CN105602962B - Application of SBP-box transcription factor of switchgrass in increasing plant biomass and fermentable sugar yield - Google Patents
Application of SBP-box transcription factor of switchgrass in increasing plant biomass and fermentable sugar yield Download PDFInfo
- Publication number
- CN105602962B CN105602962B CN201610035833.0A CN201610035833A CN105602962B CN 105602962 B CN105602962 B CN 105602962B CN 201610035833 A CN201610035833 A CN 201610035833A CN 105602962 B CN105602962 B CN 105602962B
- Authority
- CN
- China
- Prior art keywords
- switchgrass
- sbp
- fermentable sugar
- plant
- pvspl2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
- C12N15/8229—Meristem-specific, e.g. nodal, apical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
Abstract
The invention discloses application of switchgrass SBP-box transcription factors in increasing plant biomass and fermentable sugar yield, belonging to the technical field of plant genetic engineering. The main content includes gene cloning and sequencing of switchgrass SBP-box type transcription factors PvSPL1 and 2, and then on this basis, using chimeric suppressor silencing technology (CRES-T), inhibiting the transcriptional activity of the entire PvSPL1 and 2 transcription factor subfamily, thereby producing transgenic switchgrass plants with increased tillering numbers. The dry matter biomass of the obtained transgenic plants is increased by 2.0-2.3 times, and the total fermentable sugar yield is increased by 2.0-2.2 times. The transgenic energy plant generated by the invention can be integrated into a conventional breeding project, and provides valuable germplasm resources for creating a new energy plant variety with high yield and high degradation efficiency. Meanwhile, the identified switchgrass SBP-box transcription factors PvSPL1 and 2 can provide new targets for future molecular breeding and are used for the targeted molecular design of commercial energy plant products.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to switchgrass SBP-box transcription regulation factors PvSPL1 and 2 and transformation of a coding gene thereof, and improvement of switchgrass biomass and fermentable sugar yield.
Background
The excessive consumption of fossil energy necessitates the development of new energy. The biomass energy has the characteristics of rich resources, environmental protection, cleanness, renewability and the like. The research at present mostly focuses on two major categories of biodiesel and bioethanol, wherein the development and utilization of bioethanol are gradually implementedNow commercialized. Switchgrass (Panicum virgatumL.) belongs to perennial C4 high herbaceous plants, is mainly used as energy grass and pasture, and is an important fiber biomass resource. Increasing switchgrass biomass and fermentable sugar yield are important means for improving the development and utilization of biomass energy. The completion of switchgrass whole genome sequencing and the publication of an expression chip database provide resource guarantee for the mining and verification of functional genes.
SQUMOSA PROMOTER BINDING PROTEIN-like (SPL) transcription factor contains a conserved SBP-box core domain, and is a plant-specific transcription regulatory factor family. In addition, the SBP-box family genes beyond 1/2 contain the target site of miR156, and are strictly regulated by miR156 at the post-transcriptional level. Therefore, after miR156 is over-expressed, the miR156 target site is containedSPLThe gene expression level is significantly reduced. The present research shows that it is possible to determine,SPLthe gene has wide functions, participates in various stages of plant growth and development and multiple ways of physiological and biochemical reactions, and mainly relates to the processes of plant flowering phase regulation, tillering generation, plant type establishment, biotic/abiotic stress response and the like. Identified earliest in plantsSPLThe gene is isolated from Goldfish grassSBP1AndSBP2genes (Klein et al, A new family of DNA-binding proteins genes of the sameAntirrhinum majus floral meristem identity gene SQUAMOSA1996, Molecular and General Genetics, 259: 7-16). The Arabidopsis thaliana and the rice contain 17 and 19 plants respectivelySPLGenes, wherein 11 genes contain miR156 target sites. The current research is more intensiveSPLThe gene regulates the flowering time and influences the transition of plants from vegetative growth period to reproductive growth period. There is no systematic study on plant type establishment and tillering, and AtSPL9 and AtSPL15 are reported to be related to rosette leaf enlargement in Arabidopsis (Schwarz et al, The microRNA regulated SBP-box genes)SPL9 and SPL15Control shoot formation in Arabidopsis 2008, Plant Molecular Biology 67: 183-195), OsSPL14 involved in the development of rice tillering (Luo et al, Control of tillering of rice by OsSPL14 and strigolactones, while work in two indendogenous pathways Plant and Cell Physiology, 2012, 53: 1793-. After miR156 is over-expressed in switchgrass, tillering number is increased, biomass and fermentable sugar yield are increased (Fu et al, Overexpression of miR156 in switchgrass: (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production. 2012, Plant Biotechnology Journal, 10: 443-452）。
Researches on improvement of plant biomass through plant genetic engineering are relatively few, and the over-expression of miR156 to increase the number of branches or tillers of a plant is a hot spot of the current researches.SPLThe gene is used as a direct target gene of miRNA156, and the mechanism of regulating plant branching or tillering is not clear. The present invention is directed primarily to switchgrassPvSPL1And2the gene converts PvSPL1 and 2 into a high-efficiency negative regulator through a chimeric inhibitor silencing technology, finally realizes the regulation of the occurrence of switchgrass tillering, obtains transgenic switchgrass plants with increased biomass and fermentable sugar yield, and has important guiding significance for genetic breeding and directed molecule design of switchgrass and other gramineous plants.
