CN114231539B

CN114231539B - Application of switchgrass SBP-box transcription factor PvSPL6 and recombinant vector thereof

Info

Publication number: CN114231539B
Application number: CN202111671083.3A
Authority: CN
Inventors: 付春祥; 王亚梅; 刘文文; 杨瑞娟; 白史且; 姜珊珊
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-06-02
Anticipated expiration: 2041-12-31
Also published as: CN114231539A

Abstract

The invention relates to application of a switchgrass SBP-box transcription factor PvSPL6 and a recombinant vector thereof, belonging to the technical field of plant genetic engineering, wherein the switchgrass SBP-box transcription factor PvSPL6 can regulate and control flowering time, stem length and biomass yield of switchgrass. The invention also provides a recombinant vector which is used for over-expressing pANIC6B-PvSPL6 or inhibiting the expression of pANIC8B-PvSPL6-RNAi, and the recombinant vectors pANIC6B-PvSPL6 and pANIC8B-PvSPL6-RNAi have influence on flowering time, stem length and biomass yield of switchgrass after being transferred into the switchgrass. Compared with the wild type, the flowering time of the obtained transgenic plant (PvSPL 6-RNAi) with the PvSPL6 transcript inhibition expression is delayed by about 40 days, the stem node length is increased by about 50%, and the biomass is increased by 63%.

Description

Application of switchgrass SBP-box transcription factor PvSPL6 and recombinant vector thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and discloses application of an SBP-box transcription factor PvSPL6 related to unknown functions of plants in delaying flowering time of switchgrass, increasing stem node length and increasing biomass yield.

Background

Switchgrass (Panicum virgatum l.) belongs to perennial C4 tall herbaceous plants and is mainly used as energy grass and pasture. As an important fiber biomass resource, the development and utilization of the biomass energy of the switchgrass by increasing the biomass and the fermentable sugar yield of the fiber biomass resource have important practical application values. The completion of the whole genome sequencing of the switchgrass, the publication of the expression chip database and the perfection of the transformation system provide resource guarantee for the excavation and verification of functional genes.

Flowering time is an important trait that determines the biomass of energy grass. After the gramineae goes from vegetative to reproductive growth, the biomass is essentially no longer increased, and the nutrient supply now flows more to the inflorescence. Thus, regulating flowering time of switchgrass and other energy crops is a key way to increase their biomass yield. Studies have shown that molecular regulation of flowering time in plants is a complex synergistic regulation of exogenous and endogenous factors (Park et al, molecular interactions between flowering time and abiotic stress pathwax. Int Rev Cell Mol Biol,2016, 327:371-412). The age pathway has received considerable attention in recent years as a new mechanism in the five major flowering pathways (photoperiod pathway, vernalization pathway, gibberellin pathway, autonomous pathway, age pathway) that is capable of regulating plant flowering under non-induced conditions. MiR156 and its target gene SQUAMOSA PROMOTER BINDING-LIKEs (SPLs) are key regulatory hubs of the age pathway. SPL genes encode a class of plant-specific transcription factors and are conserved in monocots and dicots (Yang et al, comparative study of SBP-box gene family in Arabidopsis and Rice. Gene,2007, 407:1-11). In Arabidopsis, almost all miR 156-targeted SPL genes are acceleration factors for plant flowering transitions, e.g., atSPL3 affects flowering by modulating AP1, etc., atSPL9 modulates flowering by participating in the miR172 pathway, atSPL10 affects flowering by modulating AGL79, etc. (Gao et al, miR156/SPL10 modulates lateral root development, branching and leaf morphology in Arabidopsis by silencing AGAMOUS-LIKE 79.Front Plant Sci,2018, 8:2226). However, only PvSPL7 and 8 have been identified in switchgrass as involved in flowering regulation (Gou et al, SPL7 and SPL8 represent a novel flowering regulation mechanism in switching grass, new Phytol,2019, 222:1610-1623).

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of a switchgrass SBP-box transcription factor PvSPL6 in delaying switchgrass flowering and increasing plant biomass yield so as to solve the problems that the existing energy plant biomass improvement gene resource library is insufficient and the requirements of energy crop plant type improvement and yield increase molecular design cannot be met at the same time.

The invention is realized by the following technical scheme:

a coding gene of a switchgrass SBP-box transcription factor PvSPL 6; the nucleotide sequence is shown as SEQ ID NO.1. The amino acid sequence of SBP-box protein PvSPL6 coded by the gene is shown as SEQ ID NO. 2.

