[0010] Further, the strain overexpressing the Panicum virgatum PvCOL16 has a late flowering trait compared with a wild type strain.

[0011] Further, in the vegetative growth and reproductive growth stages of the plant, the plant height of the strain overexpressing the Panicum virgatum PvCOL16 is lower than that of the wild type strain.

[0012] Further, the flowering-related genes with changed expression levels caused by overexpression of the Panicum virgatum PvCOL16 gene include Ehd1, Ehd2, Ehd4, Hd3a, Ghd7, Ghd8, Hd16 and RCN1.

[0013] Further, the expression levels of the genes Ghd7, Ghd8, Hd16 and RCN1 in the strain overexpressing the Panicum virgatum PvCOL16 are obviously up-regulated and higher than those of the wild type plant, and the expression levels of the genes Ehd1, Ehd2, Ehd4 and Hd3a are obviously down-regulated and lower than those of the wild type plant.

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

[0015] The present application isolates and clones a PvCOL16 gene from switchgrass, uses existing plant genetic engineering technology to obtain a recombinant overexpression vector, and then uses an agrobacterium infection transformation method to transform rice callus, and through tissue culture and identification screening, a homozygous overexpression PvCOL16 gene rice strain is obtained. Compared with wild-type plants grown under the same conditions, the overexpression PvCOL16 gene rice plants exhibit a 4-6 week late flowering phenotype, delay rice flowering, and significantly reduce plant height, and regulate the expression of flowering-related genes, so the PvCOL16 gene can be applied to cultivate late-flowering or low-height varieties, and has high agricultural utilization value. BRIEF DESCRIPTION OF DRAWINGS

[0016] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which like reference characters refer to like parts throughout the figures, and wherein:

[0017] Figure 1 : Phylogenetic tree of amino acids encoded by switchgrass PvCOL16 gene and proteins in Arabidopsis (AtCOL16), rice (OsCOL16), soybean (GmCOL16), wheat (TaCOL16), sorghum (SbCOL16), and Brachypodium (BdCOL16).

[0018] Figure 2 : Electrophoretic bands detected by PCR amplification of switchgrass PvCOL16 gene.

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

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

[0021] Figure 5 : Seedling height and biomass statistics of transgenic lines; wherein a and b are height comparison and statistical analysis, c is fresh weight statistical analysis, and d is dry weight statistical analysis.

[0022] Figure 6 : Statistical results of flowering time and biomass; wherein a, e, and f are height and heading time comparison and statistical analysis of transgenic lines, b and i are stem node length and statistical analysis, c, d, and j are main ear grain number and statistical analysis, g and h are fresh and dry weight comparison and statistical analysis.

[0023] Figure 7Quantitative expression analysis of flowering-related genes in the transgenic rice plants overexpressing PvCOL16. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific examples. The experimental methods not specified in the following examples are generally carried out according to the conventional conditions or the conditions recommended by the manufacturers; and the reagents and materials used are commercially available products if not specifically specified.

[0025] Example 1: Sequence analysis, cloning and vector construction of switchgrass PvCOL16 gene

[0026] I. Acquisition of switchgrass PvCOL16 gene

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

[0028] The amino acid sequence of switchgrass PvCOL16 was used to construct an evolutionary tree with COL16 proteins in Arabidopsis, rice, soybean, wheat, Triticum aestivum, Sorghum and Brachypodium, as shown in FIG. 1. The switchgrass PvCOL16 protein has a close homology with the Sorghum SbCOL16 protein. Figure 1

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

[0030] 1. Extraction of RNA: total RNA of switchgrass was extracted using TRIzon;

[0031] (1) Fresh plant tissues were ground in liquid nitrogen or the plant tissues were directly ground in TRIzon after being cut, 1 ml of TRIzon was added for every 30-50 mg of tissues, and mixed well.

[0032] (2) Chloroform was added to the above solution, 0.2 ml of chloroform was added for every 1 ml of TRIzon, the tube cap was covered, and the mixture was vigorously shaken for 15 seconds and left at room temperature for 2-3 minutes.

[0033] (3) Centrifugation at 12000 rpm for 15 minutes at 4°C, and the upper aqueous phase was transferred to a new RNase-Free centrifuge tube.