Disclosure of Invention
The invention aims to provide an SBP-box type transcription factor of switchgrassPvSPL1And2the coding gene of (1); the nucleotide sequence and the amino acid sequence are respectively shown in SEQ ID NO.1 and 2 and SEQ ID NO.3 and 4. Considering the degeneracy of the codon, the synonymous mutation of the nucleotide sequence without changing the amino acid sequence also falls into the protection scope of the present invention.
The second purpose of the invention is to provide an SRDX fusion fragment aiming at switchgrass SBP-box type transcription factors PvSPL1 and 2, and the nucleotide sequences are shown as SEQ ID NO.5 and 6. The utilization of the fusion fragment can obviously increase the tillering number of the switchgrass so as to increase the biomass thereof.
The third object of the present invention is to provide a chimeric suppressor silencing expression vector of switchgrass SBP-box type transcription factors PvSPL1 and 2, which is cloned the above fusion fragment as shown in SEQ ID NO.5 and 6.
The fourth purpose of the invention is to provide the application of the switchgrass SBP-box transcription factors PvSPL1 and 2 in the aspect of regulating and controlling the tillering of the switchgrass.
The fifth purpose of the invention is to provide the application of the SRDX fusion segment aiming at the switchgrass SBP-box transcription factors PvSPL1 and 2 in increasing the biomass and the fermentable sugar yield of the switchgrass.
The SRDX fusion fragments of the switchgrass PvSPL1 and 2 are recombined and integrated into an overexpression vector pANIC6B based on Gateway technology; by using AgrobacteriumAGL1The mediated genetic transformation method comprises the steps of introducing an overexpression vector into switchgrass embryogenic callus cells, obtaining resistance regeneration plants through hygromycin resistance screening, and finally determining positive transgenic plants through PCR analysis; the counting of tillering number, the measurement results of biomass and fermentable sugar yield show that the PvSPL1 and 2 can obviously increase the tillering number, the biomass and the fermentable sugar yield of switchgrass.
The core characteristics and the inventive concept of the invention comprise:
1. the chimeric inhibitor silencing technology is used for realizing the transformation of SBP-box transcription factors PvSPL1 and 2 of switchgrass, so that the switchgrass becomes a high-efficiency negative regulator, and the problem of insignificant inhibition effect caused by the existence of functional redundant genes in the switchgrass can be solved;
2. aiming at the main character of switchgrass tillering number which influences the biomass height, the invention regulates and controls the switchgrass tillering number by advanced genetic engineering technology and can obtain obvious effect in a short time.
The invention has the following beneficial effects:
1. switchgrass SBP-box-like gene obtained in the inventionPvSPL1And2the gene is a key gene for regulating and controlling the tillering of switchgrass, and has important contribution to obtaining ideal energy plant types through molecular orientation design;
2. the molecular regulation and control of the PvSPL1 and the PvSPL 892 can obviously increase the biomass and the fermentable sugar yield of the switchgrass, and has important reference significance for the genetic improvement of the biomass and the fermentable sugar yield of energy plants and gramineous forage grasses;
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 is a schematic diagram of an overexpression vector of a fusion fragment of pANIC6B-PvSPL2-SRDX from switchgrass;
FIG. 2 PCR identification of PvSPL2-SRDX gene transformed plants. M represents DNA Marker, 1-16 represents pANIC6B-PvSPL 2-SRDX-transformed switchgrass plants, WT-represents non-transgenic wild-type switchgrass plants.hphAnd (3) selecting a marker gene for plant resistance carried on the PvSPL2-SRDX vector.
FIG. 3 qRT-PCR analysis of PvSPL2-SRDX gene transferred plants. CtrlP indicates pANIC6B empty vector plant, TSPL2-1/-2/-3 indicates three independent positive transgenic lines, respectively.
FIG. 4 phenotype (A) and tillering number (B) of transgenic positive plants. CtrlP indicates pANIC6B empty vector plant, TSPL2-1/-2/-3 indicates three independent positive transgenic lines, respectively.
FIG. 5 determination of dry matter biomass of transgenic plants. CtrlP indicates pANIC6B empty vector plant, TSPL2-1/-2/-3 indicates three independent positive transgenic lines, respectively.
FIG. 6 fermentable sugar yield assay of transgenic plants. CtrlP indicates pANIC6B empty vector plant, TSPL2-1/-2/-3 indicates three independent positive transgenic lines, respectively.
