CN114231539A

CN114231539A - Application of switchgrass SBP-box transcription factor PvSPL6 and recombinant vector thereof

Info

Publication number: CN114231539A
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: 2022-03-25
Anticipated expiration: 2041-12-31
Also published as: CN114231539B

Abstract

The invention relates to the application of switchgrass SBP-box transcription factor PvSPL6 and a recombinant vector thereof, belonging to the technical field of plant genetic engineering. The switchgrass SBP-box transcription factor PvSPL6 can regulate the flowering time, stem node length and biomass yield of switchgrass. The present invention also provides that the recombinant vector overexpresses pANIC6B-PvSPL6 or inhibits the expression of pANIC8B-PvSPL6-RNAi, and the recombination vectors pANIC6B-PvSPL6 and pANIC8B-PvSPL6-RNAi are transformed into switchgrass for the flowering time, stem node length and biomass yield of switchgrass all have influence. Compared with wild type, the obtained transgenic plants with suppressed expression of PvSPL6 transcripts (PvSPL6-RNAi) showed delayed flowering by about 40 days, increased stem node length by about 50%, and increased biomass 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 the flowering time of switchgrass, increasing the length of stem nodes and increasing the biomass yield.

Background

Switchgrass (Panicum virgatum L.), which belongs to the perennial C4 tall grass, is used mainly as an energy grass and pasture. As an important fiber biomass resource, increasing the biomass and the yield of fermentable sugar to improve the development and utilization of the switchgrass biomass energy has important practical application value. The completion of switchgrass whole genome sequencing, the publication of an expression chip database and the perfection of a transformation system provide resource guarantee for the mining and verification of functional genes.

Flowering time is an important trait that determines energy grass biomass. After the gramineous plants have grown from vegetative to reproductive growth, the biomass is not substantially increased any more, and the supply of nutrients is more directed towards the inflorescence. Therefore, regulating the flowering time of switchgrass and other energy crops is a key approach to increasing their biomass production. Studies have shown that Molecular regulation of plant flowering time is a complex coordinated regulation of exogenous and endogenous factors (Park et al, Molecular interactions between flowering time and ecological stress pathways. int Rev Cell Mol Biol,2016,327: 371-. The age pathway has recently attracted much attention as a new mechanism capable of regulating flowering of plants under non-induced conditions in five major flowering pathways (photoperiod pathway, vernalization pathway, gibberellin pathway, autonomic pathway, age pathway). MiR156 and its target gene SQUAMOSA PROMOTER BINDING-LIKEs (SPLs) are key regulatory pivots in the age pathway. The SPL gene encodes a class of plant-specific transcription factors and is conserved in both monocots and dicots (Yang et al, synthetic study of SBP-box gene family in Arabidopsis and rice, 2007,407: 1-11). In Arabidopsis, almost all of the miR 156-targeted SPL genes are accelerators of Plant flowering transitions, e.g., AtSPL3 influences flowering by regulating AP1 and the LIKE, AtSPL9 regulates flowering by participating in the miR172 pathway, AtSPL10 influences flowering by regulating AGL79 and the LIKE (Gao et al, miR156/SPL10 modulated flowering errors and maintenance, broad and leaf morphology in Arabidopsis by spacing growing AGAMOUS-LIKE 79.Front Plant Sci, 2018: 2226). However, the only SPLs identified in switchgrass that are involved in flowering regulation are PvSPL7 and 8 (Gou et al, SPL7 and SPL8 expression a novel flowering regulation mechanism in switching grass. New Phytol,2019,222: 1610-.

Disclosure of Invention

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

The invention is realized by the following technical scheme:

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

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

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

The second purpose of the invention is to provide a recombinant vector containing the switchgrass SBP-box transcription factor PvSPL6 gene, wherein the recombinant vector is pANIC6B-PvSPL6, and the recombinant vector contains SEQ ID NO. 1.

The third object of the present invention is to provide a recombinant vector containing RNA interference segment at the level of SBP-box gene transcript of switchgrass, said recombinant vector is pANIC8B-PvSPL6-RNAi, wherein it contains SEQ ID NO.3, and said SEQ ID NO.3 is a part of SEQ ID NO. 1.

The core characteristics and the inventive concept of the invention comprise:

1. flowering time is an important factor affecting energy grass biomass. The delay of flowering time of energy grass by plant genetic engineering to increase biomass is one of important research directions. The invention utilizes genetic engineering means to regulate and functionally verify the expression level of an SBP-box type transcription factor PvSPL6 in switchgrass by overexpression and RNA interference expression technologies aiming at PvSPL6 gene of energy plant switchgrass respectively, wherein the interference of the PvSPL6 transcript level can obtain transgenic switchgrass plants with delayed flowering time, increased stem length and increased biomass yield, and the method has important guiding significance for genetic breeding and targeted molecule design of the switchgrass and other gramineous plants.