A segment for inhibiting RNA interference of switchgrass PvSPL6 gene transcript level, the nucleotide sequence of which is shown in SEQ ID No. 3.

The first object of the invention is to provide an application of a coding gene of a switchgrass SBP-box transcription factor PvSPL6, wherein the gene is shown as SEQ ID NO.1, and the application is an application of over-expressed PvSPL6 in the aspects of regulating early flowering, shortening the length of a stem and reducing biomass of the switchgrass, and an application of inhibiting the expression of the PvSPL6 in the aspects of regulating the late flowering time, increasing the length of the stem and the biomass of the switchgrass.

A second object of the present invention is to provide a recombinant vector comprising a switchgrass SBP-box transcription factor PvSPL6 gene, which is pANIC6B-PvSPL6, comprising SEQ ID NO.1.

A third object of the present invention is to provide a recombinant vector comprising an RNA interference segment at the level of SBP-box gene transcript of switchgrass, said recombinant vector being pANIC8B-PvSPL6-RNAi, comprising SEQ ID NO.3, said SEQ ID NO.3 being part of SEQ ID NO.1.

The invention has the core characteristics and the inventive concept that:

1. flowering time is an important factor affecting energy grass biomass. The improvement of biomass by delaying the flowering time of energy grass through plant genetic engineering is one of the important research directions. The invention utilizes the means of genetic engineering to regulate and verify the expression level of SBP-box transcription factor PvSPL6 in the switchgrass by over-expression and RNA interference expression technology, wherein, the transgenic switchgrass plants with delayed flowering time, increased stem node length and increased biomass yield can be obtained by interfering the transcript level of the PvSPL6, which has important guiding significance for genetic breeding and directional molecular design of switchgrass and other gramineous plants.

2. The invention starts from the related genes for regulating and controlling the flowering time of switchgrass, and simultaneously regulates and controls the biomass yield of plants through advanced genetic engineering technology, thereby providing a new target for the genetic improvement and molecular breeding of the biomass of pasture and energy crops.

Compared with the prior art, the invention has the following beneficial effects:

1. the switchgrass SBP-box transcription factor PvSPL6 gene obtained in the invention is an important gene for regulating and controlling the flowering time, the length of the stem node, the number of the stem node and other characters of the switchgrass, which has important contribution to obtaining ideal energy plant types through molecular directional design;

2. the invention over-expresses PvSPL6 to lead the flowering time of the switchgrass to be advanced, the length of the stem node to be shortened, and the biomass yield to be reduced; the transcript level of the SPL6 of the switchgrass is inhibited, the flowering time of the switchgrass can be obviously delayed, the length of the stem node and the biomass of the switchgrass are increased, and the method has important reference significance for the genetic improvement of the biomass of energy plants and gramineous pastures;

3. the genetically modified plants produced in the invention can be integrated into conventional breeding projects, thereby providing new germplasm resources for the variety cultivation of energy plants and gramineous pasture crops.

Drawings

FIG. 1 is a PCR amplification electrophoretogram of SBP-box transcription factor PvSPL6 (A) and PvSPL6-RNAi (B) in switchgrass;

FIG. 2 schematic diagrams of switchgrass pANIC6B-PvSPL6 overexpression (A) and pANIC8B-PvSPL6-RNAi interference expression (B) vectors;

FIG. 3 PCR identification of PvSPL6 over-expressed (A) and interference expressed (B) transgenic switchgrass. 1 ^# -12 ^# : pvSPL6 overexpressing transgenic switchgrass plants; 1' ^# -11’ ^# +: pvSPL6 interference expression transgenic switchgrass plants; WT: wild type switchgrass plants; m: DL 2000DNA maker.

FIG. 4PvSPL6 overexpression (A) and interference expression (B) results of qRT-PCR of the PvSPL6 gene in transgenic switchgrass plants. Control represents wild type switchgrass plants, pvSPL6OE-67/-71/-76 represents three independent positive overexpressing transgenic lines, respectively, pvSPL6RNAi-1/-6/-7 represents three independent positive interfering expressing transgenic lines, respectively.

FIG. 5PvSPL6 positive transgenic plant phenotype. Control represents wild type switchgrass plants, pvSPL6 _OE Represents a positive overexpressing strain, pvSPL6 _RNAi Positive interference expression lines are indicated.