Detailed Description
The principles and features of the present invention are described below in the examples directed to the chimeric inhibitor silencing of PvSPL2 to increase switchgrass tillering number, biomass, and fermentable sugar yield, which are intended to be illustrative only and not limiting to the scope of the invention. Materials, reagents, binary vectors, Agrobacterium, and the like used in the following examples are commercially available from companies, unless otherwise specified, and the MS minimal medium is available from Phytotechnology Laboratories (Cat. No. M519).
Example 1:PvSPL2cloning and sequencing of genes
Taking tender stem parts of low-grade switchgrass Alamo, extracting tender stem total RNA by using TriZol Reagent (Invitrogen, Cat. No. 15596026), detecting the content and purity of the total RNA by using agarose gel electrophoresis and an ultraviolet spectrophotometer, and taking 2.0 mu g of the total RNA to perform reverse transcription reaction by using M-MLV (Promega, Cat. No. M1701), wherein the steps of the reverse transcription reaction refer to the using instructions of the reverse transcriptase. First strand cDNA synthesized by reverse transcription was used as a template, and primers:
5’- ATGGGTTCATTTGGGATGGAC-3’
5’- AAGCGTCAGTGCATCAGGTCATAG-3’
carrying out conventional PCR amplification; the PCR reaction system is as follows: mu.L of cDNA, 5. mu.L of 10 XBuffer, 4. mu.L of dNTP (2.5 mM), 1. mu.L each of forward/reverse primers (10. mu.M), 0.5. mu.L of Taq enzyme (5U/. mu.L) and 36.5. mu.L of ddH2And O. Sample was added to ice and mixed well. The PCR reaction conditions are as follows: 94o C 5 min；94 oC 30 sec，56 oC 30 sec；72 oC90 sec, 32 cycles; 72oAnd C10 min. PCR amplification yielded a fragment of about 1.4 kb. After recovering the fragment by agarose gel electrophoresis, the fragment was ligated to a pMD-18T vector (TaKaRa, cat # 6011) and subjected to ligation: 4.5. mu.L of PCR product, 0.5. mu.L of pMD-18T vector and 5. mu.L of Solution I, 16oC ligation was performed overnight. Taking 5 μ L of ligation product, transforming Escherichia coli DH5 α competent cells by heat shock method, adding 800 μ L of liquid LB medium, recovering for 1 hr, spreading on LB plate coated with ampicillin (X-gal/IPTG), 37 μ LoCulturing for 14 hr. And selecting white monoclonal colonies, performing amplification culture in a liquid LB culture medium containing aminobenzene antibiotics, and sequencing. Sequencing analysis shows that the sequence contains a complete open reading frame, the total length is 1413 basic groups, the sequence is shown as SEQ ID NO.2, the coded protein contains 470 amino acid residues, and the sequence is shown as SEQ ID NO. 4.
Example 2:PvSPL2chimeric suppressor silencing expression vector construction and genetic transformation of genes
The negative regulator used in the chimeric repressor silencing expression vector used in the invention is a transcription repressor SRDX (LDLDLELRLGFA) of EAR-like motif. The coding sequence of this motif was fused into PvSPL2 prior to the stop codon (TGA) by PCR reaction using primers:
5’- CACCATGGGTTCATTTGGGATGGAC-3’；
5’-TCATTAAGCGAAACCCAAACGGAGTTCTAGATCCAGATCCAGGTGCATCAGGTCATA GTG-3’
PCR amplification is carried out, and the amplification template is the PvSPL2-pMD 18T recombinant plasmid. The PCR reaction system is as follows: mu.L template, 5. mu.L 10 XBuffer, 4. mu.L dNTP (2.5 mM), 1. mu.L each of forward/reverse primers (10. mu.M), 0.5. mu.L Taq enzyme (5U/. mu.L) and 36.5. mu.L ddH2And O. The PCR reaction conditions are as follows: 94o C 5 min；94 oC 30 sec，56 oC 30 sec；72 oC90 sec, 32 cycles; 72oAnd C10 min. The PCR amplification obtained about 1.4 kb fragment, the fragment sequence is shown in SEQ ID NO. 6. After recovering the fragment by agarose gel electrophoresis, pENTR vector (Invitrogen, cat # 450228) was ligated. The ligation reaction is described with reference to the vector instructions. mu.L of the ligation product was taken, transformed into E.coli DH 5. alpha. competent cells by heat shock, added with 800. mu.L of liquid LB medium, thawed for 1 hr, plated on kanamycin-resistant LB plates (coated with X-gal/IPTG), 37oCulturing for 14 hr. White monoclonal colonies were selected, amplified in kanamycin-resistant LB medium and sequenced. Extraction of recombinant strain plasmid with correct sequencing, EcoR V endonuclease 37, by alkaline lysis methodoC enzyme digestion for 1 hr, and transferring the recombinant plasmid enzyme digestion recovery fragment into an overexpression vector pANIC6B (FIG. 1) by using Gateway technology. The recombination reaction is as follows: 100 ng of the cleaved and recovered fragment, 50 ng of pANIC6B vector plasmid, 1. mu.L of LR enzyme (Invitrogen, cat # 11791020), and then digested with ddH2Make up to 10. mu.L of O. 16oC ligation was performed overnight. mu.L of the ligation product was taken, transformed into E.coli DH 5. alpha. competent cells by heat shock, added with 800. mu.L of liquid LB medium, thawed for 1 hr, plated on kanamycin-resistant LB plates (coated with X-gal/IPTG), 37oCulturing for 14 hr. White monoclonal colonies were selected, amplified in kanamycin-resistant LB medium and sequenced. Extracting recombinant strain plasmid with correct sequencing by alkaline lysis method, and transforming agrobacterium by electric shock methodAGL1A competent cell which is capable of expressing a cell,spread on LB plates containing kanamycin and rifampicin, 28oAnd C, dark culture for 2 days, detecting positive monoclonals by using the primers, and performing amplification culture in a liquid LB culture medium resistant to kanamycin and rifampicin for genetic transformation of switchgrass embryogenic calluses.