[0034] ​(4) Add equal volume of isopropanol to the obtained aqueous solution, invert mix, and stand at room temperature for 10 minutes. Centrifuge at 12000 rpm for 10 minutes at 4°C, and discard the supernatant.

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

[0036] (6) Centrifuge at 12000 rpm for 3 minutes at 4°C, and carefully aspirate the supernatant, taking care not to aspirate the RNA precipitate.

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

[0038] 2. Synthesis of reverse transcription cDNA first strand: after determining the concentration of the above RNA, use PrimeScript TM 1stStrand cDNA Synthesis Kit to obtain cDNA according to the instructions.

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

[0040] According to the instructions for seamless cloning, linearize the 1302 vector at two sites of KpnI and XbaI, and design a primer pair for the PvCOL16 gene according to the CDS sequence of the switchgrass PvCOL16 gene. After amplifying the fragment, perform recombination reaction, and the sequence of the primer pair with homologous sequences is (5'-3'):

[0041] upstream primer - gattatttttgcagggtaccCGGGAGAAGACGAAGTGTTC

[0042] downstream primer - aatgtttgaacgatctctagaGCAATGCACGACGAGTAGT

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

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

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

[0046] The recombination product was transformed into E. coli DH5a strain, and coated on LB plates with kanamycin resistance and grown overnight.

[0047] The white single colony was picked and grown in LB liquid medium for 8 hours, and the bacterial liquid PCR verification was performed. The correct band strain was sent to the company for sequencing, and the strain and plasmid after sequencing were preserved for subsequent experiments.

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

[0049] 1. Seed disinfection and induction of callus

[0050] (1) Prepare the induction medium, dispense into 100 ml triangular bottles, and sterilize at 121°C high temperature and high pressure for 15 min after sealing the film. The rice seeds were first washed with 75% ethanol for 1 min, then sterilized with 15% NaClO for 15-20 min, and finally washed with sterile ddH2O for 4-5 times. 8-12 sterilized rice seeds were inoculated into each bottle of induction medium, and the callus was induced by culturing at 30°C in the dark for 40-45 days.

[0051] (2) The Agrobacterium strain containing the target gene in Example 1 was streaked on a plate containing kanamycin and rifampicin, and single colonies were picked after two days and cultured in a 28°C shaker.

[0052] (3) The cultured bacterial liquid was centrifuged at 4000 rpm for 15 minutes, the supernatant was discarded, and the bacterial body was resuspended to OD=0.3 by adding the infection liquid. 100 μl AS and 2 ml 50% glucose were added to each 100 ml of infection liquid, and the culture was continued for 30 minutes.

[0053] (4) The Agrobacterium suspension was introduced into the triangular bottles containing callus, and stood for 10 minutes. The bacterial liquid was poured out, and the residual suspension in the callus was absorbed with water-absorbing paper. Finally, it was placed in a culture dish covered with filter paper and naturally dried for 1-2 hours.

[0054] (5) The dried callus particles were transferred to the co-culture medium with forceps, sealed with sealing glue, and co-cultured at 19°C in the dark for 3 days.

[0055] (6) The callus was transferred to the screening medium, and the resistant callus was obtained after 40-60 days.

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

[0057] 2. Identification of transgenic positive seedlings

[0058] (1) The transgenic plants overexpressing PvCOL16 obtained above were used as the T0 generation. Genomic DNA was extracted from their leaves and identified by PCR. The results were as follows: Figure 4 The selected plants are all positive plants.

[0059] (2) The harvested T0 generation seeds were used as T1 generation after germination. Genomic DNA was extracted from their leaves and identified by PCR. The results showed that the positive: negative ratio of T1 generation plants was between 2:1 and 6:1, and the positive line was a single copy.

[0060] (3) The harvested T1 generation positive seeds are used as T2 generation after germination. Genomic DNA is extracted from their leaves and identified by PCR. If the results show that T-DNA segregation does not occur in the T2 generation of a certain line, the line is identified as a homozygous line and the homozygous line is selected for subsequent experiments.

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

[0062] The plant height, fresh weight, and dry weight of rice seedlings after 30 days of growth were recorded. The flowering duration and plant height of T2 generation homozygous rice plants were also recorded: the flowering duration was the number of days from the start of germination to the emergence of awns under light conditions using wet filter paper; the plant height was the height of the above-ground part from the base to the end of the main stem.