FIG. 6 flowering time (A) and stem length (B) measurements of PvSPL6 positive transgenic plants. Control represents wild type switchgrass plants, pvSPL6OE-67/-71/-76 represents three independent positive overexpressing transgenic lines, respectively, pvSPL6RNAi-1/-6/-7 represents three independent positive interfering expressing transgenic lines, respectively.

FIG. 7PvSPL6 positive transgenic plant biomass assay. Control represents wild type switchgrass plants, pvSPL6OE-67/-71/-76 represents three independent positive overexpressing transgenic lines, respectively, pvSPL6RNAi-1/-6/-7 represents three independent positive interfering expressing transgenic lines, respectively.

Detailed Description

The invention will be described in further detail with reference to specific embodiments and drawings. Materials, reagents, molecular labeled probes, and the like used in the examples described below are commercially available from the company unless otherwise specified.

Example 1: amplification of PvSPL6 and PvSPL6-RNAi sequences

According to the published genome information of switchgrass in the Phytozome (https:// Phytozome. Jgi. Doe. Gov) website, primers (PvSPL 6-F and PvSPL 6-R) are designed on both sides of the full-length sequence of PvSPL6, respectively, and primers (PvSPL 6-RNAi-F and PvSPL 6-RNAi-R) are designed in non-conserved regions of the PvSPL6 gene, and PCR amplification is performed using the primers as templates.

The primer sequences are as follows:

PvSPL6-F：atgagagctaagcaagctagc

PvSPL6-R：ttatctgatctggaagtggttccgt

PvSPL6-RNAi-F：cccttgcttcgtgtcatcgt

PvSPL6-RNAi-R：tgccgtagcagggttctgtc

the PCR reaction system is as follows: mu.L of cDNA, 25. Mu.L of 2 XBuffer, 4. Mu.L of 10pM dNTP, 10. Mu.M of forward/reverse primer 2. Mu.L each, 0.5. Mu.L of 5U/. Mu. L PrimerSTAR HS DNA were polymerizedEnzyme and 14.5. Mu.L ddH ₂ O. Mixing the materials on ice. The PCR reaction conditions were: 98 ℃ for 3min;98℃for 5sec and 56℃for 15sec; 30sec at 72℃for 35 cycles; and at 72℃for 5min.

The PCR amplified products were subjected to 1% agarose gel electrophoresis to obtain fragments of approximately 650bp (FIG. 1A) and 250bp (FIG. 1B), and the amplified fragments were subjected to gel recovery (using Promega gel recovery kit) and conventional sequencing (Beijing Liuhua Dairy Gene technologies Co., ltd.). Sequencing results show that the amplified long fragment contains a complete open reading frame with the total length of 642 bases, the sequence is shown as SEQ ID NO.1, and the encoded protein contains 243 amino acid residues, and the sequence is shown as SEQ ID NO. 2. The short fragment has a length of 275 bases and a sequence shown in SEQ ID NO. 3.

The coding gene of PvSPL 6:

atgagagctaagcaagctagcaagcgcggctcccgaactgcacctccacctcgccggctctcctcgatcggctcgtcccc cgacggcgccgccatggaccgcaagggcacctcgagctcggcggcggcgtccatggccgcgctcgccgccgccgccgcggccggccagggccagccgacctccggccaggccaacggggcgctgtcgtcgccgcatgcggaggaggacgag aaccctgctacggcagccgccgtgagcggtggcggcgcctccggctcctcggacccggtggccgcgaggaggggagcggcgggcggcggcccgagctgccaggtggagcggtgcgccgccgacctacacgatgcgaggcggtactaccgga ggcacaaggtgtgcgagccgcactccaaggagctcgccgtgctcgtcgccggcctccgccagcgcttctgccagcaatgcagccggttccatgagctgttggagttcgacggcgacaagcgcagctgccgccggcgcctggaggggcacaacgca cggcgccggaggagctcggcggataggcacggcggcagcggcggcgaccaggacggccggagccacccagggaacccgtcacggaaccacttccagatcagataa

amino acid sequence of protein PvSPL 6:

MRAKQASKRGSRTAPPPRRLSSIGSSPDGAAMDRKGTSSSAAASMAALAAA AAAGQGQPTSGQANGALSSPHAEEDENPATAAAVSGGGASGSSDPVAARRGA AGGGPSCQVERCAADLHDARRYYRRHKVCEPHSKELAVLVAGLRQRFCQQCSRFHELLEFDGDKRSCRRRLEGHNARRRRSSADRHGGSGGDQDGRSHPGNPS RNHFQIR

nucleotide sequence of the interfering fragment of PvSPL 6:

cccttgcttcgtgtcatcgtcctccgctcatgagagctaagcaagctagcaagcgcggctcccgaactgcacctccacctc gccggctctcctcgatcggctcgtcccccgacggcgccgccatggaccgcaagggcacctcgagctcggcggcggcgtccatggccgcgctcgccgccgccgccgcggccggccagggccagccgacctccggccaggccaacggggcgctgt cgtcgccgcatgcggaggaggacgagaaccctgctacggca

example 2: recombinant vector construction and transient expression in tobacco cells for observing subcellular localization

Using the obtained full-length sequence fragment as a template, designing a PvSPL6 with a joint primer which is in seamless connection with an expression vector pCABIA1300-cGFP, and amplifying the fragment by using high-fidelity enzyme; the expression vector pCABIA1300-cGFP was digested with the restriction enzyme HindIII. The PvSPL6 gene fragment and the pCABIA1300-cGFP vector fragment were recovered. The two recovered fragments were then subjected to homologous recombination ligation using a seamless ligase (available from Vazyme). The ligation product was transformed into E.coli DH 5. Alpha. Competent cells by heat shock. And (3) picking a monoclonal colony, performing amplification culture in a liquid LB culture medium containing the kanamycin, and performing PCR amplification detection and sequencing verification to obtain a recombinant plasmid pCABIA1300-PvSPL6-cGFP.

The recombinant vector pCABIA1300-PvSPL6-cGFP which was successfully constructed was transformed into Agrobacterium EHA105 and the strain was preserved. Injecting the bacterial liquid into tobacco through a tobacco transient expression technology for subcellular localization observation. Unlike typical transcription factors, the fluorescent confocal results show that PvSPL6 can localize to the cell membrane in addition to the tobacco nucleus, suggesting that this gene may have other important biological functions in addition to functioning as a transcription factor.

Example 3: acquisition of PvSPL6 transgenic switchgrass plants

And (3) respectively designing primers of the linkage entry vector in the overexpression and interference expression vector: the ends of the primers were introduced with AhdI cleavage site and 18 bases (seamless junction linker sequence) after entry of the vector pGWC cleavage site, and PCR amplification was performed using the resulting full-length PvSPL6 sequence as a template, using the primers described above.

The primer sequences are as follows:

PvSPL6-pGWC-F：aaagcaggctttgactttatgagagctaagcaagctagc

PvSPL6-pGWC-R：gctgggtctagagacttttatctgatctggaagtggttccgt；

PvSPL6-RNAi-pGWC-F：aaagcaggctttgactttcccttgcttcgtgtcatcgt

PvSPL6-RNAi-pGWC-R：gctgggtctagagactttgccgtagcagggttctgtc；

wherein the seamless junction sequences are underlined.

Recovering the amplified fragment. pGWC vectors were digested with restriction enzyme AhdI and recovered. The two recovered fragments were subjected to homologous recombination ligation using a seamless ligase (available from Vazyme Co.) and E.coli DH 5. Alpha. Competent cells were transformed by heat shock. Monoclonal colonies were picked, grown by amplification in kanamycin-resistant LB medium, and sequenced. The kit extracts recombinant strain plasmids with correct sequencing, and uses Gateway technology to transfer the recombinant plasmid enzyme digestion recovery fragments into an excessive expression vector pANIC6B and an interference expression vector pANIC8B (figure 2) respectively. The recombination reaction is as follows: 100ng of the fragment was recovered by cleavage, 50ng of pANIC6B/pANIC8B vector plasmid, 1. Mu.L of LR enzyme (Invitrogen, cat. No. 11791020), followed by ddH ₂ O was made up to 10. Mu.L. Culturing at 25 ℃ for 6 hours, transforming escherichia coli DH5 alpha competent cells, obtaining positive recombinant strain plasmids pANIC6B-PvSPL6 and pANIC8B-PvSPL6-RNAi which are correctly sequenced, and transforming agrobacterium EHA105.