Genetic transformation of switchgrass embryogenic callus mediated by agrobacterium was used (Xi et al,Agrobacterium-mediated transformation of switchgrass and affinity of the transformations 2009, Bioenergy Research, 2: 275-hphGenes, ultimately positive transgenic lines were determined (fig. 2).
Example 3: molecular identification of transgenic plants
Taking shoot tissue of the above identified transgenic positive plant, extracting total RNA by TriZol (Invitrogen, cat # 15596026), synthesizing first strand cDNA by reverse transcriptase (Promega, cat # M1701), and collecting the first strand cDNA from switchgrassUbiquitinThe gene is used as an internal reference gene, and primers PvSPL2-qF1/qR1 and PvSPL2-qF2/qR2 are respectively adopted to detect the endogenesis and the total content of a positive transgenic plantPvSPL2The expression of the gene (FIG. 3), the primer sequences were as follows:
PvUbiquitin-qF: 5’-TTCGTGGTGGCCAGTAAG-3’
PvUbiquitin-qR: 5’-AGAGACCAGAAGACCCAGGTACAG-3’
PvSPL2-qF1: 5’-GCGCGGTTTCAGGCTCTCG-3’
PvSPL2-qR1: 5-CCTCGCAACCGGACAATGGA-3’
PvSPL2-qF2: 5’-GTGTGGAGCGGCGGTTTT-3’
PvSPL2-qR2: 5’-TCCGTCTGCGGGCATTGT-3’
as shown in FIG. 3, endogenous to the transgenic plants (labeled TSPL 2-1/-2/-3)PvSPL2The expression level of (D) was not significantly different from that of the control (pANIC 6B empty vector, labeled CtrlP), while the total expression level wasPvSPL2The expression level is obviously increased, which indicates that the gene is transferred from the external sourcePvSPL2The overexpression effect is achieved.
Example 4: transgenic plant tillering number, biomass and fermentable sugar yield analysis
When the tillering number and biomass of the plants growing for six months were measured, the tillering number of the transgenic plants was increased to 1.6-2.0 times as compared with the control plants (FIG. 4). Further collecting CtrlP and TSPL2-1/-2/-3 overground parts of six-month-size plants, placing on 40oC oven for 1 week, the dry matter weight was measured, and the results showed that the biomass of transgenic plants increased 2.0-2.3 times that of the control (FIG. 5). The method for measuring the yield of the fermentable sugar adopts a phenol sulfate method, and comprises the following specific steps: directly enzymolyzing cell wall residue with cellulase and cellobiase mixture for 72 hr as control; 1.5% H was used2SO4At 121oPretreatment for 60 min under C condition, and enzymolysis of cell wall residue with the same amount of cellulase and cellobiase mixture for 72 hr as treatment group. The enzymatic products were assayed for fermentable sugar content using the phenol sulfate method (Dubois et al, Colorimetric method for determination of sugars and related issues, 1956, Analytical Chemistry, 28: 350-. The saccharification efficiency is the ratio of the difference of the fermentable sugar content before and after enzymolysis to the fermentable sugar content before enzymolysis. Thus, according to the formula: the fermentable sugar yield (g/plant) = overground cell wall carbohydrate yield (g/plant) × saccharification efficiency, and the fermentable sugar yield of the transgenic plant was calculated to increase 2.0-2.2 times that of the control plant (fig. 6).