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

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

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

2. the overexpression of PvSPL6 in the invention leads to the advancing of the flowering time of switchgrass, the shortening of the length of stem nodes and the reduction of biomass yield; the inhibition of the transcript level of the SPL6 of the switchgrass can obviously delay the flowering time of the switchgrass and increase the stem length and biomass of the switchgrass, and has important reference significance for the genetic improvement of the biomass of energy plants and gramineous forage grass;

3. the genetically improved plant produced by the invention can be integrated into a conventional breeding project, thereby providing a new germplasm resource for variety cultivation of energy plants and gramineous forage grass crops.

Drawings

FIG. 1 PCR amplification electropherograms of SBP-box type transcription factors PvSPL6(A) and PvSPL6-RNAi (B) in switchgrass;

FIG. 2 simplified diagram of pANIC6B-PvSPL6 overexpression and pANIC8B-PvSPL6-RNAi interference expression (B) vectors for switchgrass;

fig. 3PvSPL6 overexpression (a) and interference expression (B) transgenic switchgrass PCR identification results. 1^#-12^#: PvSPL6 overexpresses transgenic switchgrass plants; 1'^#-11’^#+: PvSPL6 interfering with expression of transgenic switchgrass plants; WT: wild-type switchgrass plants; m: DL 2000DNA maker.

FIG. 4PvSPL6 overexpression (A) and interference expression (B) PvSPL6 gene qRT-PCR results in transgenic switchgrass plants. Control represents wild-type switchgrass plants, PvSPL6OE-67/-71/-76 represents three independent positive overexpression transgenic lines, respectively, and PvSPL6RNAi-1/-6/-7 represents three independent positive interference expression transgenic lines, respectively.

FIG. 5PvSPL6 positive transgenic plant phenotype. Control denotes wild type switchgrass plants, PvSPL6_OERepresenting a positive overexpression line, PvSPL6_RNAiRepresenting positive interfering expression strains.

FIG. 6 flowering time (A) and stem node length (B) measurements of PvSPL6 positive transgenic plants. Control represents wild-type switchgrass plants, PvSPL6OE-67/-71/-76 represents three independent positive overexpression transgenic lines, respectively, and PvSPL6RNAi-1/-6/-7 represents three independent positive interference expression transgenic lines, respectively.

FIG. 7PvSPL6 positive transgenic plant biomass assay. Control represents wild-type switchgrass plants, PvSPL6OE-67/-71/-76 represents three independent positive overexpression transgenic lines, respectively, and PvSPL6RNAi-1/-6/-7 represents three independent positive interference expression transgenic lines, respectively.

Detailed Description

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

Example 1: amplification of PvSPL6 and PvSPL6-RNAi sequences

Based on the published genomic information of switchgrass in the website of Phytozome (https:// Phytozome.jgi.doe.gov), primers (PvSPL6-F and PvSPL6-R) and primers (PvSPL6-RNAi-F and PvSPL6-RNAi-R) designed on both sides of the full-length sequence of PvSPL6 and on the non-conserved region of PvSPL6 gene were PCR amplified using cDNA of switchgrass as a template.

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 cDNA, 25. mu.L 2 XBuffer, 4. mu.L 10pM dNTPs, 2. mu.L each of 10. mu.M forward/reverse primers, 0.5. mu.L 5U/. mu.L PrimerSTAR HS DNA polymerase and 14.5. mu.L ddH₂And O. Sample was added to ice and mixed well. The PCR reaction conditions are as follows: 3min at 98 ℃; 5sec at 98 ℃ and 15sec at 56 ℃; 30sec at 72 ℃ for 35 cycles; 5min at 72 ℃.

Detecting the PCR amplification product by 1% agarose gel electrophoresis to obtain fragments with the sizes of about 650bp (figure 1A) and 250bp (figure 1B), respectively, carrying out gel recovery (using a Promega gel recovery kit) on the amplification fragments, and carrying out conventional sequencing (Beijing Liuhe Huada Dagenescience and technology Co., Ltd.). Sequencing results show that the long fragment obtained by amplification contains a complete open reading frame, the total length is 642 basic groups, the sequence is shown as SEQ ID NO.1, the coded protein contains 243 amino acid residues, and the sequence is shown as SEQ ID NO. 2. The short fragment has the length of 275 basic groups, and the sequence is shown in SEQ ID NO. 3.