The genetic transformation method of Agrobacterium-mediated switchgrass embryogenic callus (Xi et al, agrobacterium-mediated transformation of switchgrass and inheritance of the transgenes.bioenergy Research,2009, 2:275-283) was used to introduce pANIC6B-PvSPL6 and pANIC8B-PvSPL6-RNAi into the low-ground wild-type switchgrass Alamo, respectively, to obtain resistant seedlings. Full-length genes were detected using the overexpression vector universal primer ZmUbq-F and the full-length gene downstream primer PvSPL6-R, interference fragments were detected using the interference expression vector universal primer Guslink-F and the interference fragments downstream primer PvSPL6-RNAi-R, guslink-R and PvSPL6-RNAi-R, hygromycin genes were detected using the anti-hygromycin gene upstream and downstream primer (hph3+hph4), and finally positive transgenic lines were determined (FIG. 3).

Example 4: identification of transgenic plant molecules

The tender stem tissue of the above identified transgenic positive plant was taken, total RNA was extracted with TriZol Reagent kit (Invitrogen Co., product No. 15596026), the content and purity of total RNA was detected by agarose gel electrophoresis and a nucleic acid analyzer (NanoDrop), 1.0. Mu.g of total RNA was taken for reverse transcription reaction, reverse transcription was performed using reverse transcriptase (Promega Co., product No. M1701) to cDNA, and the reverse transcription reaction procedure was referred to the instructions. The cDNA is used as a template, and primers PvSPL6-qRT-F and PvSPL 6-qRT-R are used for fluorescence quantitative PCR detection, and the reference gene is switchgrass Ubiquitin (UBQ) gene. The primer sequences were as follows:

PvUBQ-F：ttcgtggtggccagtaag

PvUBQ-R：agagaccagaagacccaggtacag

PvSPL6-qRT-F：caggtgattaagcaggtaccct

PvSPL6-qRT-R：ggcacaggcgaacgaattac

the real-time fluorescent quantitative PCR reaction system was 20. Mu.L, in which each of the forward/reverse primers was 1. Mu.L, the cDNA template was 2. Mu.L, SYBR Green qRT Master Mix (available from Takara Bio-engineering Co., ltd.) was 10. Mu.L, and ddH ₂ O was made up to 20. Mu.L. The real-time fluorescent quantitative PCR instrument was performed using Roche480 using a two-step method. The detection result shows that the expression level of PvSPL6 in the over-expression plants PvSPL6OE-67, pvSPL6OE-71 and PvSPL6OE-76 is obviously increased compared with the wild type Control (A in FIG. 4); in contrast, the expression level of PvSPL6 in the interference-expressed plants PvSPL6RNAi-1, pvSPL6RNAi-6 and PvSPL6RNAi-7 was significantly decreased (B in FIG. 4).

Example 5: transgenic plant flowering time, stem node length and biomass determination

The flowering time, the stem length and the biomass of switchgrass plants grown for six months were measured. Compared with the wild type, the PvSPL6 over-expressed plant has obvious flowering time which is about 30 days earlier, the length of the stem node is reduced by about 30%, the number of the stem node is reduced, and the plant is dwarfed (figure 5); while PvSPL6 interference-expressed plants exhibited a significant delay in flowering time compared to wild-type switchgrass (about 40 days), with an increase in stem length of about 50%, an increase in stem number, and a corresponding increase in plant height (FIG. 5). Plant aerial parts of Control, pvSPL OE-67/-71/-76 and PvSPL6RNAi-1/6/7 grown for six months were collected and fresh weights were measured. The results showed that the dry matter biomass yield of the PvSPL6 overexpressing strain was reduced by about 56% compared to the wild-type and the dry matter biomass yield of the PvSPL6 interference expressing strain was increased by about 63% (fig. 7).

The generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> yellow sea aquatic institute of China aquatic science institute

<120> application of SBP-box transcription factor PvSPL6 of switchgrass and recombinant vector thereof

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 642

<212> DNA

<213> Panicum virgatum L. (switchgrass)

