Application of SBP-box type transcription factors of switchgrass in aspect of increasing plant biomass and fermentable sugar yields

A transcription factor, sbp-box technology, applied in the field of plant genetic engineering, can solve problems such as unclear mechanism, and achieve the effect of increasing biomass and fermentable sugar production

Active Publication Date: 2016-05-25
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a direct target gene of miRNA156, SPL gene regulates branching or tillering in plants, and its mechanism is still unclear

Method used

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  • Application of SBP-box type transcription factors of switchgrass in aspect of increasing plant biomass and fermentable sugar yields
  • Application of SBP-box type transcription factors of switchgrass in aspect of increasing plant biomass and fermentable sugar yields
  • Application of SBP-box type transcription factors of switchgrass in aspect of increasing plant biomass and fermentable sugar yields

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning and sequence determination of PvSPL2 gene

[0026] Take the tender stem parts of lowland switchgrass Alamo, extract the total RNA from tender stems with TriZolReagent (Invitrogen, Cat. For the reverse transcription reaction, the reverse transcriptase used was M-MLV (Promega, Cat. No. M1701). For the steps of the reverse transcription reaction, refer to the instructions for use of the reverse transcriptase. Using the first-strand cDNA synthesized by reverse transcription reaction as a template, use primers:

[0027] 5'-ATGGGTTCATTTGGGATGGAC-3'

[0028] 5'-AAGCGTCAGTGCATCAGGTCATAG-3'

[0029]Carry out routine PCR amplification; PCR reaction system: 2 μL cDNA, 5 μL 10×Buffer, 4 μL dNTP (2.5 mM), forward / reverse primer (10 μM) 1 μL each, 0.5 μL Taq enzyme (5U / μL) and 36.5 μL ddH 2 O. Mix well after adding the sample on ice. The PCR reaction conditions are: 94 o C5min; 94 o C30sec, 56 o C30sec; 72 o C90sec, 32 cycles; 72 o C10min. A fragment of ...

Embodiment 2

[0030] Example 2: Construction and genetic transformation of chimeric suppressor silencing expression vector of PvSPL2 gene

[0031] The negative regulatory factor used in the chimeric suppressor silencing expression vector used in the present invention is a kind of EAR-like motif transcriptional suppressor SRDX (LDLDLELRLGFA). The coding sequence of this motif was fused by PCR reaction before the stop codon (TGA) of PvSPL2 using primers:

[0032] 5'-CACCATGGGTTCATTTGGGATGGAC-3';

[0033] 5'-TCATTAAGCGAAACCCAAACGGAGTTCTAGATCCAGATCCAGGTGCATCAGGTCATAGTG-3'

[0034] Perform PCR amplification, and the amplification template is the above-mentioned PvSPL2-pMD18T recombinant plasmid. The PCR reaction system is: 2 μL template, 5 μL 10×Buffer, 4 μL dNTP (2.5 mM), 1 μL forward / reverse primer (10 μM), 0.5 μL Taq enzyme (5U / μL) and 36.5 μL ddH 2 O. The PCR reaction conditions are: 94 o C5min; 94 o C30sec, 56 o C30sec; 72 o C90sec, 32 cycles; 72 o C10min. A fragment of about 1.4 ...

Embodiment 3

[0036] Example 3: Molecular Identification of Transgenic Plants

[0037] Take the tender stem tissue of the positive transgenic plants identified above, use the TriZol (Invitrogen Company, product number 15596026) method to extract total RNA, reverse transcriptase (Promega Company, product number M1701) to synthesize the first-strand cDNA, and switchgrass Ubiquitin gene as an internal reference gene. Primers PvSPL2-qF1 / qR1 and PvSPL2-qF2 / qR2 detect the expression of endogenous and total PvSPL2 genes in positive transgenic plants ( image 3 ), the primer sequences are as follows:

[0038] PvUbiquitin-qF:5'-TTCGTGGTGGCCAGTAAG-3'

[0039] PvUbiquitin-qR:5'-AGAGACCAGAAGACCCAGGTACAG-3'

[0040] PvSPL2-qF1:5'-GCGCGGTTTCAGGCTCTCG-3'

[0041] PvSPL2-qR1:5-CCTCGCAACCGGACAATGGA-3'

[0042] PvSPL2-qF2:5'-GTGTGGAGCGGCGGTTTT-3'

[0043] PvSPL2-qR2:5'-TCCGTCTGCGGGCATTGT-3'

[0044] Such as image 3 As shown, the expression level of endogenous PvSPL2 in transgenic plants (marked as TS...

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Abstract

The invention discloses application of SBP-box type transcription factors of switchgrass in the aspect of increasing plant biomass and fermentable sugar yields, and belongs to the technical field of plant genetic engineering. The application mainly includes cloning genes of the SBP-box type transcription factors PvSPL1 and 2 of the switchgrass and determining sequences of the SBP-box type transcription factors of the switchgrass; inhibiting the transcription activity of integral subfamilies of the transcription factors PvSPL1 and 2 on the basis of the genes and the sequences by the aid of chimeric repressor silencing technologies (CRES-T) so as to generate transgenic switchgrass plants with increased tiller quantities. The application has the advantages that dry matter biomass of the obtained transgenic switchgrass plants can be increased by 2.0-2.3 times, and the total fermentable sugar yields can be increased by 2.0-2.2 times; the generated transgenic switchgrass energy plants can be integrated in conventional breeding projects, and accordingly precious germplasm resources can be provided for creating novel varieties of high-yield and efficiently degradable energy plants; novel targets for molecular breeding in future can be provided by the authenticated SBP-box type transcription factors PvSPL1 and 2 of the switchgrass, and the SBP-box type transcription factors PvSPL1 and 2 of the switchgrass can be used for designing oriented molecules of commercialized energy plant products.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the transformation of switchgrass SBP-box transcription regulators PvSPL1 and 2 and their coding genes, which improve the biomass of switchgrass and the yield of fermentable sugar. Background technique [0002] Excessive consumption of fossil energy makes the development of new energy sources necessary. Among them, biomass energy has the characteristics of abundant resources, environmental protection, cleanness and renewable. The current research focuses on biodiesel and bioethanol, among which, the development and utilization of bioethanol has been gradually commercialized. Switchgrass (Panicumvirgatum L.) is a perennial C4 tall herbaceous plant, which is mainly used as energy grass and pasture, and is an important fiber biomass resource. Increasing switchgrass biomass and fermentable sugar production is an important means to improve its biomass e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/62C07K14/415C12N15/82A01H5/00
CPCC07K14/415C07K2319/00C12N15/8205C12N15/8218C12N15/8229C12N15/8261C12N2830/00
Inventor 付春祥吴振映曹英萍杨瑞娟齐天雄刘金丽
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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