Gene encoding PvSPL 6:

atgagagctaagcaagctagcaagcgcggctcccgaactgcacctccacctcgccggctctcctcgatcggctcgtcccc cgacggcgccgccatggaccgcaagggcacctcgagctcggcggcggcgtccatggccgcgctcgccgccgccgcc gcggccggccagggccagccgacctccggccaggccaacggggcgctgtcgtcgccgcatgcggaggaggacgag aaccctgctacggcagccgccgtgagcggtggcggcgcctccggctcctcggacccggtggccgcgaggaggggag cggcgggcggcggcccgagctgccaggtggagcggtgcgccgccgacctacacgatgcgaggcggtactaccgga ggcacaaggtgtgcgagccgcactccaaggagctcgccgtgctcgtcgccggcctccgccagcgcttctgccagcaat gcagccggttccatgagctgttggagttcgacggcgacaagcgcagctgccgccggcgcctggaggggcacaacgca cggcgccggaggagctcggcggataggcacggcggcagcggcggcgaccaggacggccggagccacccaggga acccgtcacggaaccacttccagatcagataa

amino acid sequence of protein PvSPL 6:

MRAKQASKRGSRTAPPPRRLSSIGSSPDGAAMDRKGTSSSAAASMAALAAA AAAGQGQPTSGQANGALSSPHAEEDENPATAAAVSGGGASGSSDPVAARRGA AGGGPSCQVERCAADLHDARRYYRRHKVCEPHSKELAVLVAGLRQRFCQQC SRFHELLEFDGDKRSCRRRLEGHNARRRRSSADRHGGSGGDQDGRSHPGNPS RNHFQIR

nucleotide sequence of interfering fragment of PvSPL 6:

cccttgcttcgtgtcatcgtcctccgctcatgagagctaagcaagctagcaagcgcggctcccgaactgcacctccacctc gccggctctcctcgatcggctcgtcccccgacggcgccgccatggaccgcaagggcacctcgagctcggcggcggcg tccatggccgcgctcgccgccgccgccgcggccggccagggccagccgacctccggccaggccaacggggcgctgt cgtcgccgcatgcggaggaggacgagaaccctgctacggca

example 2: recombinant vector construction and transient expression observation of subcellular localization in tobacco cells

Using the obtained full-length sequence fragment as a template, designing a PvSPL6 with a joint primer seamlessly connected 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 recovered two fragments were then ligated by homologous recombination using a seamless ligase (available from Vazyme). The ligation products were transformed into E.coli DH 5. alpha. competent cells by heat shock. And (3) selecting a monoclonal colony, carrying out amplification culture in a liquid LB culture medium containing kanamycin, and carrying out PCR amplification detection and sequencing verification to obtain the recombinant plasmid pCABIA1300-PvSPL 6-cGFP.

The successfully constructed recombinant vector pCABIA1300-PvSPL6-cGFP was transformed into Agrobacterium EHA105 and the strain was preserved. And injecting the bacterial liquid into tobacco by a tobacco transient expression technology to perform subcellular localization observation. Fluorescence confocal results show that, unlike typical transcription factors, PvSPL6 can be localized to the cell membrane in addition to the tobacco cell nucleus, suggesting that the gene may have other important biological functions in addition to functioning as a transcription factor.

Example 3: acquisition of PvSPL6 transgenic switchgrass plants

Respectively designing primers for connecting an entry vector in an overexpression vector and an interference expression vector: PvSPL6-pGWC-F and PvSPL6-pGWC-R, PvSPL6-RNAi-pGWC-F and PvSPL6-RNAi-pGWC-R, 18 bases (seamless connection joint sequence) after AhdI enzyme cutting sites and entry vector pGWC enzyme cutting sites are introduced into the tail ends of the primers, and the obtained PvSPL6 full-length sequence is used as a template to carry out PCR amplification by using the primers.

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 underlined is the seamless connection joint sequence.

Recovering the amplified fragment. The pGWC vector was digested with the restriction enzyme AhdI and recovered. The recovered two fragments were ligated by homologous recombination using a seamless ligase (purchased from Vazyme) and transformed into E.coli DH 5. alpha. competent cells by heat shock method. And (4) selecting a monoclonal colony, performing amplification culture in a kanamycin-resistant LB culture medium, and sequencing. The kit extracts recombinant strain plasmids with correct sequencing, and the recombinant plasmid enzyme digestion recovery fragments are respectively transferred into an overexpression vector pANIC6B and an interference expression vector pANIC8B (figure 2) by using Gateway technology. The recombination reaction is as follows: 100ng of the cleaved and recovered fragment, 50ng of pANIC6B/pANIC8B vector plasmid, 1. mu.L of LR enzyme (Invitrogen, cat. No. 11791020), followed by ddH₂Make up to 10. mu.L of O. Culturing at 25 ℃ for 6h, transforming Escherichia coli DH5 alpha competent cells, obtaining positive recombinant strain plasmids pANIC6B-PvSPL6 and pANIC8B-PvSPL6-RNAi with correct sequencing, and transforming agrobacterium EHA 105.