SEQ ID NO.1
ATGGAGTGGACGGCCCCGAAGCCCGCCGCTTCGCCCTCCTCGCCTCCCCTGCTCTGGGACTGGGGCAACCACGCCGCCGCGGGCTCGGGCTGCTCCGGCGACGCGCCAGCGCGGCGCGGCGGGAAGGAGCGGGAGGCGAAGCGTACCAAGGGCGAGGAAGGCGGGGGAGCGGAAGTTAGGTGCCAGGTCGAGGGGTGCGGGCTGGAGCTCGGCACGGCCAAGGACTACCACCGGAAGCACCGCGTCTGCGAGGCCCACACCAAGTGCCCCCGCGTCGTCGTCGCCGGCCAGGAGCGCCGCTTCTGCCAGCAGTGCAGCAGGTTCCATGCTCTGTCCGAGTTTGATCAGAAGAAGAGGAGCTGCAGGAGGCGTCTGTCTGATCACAATGCCCGCCGGCGGAAGCCTCAGCCAGATGCATACGCCTTTGCCTCTGCGAGGCTGCCTTCATCATTGTTTGATGATAGGCGGCAAATAAGTTTTGTCTGGAATAGAGCTCCTCTTAGCCATGTAAGACCTTTCACTTCTCCATGTGACAGCTCATCTGACTTCAAGCTCTCACATGCCAAAGAAATAAGTGAGCTATCAACAAAAGTTGGGACGATAACTGGACAAGTTCATTTTGATAAATCTCACCTGTCCAATGCCATTCCAACACTTAGCCATGGCAAAGATGAGCTGTTGCCAATGAAAGGTCCGGACACATCAATAACTGCTTCAAAATTCGATGGAGCACCGGATCTTCAGCGTGCTCTCTCTCTTCTGTCAGCTGGCTCTTGTGGATTCCCCGATCCTGTACCGCAAGCATCTTGCCTTATCCAGTTCACTGGTGCCAGTGAGAACAGCGGTGACCTTCACTTATCGCATGGAGGGAACTCTGGTCCAGCTTCATGTGCCGATGAACAGCATGTAGCACCTCAGCCTCAGCTGGTTCGTTTTACCATGGATACCAGTAGCAATGTCTATGAGCCCACTTTCTTCGGTGTAAACCAGATAAATTAA
SEQ ID NO.2
ATGGGTTCATTTGGGATGGACTGGAACCAGAAGGGTTCTGTGTTGTGGGACTGGGAGAATTTGCCGCTGATAGGCACAAGTGGAAGCGAGAGCGCTAAGGCGATCGCGACTCAGGCTGAGGCCAAGTTTTCAGCCACTGAGGTCACAAGGCATGGATCAGTGCATTCTTCCTGTGGTACTTTCTCTTCCAACTCGGAGATGGGGTATGGCTCATCTAAGAGCTCCATATCCGCGTCGATTGATTCTTCACCCAAGACGGGGAAGAACATGGAGCTCAATTTTGCACCTGCCAGAGTGTCTGACAAGAACACTGTTTTGGGAAAGGTTTATGATGCCAGAACCTCCCCATCATCAGTGATAGCCGTAAGTAGTGGAGAGCCAGTGCTTAGCCTGAAACTTGGCAAGAGAACCTATTTTGAAGACGTCTGTGGAGGGCAGAGTGTCAAGAGTTCTCCGTCGGATACAAGTGCAGTGACTCCTGCTTTGGTGAAGAAGGCAAAGGCAGCACAAAACGCACAGAACACCTACTGTCAGGTTGAAGGTTGCAAGATTGATCTCTCTTCTGCTAAAGATTACCATCGAAAGCACAAAGTCTGTGAAGCTCACTCTAAGGCTCCCAAGGTGGTTGTTGCTGGTGTGGAGCGGCGGTTTTGCCAACAGTGTAGCCGGTTCCATGGTTTAGATCTCTTCGACCAGAAAAAACGAAGCTGCCGCAGGCGTCTCAATGATCACAATGCCCGCAGACGGAAGCCACAGCCTGAAGCAATTTCTTTTGGTTCATCAAGGCTCTCTGCAATGTTCTATGATGCAAGGCAACAGACAACTCTTCATTTTGGTCAAGCCCCTTATGGTCAAATGAGAAGCTGTGCAAGTTCTTCATGGGATAACCCAGGAGGAGCCTTCAAATTTGCAGAAACTAAAGCCCCTTGGTTAAAGCCAGCAAGAGCTGCTGGTATTGATGCCTTGCATTTATCAAGTCAGCAGGTATGGAACAACATTATGCCACATGGTGGCCATCAAGATTTTGATGGGTTCATGGGTTTCAAAGGAACCAGTGCAAAGGTCCTCAATCAAGGCGTTGAAGCTTCTGCGGCCATCTCCGATTCAAATGGAAACCCGGATCTTCAGCGTGCTCTCTCTCTTCTGTCAAACAATTCAGCTGGTGCTGGCAACAACCACCCGACCACTCAGCCGCACCCTGGCCTGAGCACCCTCGCCAGCACCTCCAACGCAGTGATGCAAGCTTCATCACAAGGGCTCTGGCAAGACGGCACAGCGCTTGATCTTCATGCGCGGTTTCAGGCTCTCGATCCCCTGGGCAGTGGCAGCGCCATCCCAACAACTCATGAGGTCCAGCTCCCGAAACCGTCCTTGTTCGACGACTCCTCTTCCCACTATGACCTGATGCACTGA