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

[0064] Heading time, agronomic traits, and statistical analysis of wild-type control plants and transgenic plants are as follows: Figure 6 As shown, the L1 line was approximately 23 days later than the wild-type control line, and the L2 line was approximately 46 days later than the wild-type control line. Figure 6 a, e). The transgenic plants were significantly shorter than the wild-type control plants. The average plant height of lines L1 and L2 was 72.7 cm and 57.3 cm, respectively, which were 69.9% and 55.1% of the WT plants.Figure 6 a, f); the internode length of transgenic lines L1 and L2 was significantly lower than that of the WT control plants, being 71.1% and 62.6% of the 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 lines, the fresh weight and dry weight of the L1 line being 62.9% and 69.0% of the wild type plants, respectively, and the fresh weight and dry weight of the L2 line being 71.1% and 67.4% of the wild type plants, respectively Figure 6 g, h). The average spike grain number of the main spike of lines L1 and L2 was 29.8% and 42.1% of the control, respectively Figure 7 c, d, j).

[0065] Example 4: Detection of the expression amount of flowering-related genes of the transgenic lines overexpressing PvCOL16

[0066] According to the requirements of qRT-PCR primer design, multiple specific primers of flowering-related genes were designed using Primer Premier 6.0 software. The sequences of the primer pairs were as follows:

[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-F 5'-ACTGTCTGCTCCTCTAAACA-3'

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

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

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

[0087] TB Green Premix Ex Taq TM Premix Ex Taq TM II reagents were quantitatively analyzed by QuantStudio 5 Real-Time PCR system, three biological replicates and two technical replicates were performed for each sample. The reaction conditions were as follows: 95℃ for 10 min; 95℃ for 5 s, 60℃ for 31 s, 40 cycles.

[0088] The results are shown in ​ Compared with the wild type, the expression levels of Ghd7, Ghd8, Hd16 and RCN1 involved in flowering-related genes in the overexpression PvCOL16 transgenic lines were significantly up-regulated 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 could explain the cause of the late flowering phenotype of the overexpression PvCOL16 transgenic lines.

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

[0090] Embodiments of the application have been described above, with the understanding that these embodiments are exemplary, but not exhaustive, and are not limited to the embodiments disclosed. Many modifications and variations 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 application, or improvement to the technology in the market, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

Claims

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

1. The strain overexpressing the switchgrass PvCOL16 gene has a late flowering trait compared with the wild type strain; The plant is rice.

2. The use of the switchgrass PvCOL16 gene in regulating flowering time in plants according to claim 1, characterized in that, The strain overexpressing the switchgrass PvCOL16 gene has a lower plant height than the wild type strain at the vegetative growth and reproductive growth stages of the plant.

3. The use of the switchgrass PvCOL16 gene in regulating flowering time in plants according to claim 1, wherein, The flowering-related genes whose expression levels change after overexpression of the switchgrass PvCOL16 gene include Ghd7, Ghd8, Hd16, RCN1, Hd3a, Ehd1, Ehd2 and Ehd4.

4. The use of the switchgrass PvCOL16 gene in regulating flowering time in plants according to claim 1, characterized in that, The expression levels of genes Ghd7, Ghd8, Hd16 and RCN1 in the strain overexpressing the switchgrass PvCOL16 gene are significantly up-regulated and higher than those of the wild type plant; the expression levels of genes Hd3a, Ehd1, Ehd2 and Ehd4 are significantly down-regulated and lower than those of the wild type plant. The nucleotide sequence of the switchgrass PvCOL16 gene is shown as SEQ ID NO.

1. The strain overexpressing the switchgrass PvCOL16 gene has a late flowering trait compared with the wild type strain; The plant is rice. The strain overexpressing the switchgrass PvCOL16 gene has a lower plant height than the wild type strain at the vegetative growth and reproductive growth stages of the plant. The flowering-related genes whose expression levels change after overexpression of the switchgrass PvCOL16 gene include Ghd7, Ghd8, Hd16, RCN1, Hd3a, Ehd1, Ehd2 and Ehd4. The expression levels of genes Ghd7, Ghd8, Hd16 and RCN1 in the strain overexpressing the switchgrass PvCOL16 gene are significantly up-regulated and higher than those of the wild type plant; the expression levels of genes Hd3a, Ehd1, Ehd2 and Ehd4 are significantly down-regulated and lower than those of the wild type plant.

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