A genetic transformation method of Agrobacterium-mediated embryogenic callus of switchgrass (Xi et al, Agrobacterium-mediated transformation of switchgrass and origin of the genes. bioenergy Research,2009,2:275-283) was used to introduce pANIC6B-PvSPL6 and pANIC8B-PvSPL6-RNAi, respectively, into lowland type wild type switchgrass Alamo to obtain resistant seedlings. The over-expression vector universal primer ZmUbq-F and the downstream primer PvSPL6-R of the full-length gene are used for detecting the full-length gene, the interference expression vector universal primer Guslink-F and the downstream primer PvSPL6-RNAi-R, Guslink-R and PvSPL6-RNAi-R of the interference fragment are used for detecting the interference fragment, the upstream and downstream primers (hph3+ hph4) of the hygromycin resistant gene are used for detecting the hygromycin gene, and finally a positive transgenic strain is determined (FIG. 3).

Example 4: molecular identification of transgenic plants

Taking the tender stem tissue of the identified transgenic positive plant, extracting total RNA by using a TriZol Reagent kit (Invitrogen company, product number 15596026), detecting the content and purity of the total RNA by using agarose gel electrophoresis and a nucleic acid analyzer (NanoDrop), taking 1.0 mu g of the total RNA for reverse transcription reaction, reversing the total RNA into cDNA by using reverse transcriptase (Promega company, product number M1701), and referring to the use instruction in the reverse transcription reaction step. The cDNA is taken as a template, primers PvSPL6-qRT-F and PvSPL 6-qRT-R are used for carrying out fluorescent quantitative PCR detection, and the internal reference gene is switchgrass Ubiquitin (UBQ) gene. The primer sequences are as follows:

PvUBQ-F：ttcgtggtggccagtaag

PvUBQ-R：agagaccagaagacccaggtacag

PvSPL6-qRT-F：caggtgattaagcaggtaccct

PvSPL6-qRT-R：ggcacaggcgaacgaattac

the real-time fluorescent quantitative PCR reaction system is 20 mu L, wherein each of the forward/reverse primers is 1 mu L, the cDNA template is 2 mu L, the SYBR Green qRT Master Mix (purchased from Takara Bio-engineering Co., Ltd.) is 10 mu L, and ddH₂Make up to 20. mu.L of O. Real-time fluorescent quantitative PCR instrumentThe reaction was performed using a two-step process with Roche 480. The detection result shows that compared with wild type Control, the expression level of PvSPL6 in over-expressed plants PvSPL6OE-67, PvSPL6OE-71 and PvSPL6OE-76 is obviously increased (A in figure 4); in contrast, the expression level of PvSPL6 in the interfering expression plants PvSPL6RNAi-1, PvSPL6RNAi-6 and PvSPL6RNAi-7 was significantly decreased (B in fig. 4).

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

Switchgrass plants grown for six months were taken for flowering time, stem length and biomass measurements. Compared with the wild type, the obvious flowering time of the PvSPL6 overexpression plant is advanced by about 30 days, 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); whereas PvSPL6 interfered with expression that plant flowering time showed a significant delay (about 40 days) compared to wild-type switchgrass, and stem length increased by about 50%, stem number increased, and plant height increased accordingly (fig. 5). Overground parts of plants with six months of growth size of Control, PvSPL6OE-67/-71/-76 and PvSPL6RNAi-1/6/7 were collected respectively, and fresh weights thereof were determined. The results show that the dry matter biomass yield of the PvSPL6 overexpression line is reduced by about 56% and that the dry matter biomass yield of the PvSPL6 interfering expression line is increased by about 63% compared to the wild type (FIG. 7).

The general principles defined herein may be implemented in 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> research institute for aquatic products in yellow sea of China institute for aquatic science

Application of <120> switchgrass SBP-box transcription factor PvSPL6 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

<211> 42

<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 a gene for coding a switchgrass SBP-box type transcription factor PvSPL6 is shown as SEQ ID NO.1, and the application is the application of over-expressing PvSPL6 in regulating the early flowering, shortening the stem length and reducing the biomass of switchgrass and the application of inhibiting the expression of PvSPL6 in regulating the delayed flowering time of the switchgrass and increasing the stem length and the biomass.

2. A recombinant vector containing switchgrass SBP-box transcription factor PvSPL6 gene, which is characterized in that the recombinant vector is pANIC6B-PvSPL6, wherein the recombinant vector contains SEQ ID NO. 1.

3. A recombinant vector comprising an RNA interference segment at the level of switchgrass SBP-box gene transcript, characterized in that said recombinant vector is panac 8B-PvSPL6-RNAi comprising SEQ ID No.3, said SEQ ID No.3 being a portion of SEQ ID No. 1.