SEQ ID NO.3
MEWTAPKPAASPSSPPLLWDWGNHAAAGSGCSGDAPARRGGKEREAKRTKGEEGGGAEVRCQVEGCGLELGTAKDYHRKHRVCEAHTKCPRVVVAGQERRFCQQCSRFHALSEFDQKKRSCRRRLSDHNARRRKPQPDAYAFASARLPSSLFDDRRQISFVWNRAPLSHVRPFTSPCDSSSDFKLSHAKEISELSTKVGTITGQVHFDKSHLSNAIPTLSHGKDELLPMKGPDTSITASKFDGAPDLQRALSLLSAGSCGFPDPVPQASCLIQFTGASENSGDLHLSHGGNSGPASCADEQHVAPQPQLVRFTMDTSSNVYEPTFFGVNQIN
SEQ ID NO.4
MGSFGMDWNQKGSVLWDWENLPLIGTSGSESAKAIATQAEAKFSATEVTRHGSVHSSCGTFSSNSEMGYGSSKSSISASIDSSPKTGKNMELNFAPARVSDKNTVLGKVYDARTSPSSVIAVSSGEPVLSLKLGKRTYFEDVCGGQSVKSSPSDTSAVTPALVKKAKAAQNAQNTYCQVEGCKIDLSSAKDYHRKHKVCEAHSKAPKVVVAGVERRFCQQCSRFHGLDLFDQKKRSCRRRLNDHNARRRKPQPEAISFGSSRLSAMFYDARQQTTLHFGQAPYGQMRSCASSSWDNPGGAFKFAETKAPWLKPARAAGIDALHLSSQQVWNNIMPHGGHQDFDGFMGFKGTSAKVLNQGVEASAAISDSNGNPDLQRALSLLSNNSAGAGNNHPTTQPHPGLSTLASTSNAVMQASSQGLWQDGTALDLHARFQALDPLGSGSAIPTTHEVQLPKPSLFDDSSSHYDLMH
SEQ ID NO.5
ATGGAGTGGACGGCCCCGAAGCCCGCCGCTTCGCCCTCCTCGCCTCCCCTGCTCTGGGACTGGGGCAACCACGCCGCCGCGGGCTCGGGCTGCTCCGGCGACGCGCCAGCGCGGCGCGGCGGGAAGGAGCGGGAGGCGAAGCGTACCAAGGGCGAGGAAGGCGGGGGAGCGGAAGTTAGGTGCCAGGTCGAGGGGTGCGGGCTGGAGCTCGGCACGGCCAAGGACTACCACCGGAAGCACCGCGTCTGCGAGGCCCACACCAAGTGCCCCCGCGTCGTCGTCGCCGGCCAGGAGCGCCGCTTCTGCCAGCAGTGCAGCAGGTTCCATGCTCTGTCCGAGTTTGATCAGAAGAAGAGGAGCTGCAGGAGGCGTCTGTCTGATCACAATGCCCGCCGGCGGAAGCCTCAGCCAGATGCATACGCCTTTGCCTCTGCGAGGCTGCCTTCATCATTGTTTGATGATAGGCGGCAAATAAGTTTTGTCTGGAATAGAGCTCCTCTTAGCCATGTAAGACCTTTCACTTCTCCATGTGACAGCTCATCTGACTTCAAGCTCTCACATGCCAAAGAAATAAGTGAGCTATCAACAAAAGTTGGGACGATAACTGGACAAGTTCATTTTGATAAATCTCACCTGTCCAATGCCATTCCAACACTTAGCCATGGCAAAGATGAGCTGTTGCCAATGAAAGGTCCGGACACATCAATAACTGCTTCAAAATTCGATGGAGCACCGGATCTTCAGCGTGCTCTCTCTCTTCTGTCAGCTGGCTCTTGTGGATTCCCCGATCCTGTACCGCAAGCATCTTGCCTTATCCAGTTCACTGGTGCCAGTGAGAACAGCGGTGACCTTCACTTATCGCATGGAGGGAACTCTGGTCCAGCTTCATGTGCCGATGAACAGCATGTAGCACCTCAGCCTCAGCTGGTTCGTTTTACCATGGATACCAGTAGCAATGTCTATGAGCCCACTTTCTTCGGTGTAAACCAGATAAATCTGGATCTGGATCTAGAACTCCGTTTGGGTTTCGCTTAATAA