<400> 1

atgagagcta agcaagctag caagcgcggc tcccgaactg cacctccacc tcgccggctc 60

tcctcgatcg gctcgtcccc cgacggcgcc gccatggacc gcaagggcac ctcgagctcg 120

gcggcggcgt ccatggccgc gctcgccgcc gccgccgcgg ccggccaggg ccagccgacc 180

tccggccagg ccaacggggc gctgtcgtcg ccgcatgcgg aggaggacga gaaccctgct 240

acggcagccg ccgtgagcgg tggcggcgcc tccggctcct cggacccggt ggccgcgagg 300

aggggagcgg cgggcggcgg cccgagctgc caggtggagc ggtgcgccgc cgacctacac 360

gatgcgaggc ggtactaccg gaggcacaag gtgtgcgagc cgcactccaa ggagctcgcc 420

gtgctcgtcg ccggcctccg ccagcgcttc tgccagcaat gcagccggtt ccatgagctg 480

ttggagttcg acggcgacaa gcgcagctgc cgccggcgcc tggaggggca caacgcacgg 540

cgccggagga gctcggcgga taggcacggc ggcagcggcg gcgaccagga cggccggagc 600

cacccaggga acccgtcacg gaaccacttc cagatcagat aa 642

<210> 2

<211> 213

<212> PRT

<213> switchgrass (Panicum virgatum l.)

<400> 2

Met Arg Ala Lys Gln Ala Ser Lys Arg Gly Ser Arg Thr Ala Pro Pro

1 5 10 15

Pro Arg Arg Leu Ser Ser Ile Gly Ser Ser Pro Asp Gly Ala Ala Met

20 25 30

Asp Arg Lys Gly Thr Ser Ser Ser Ala Ala Ala Ser Met Ala Ala Leu

35 40 45

Ala Ala Ala Ala Ala Ala Gly Gln Gly Gln Pro Thr Ser Gly Gln Ala

50 55 60

Asn Gly Ala Leu Ser Ser Pro His Ala Glu Glu Asp Glu Asn Pro Ala

65 70 75 80

Thr Ala Ala Ala Val Ser Gly Gly Gly Ala Ser Gly Ser Ser Asp Pro

85 90 95

Val Ala Ala Arg Arg Gly Ala Ala Gly Gly Gly Pro Ser Cys Gln Val

100 105 110

Glu Arg Cys Ala Ala Asp Leu His Asp Ala Arg Arg Tyr Tyr Arg Arg

115 120 125

His Lys Val Cys Glu Pro His Ser Lys Glu Leu Ala Val Leu Val Ala

130 135 140

Gly Leu Arg Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe His Glu Leu

145 150 155 160

Leu Glu Phe Asp Gly Asp Lys Arg Ser Cys Arg Arg Arg Leu Glu Gly

165 170 175

His Asn Ala Arg Arg Arg Arg Ser Ser Ala Asp Arg His Gly Gly Ser

180 185 190

Gly Gly Asp Gln Asp Gly Arg Ser His Pro Gly Asn Pro Ser Arg Asn

195 200 205

His Phe Gln Ile Arg

210

<210> 4

<211> 275

<212> DNA

<213> switchgrass (Panicum virgatum l.)

<400> 4

cccttgcttc gtgtcatcgt cctccgctca tgagagctaa gcaagctagc aagcgcggct 60

cccgaactgc acctccacct cgccggctct cctcgatcgg ctcgtccccc gacggcgccg 120

ccatggaccg caagggcacc tcgagctcgg cggcggcgtc catggccgcg ctcgccgccg 180

ccgccgcggc cggccagggc cagccgacct ccggccaggc caacggggcg ctgtcgtcgc 240

cgcatgcgga ggaggacgag aaccctgcta cggca 275

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atgagagcta agcaagctag c 21

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ttatctgatc tggaagtggt tccgt 25

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cccttgcttc gtgtcatcgt 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tgccgtagca gggttctgtc 20

<210> 8

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

aaagcaggct ttgactttat gagagctaag caagctagc 39

<210> 9

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gctgggtcta gagactttta tctgatctgg aagtggttcc gt 42

<210> 10

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

aaagcaggct ttgactttcc cttgcttcgt gtcatcgt 38

<210> 11

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gctgggtcta gagactttgc cgtagcaggg ttctgtc 37

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ttcgtggtgg ccagtaag 18

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

agagaccaga agacccaggt acag 24

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

caggtgatta agcaggtacc ct 22

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ggcacaggcg aacgaattac 20

Claims

1. The application of the coding gene of the switchgrass SBP-box transcription factor PvSPL6 is characterized in that the gene is shown as SEQ ID NO.1, and the application is the application of over-expression of the PvSPL6 in the aspects of regulating early flowering, shortening the length of a stem node and reducing the biomass of the switchgrass, and the application of inhibiting expression of the PvSPL6 in the aspects of regulating the delay flowering time of the switchgrass and increasing the length of the stem node and the biomass.