SEQ ID NO.6
ATGGGTTCATTTGGGATGGACTGGAACCAGAAGGGTTCTGTGTTGTGGGACTGGGAGAATTTGCCGCTGATAGGCACAAGTGGAAGCGAGAGCGCTAAGGCGATCGCGACTCAGGCTGAGGCCAAGTTTTCAGCCACTGAGGTCACAAGGCATGGATCAGTGCATTCTTCCTGTGGTACTTTCTCTTCCAACTCGGAGATGGGGTATGGCTCATCTAAGAGCTCCATATCCGCGTCGATTGATTCTTCACCCAAGACGGGGAAGAACATGGAGCTCAATTTTGCACCTGCCAGAGTGTCTGACAAGAACACTGTTTTGGGAAAGGTTTATGATGCCAGAACCTCCCCATCATCAGTGATAGCCGTAAGTAGTGGAGAGCCAGTGCTTAGCCTGAAACTTGGCAAGAGAACCTATTTTGAAGACGTCTGTGGAGGGCAGAGTGTCAAGAGTTCTCCGTCGGATACAAGTGCAGTGACTCCTGCTTTGGTGAAGAAGGCAAAGGCAGCACAAAACGCACAGAACACCTACTGTCAGGTTGAAGGTTGCAAGATTGATCTCTCTTCTGCTAAAGATTACCATCGAAAGCACAAAGTCTGTGAAGCTCACTCTAAGGCTCCCAAGGTGGTTGTTGCTGGTGTGGAGCGGCGGTTTTGCCAACAGTGTAGCCGGTTCCATGGTTTAGATCTCTTCGACCAGAAAAAACGAAGCTGCCGCAGGCGTCTCAATGATCACAATGCCCGCAGACGGAAGCCACAGCCTGAAGCAATTTCTTTTGGTTCATCAAGGCTCTCTGCAATGTTCTATGATGCAAGGCAACAGACAACTCTTCATTTTGGTCAAGCCCCTTATGGTCAAATGAGAAGCTGTGCAAGTTCTTCATGGGATAACCCAGGAGGAGCCTTCAAATTTGCAGAAACTAAAGCCCCTTGGTTAAAGCCAGCAAGAGCTGCTGGTATTGATGCCTTGCATTTATCAAGTCAGCAGGTATGGAACAACATTATGCCACATGGTGGCCATCAAGATTTTGATGGGTTCATGGGTTTCAAAGGAACCAGTGCAAAGGTCCTCAATCAAGGCGTTGAAGCTTCTGCGGCCATCTCCGATTCAAATGGAAACCCGGATCTTCAGCGTGCTCTCTCTCTTCTGTCAAACAATTCAGCTGGTGCTGGCAACAACCACCCGACCACTCAGCCGCACCCTGGCCTGAGCACCCTCGCCAGCACCTCCAACGCAGTGATGCAAGCTTCATCACAAGGGCTCTGGCAAGACGGCACAGCGCTTGATCTTCATGCGCGGTTTCAGGCTCTCGATCCCCTGGGCAGTGGCAGCGCCATCCCAACAACTCATGAGGTCCAGCTCCCGAAACCGTCCTTGTTCGACGACTCCTCTTCCCACTATGACCTGATGCACCTGGATCTGGATCTAGAACTCCGTTTGGGTTTCGCTTAATGA
Claims (6)
1. The full-length CDS of switchgrass SBP-box class transcription factor PvSPL2, characterized by: the nucleotide sequence is shown as SEQ ID NO. 2.
2. The full-length CDS-encoded amino acid sequence of PvSPL2 of claim 1, wherein: the amino acid sequence is shown as SEQ ID NO. 4.
3. The fusion fragment PvSPL2-SRDX formed by chimeric inhibitor SRDX from the nucleotide sequence of PvSPL2 of claim 1, and the sequence thereof is shown in SEQ ID NO. 6.
4. A plant expression vector comprising the PvSPL2-SRDX fusion fragment of claim 3.
5. Use of the plant expression vector of claim 4 for modulating switchgrass tillering number, biomass, and fermentable sugar yield.
6. Use of the plant expression vector of claim 4 for genetic improvement and molecular breeding of switchgrass.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610035833.0A CN105602962B (en) | 2016-01-20 | 2016-01-20 | Application of SBP-box transcription factor of switchgrass in increasing plant biomass and fermentable sugar yield |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610035833.0A CN105602962B (en) | 2016-01-20 | 2016-01-20 | Application of SBP-box transcription factor of switchgrass in increasing plant biomass and fermentable sugar yield |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105602962A CN105602962A (en) | 2016-05-25 |
| CN105602962B true CN105602962B (en) | 2021-04-13 |
Family
ID=55983305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610035833.0A Active CN105602962B (en) | 2016-01-20 | 2016-01-20 | Application of SBP-box transcription factor of switchgrass in increasing plant biomass and fermentable sugar yield |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105602962B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531506B (en) * | 2018-04-20 | 2021-07-13 | 中国农业科学院蔬菜花卉研究所 | Method for regulating and controlling plant type structure of crop and method for obtaining compact plant type crop |
| CN109053870B (en) * | 2018-08-08 | 2021-11-12 | 中国科学院植物研究所 | Application of AtERF49 gene in plant response high-temperature stress process |
| CN112359044B (en) * | 2020-11-16 | 2021-10-15 | 中国科学院青岛生物能源与过程研究所 | Plant SOSEKI protein SOK2, encoding gene and application |
| CN114231539B (en) * | 2021-12-31 | 2023-06-02 | 中国科学院青岛生物能源与过程研究所 | Application of switchgrass SBP-box transcription factor PvSPL6 and recombinant vector thereof |
| CN116042894B (en) * | 2022-12-05 | 2023-06-16 | 中国科学院青岛生物能源与过程研究所 | Application of PvCKX4 and miR156-SPL-CKX4 molecular module in switchgrass trait improvement |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1382682A3 (en) * | 2002-07-17 | 2004-06-30 | Expressive Research B.V. | Modulating developmental pathways in plants |
| EP2441844A1 (en) * | 2006-03-31 | 2012-04-18 | BASF Plant Science GmbH | Plants having enhanced yield-related traits and a method for making the same |
| CN103215280B (en) * | 2013-05-08 | 2014-08-20 | 山东省农业科学院高新技术研究中心 | Peanut SPL (squamosa promoter-binding protein-like) transcription factor gene, as well as encoded protein and application thereof |
| CN104975028A (en) * | 2014-04-01 | 2015-10-14 | 中国科学院青岛生物能源与过程研究所 | Application of Miscanthus WRKY transcription factor in increase of biomass of plant fiber |
| CN103937812B (en) * | 2014-04-09 | 2016-01-13 | 武汉大学 | Paddy rice spot leaf proterties controlling gene SPL29 old and feeble and disease-resistant on purposes |
| US11584937B2 (en) * | 2014-04-28 | 2023-02-21 | The Trustees Of The University Of Pennsylvania | Compositions and methods for controlling plant growth and development |
| CN104450744B (en) * | 2014-12-31 | 2017-07-07 | 福建农林大学 | A kind of paddy rice SBP box transcription factor genes and its application |
-
2016
- 2016-01-20 CN CN201610035833.0A patent/CN105602962B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105602962A (en) | 2016-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | A transcription factor OsbHLH156 regulates Strategy II iron acquisition through localising IRO2 to the nucleus in rice | |
| CN105602962B (en) | Application of SBP-box transcription factor of switchgrass in increasing plant biomass and fermentable sugar yield | |
| US7148406B2 (en) | Genes encoding enzymes for lignin biosynthesis and uses thereof | |
| US20050223426A1 (en) | Maize cellulose synthases and uses thereof | |
| CN108130334B (en) | Application of switchgrass S-adenosylmethionine synthetase gene SAMS1 in regulation and control of lignin synthesis | |
| US20200399637A1 (en) | LOSS OF FUNCTION ALLELES OF PtEPSP-TF AND ITS REGULATORY TARGETS IN RICE | |
| CN101585870B (en) | Protein related to plant heat resistance property and coding gene and application thereof | |
| MXPA02004482A (en) | Genes encoding enzymes for lignin biosynthesis and uses thereof. | |
| CN108546705B (en) | Arabidopsis flowering time regulating gene SSF and application thereof | |
| CN114231539B (en) | Application of switchgrass SBP-box transcription factor PvSPL6 and recombinant vector thereof | |
| AU760787C (en) | Novel maize orthologues of bacterial RecA proteins | |
| CN101061228A (en) | Isopentenyl transferase sequences and methods of use | |
| JP6856639B2 (en) | Dorimenol synthase III | |
| US11629356B2 (en) | Regulating lignin biosynthesis and sugar release in plants | |
| CN108085326B (en) | Application of adenosine homocysteine of switchgrass in changing lignin monomer and improving cell wall degradation efficiency | |
| CN112359044B (en) | Plant SOSEKI protein SOK2, encoding gene and application | |
| CN112553203A (en) | Long-chain non-coding RNA-lnc5 for regulating growth and development of poplar and application thereof | |
| CN102533756B (en) | Promoter and application thereof | |
| CN112094858B (en) | SsCBL01 gene for regulating and controlling potassium absorption efficiency of sugarcane and application thereof | |
| AU765102B2 (en) | Maize DNA ligase I orthologue and uses thereof | |
| Reyna-Llorens et al. | Ancient coding sequences underpin the spatial patterning of gene expression in C4 leaves | |
| EP1078087A2 (en) | Zea mays phosphate transporter genes and uses thereof | |
| WO2000009723A1 (en) | Maize rad6 genes and uses thereof | |
| AU772028C (en) | KIN17 orthologues and uses thereof | |
| CN116716335A (en) | Application of dandelion TmABI5 gene in promoting plant to synthesize chicoric acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |