Method for improving plant saccharification efficiency by down-regulating UXE gene and application thereof
阅读说明：本技术 通过下调uxe基因提高植物糖化效率的方法及其应用 (Method for improving plant saccharification efficiency by down-regulating UXE gene and application thereof ) 是由 吴蔼民 陈辰 李慧玲 赵先海 王旭川 于 2020-06-22 设计创作，主要内容包括：本发明公开一种通过下调UXE基因提高植物糖化效率的方法及其应用,属于生物质能源领域。本发明通过使植物的UXE基因功能缺失或下调,来提高植物的糖化效率。通过本发明的方法使得阿拉伯糖含量降低,减少了半纤维素分支度,减弱了其与木质素和纤维素交联的紧密,进一步导致突变体材料的糖化效率提高,降低了材料处理成本。因此,本发明从根本上改变了材料的生物质特性,使糖化效率有了根本上的提高,可以节约能源,降低劳动强度。通过本发明的方法获得材料可以应用在生物质转化、纸浆和造纸工业中。(The invention discloses a method for improving plant saccharification efficiency by down-regulating a UXE gene and application thereof, belonging to the field of biomass energy. The invention improves the saccharification efficiency of plants by deleting or down-regulating the UXE gene function of the plants. The method of the invention reduces the arabinose content, reduces the branching degree of hemicellulose, weakens the compact crosslinking of the hemicellulose with lignin and cellulose, further leads to the improvement of the saccharification efficiency of mutant materials and reduces the material treatment cost. Therefore, the invention fundamentally changes the biomass characteristics of the material, fundamentally improves the saccharification efficiency, can save energy and reduce the labor intensity. The material obtained by the process of the invention can be applied in the biomass conversion, pulp and paper industry.)
1. A method of increasing plant saccharification efficiency by down-regulating the UXE gene, comprising: the saccharification efficiency of the plant is improved by deleting or down-regulating the UXE gene function of the plant.
2. The method of improving plant saccharification efficiency by downregulating the UXE gene of claim 1, wherein:
the UXE gene function is down-regulated to down-regulate the UXE activity in the plant body by a genome editing technology, so that the aim of improving the saccharification efficiency is fulfilled.
3. The method of increasing plant saccharification efficiency by downregulating the UXE gene according to claims 1 or 2, characterized in that:
the plant is rice, arabidopsis, poplar, eucalyptus, pine or jatropha curcas.
4. The method of increasing plant saccharification efficiency by downregulating the UXE gene according to claims 1 or 2, characterized in that:
the plant is rice.
5. The method of increasing plant saccharification efficiency by downregulating the UXE gene of claim 4, wherein:
the UXE gene is at least one of OsUXE1, OsUXE2 and OsUXE 3.
6. The method of increasing plant saccharification efficiency by downregulating the UXE gene of claim 5, wherein:
the UXE gene is at least two of OsUXE1, OsUXE2 and OsUXE 3.
7. Use of a UXE downregulated plant obtained by the method of increasing plant saccharification efficiency by downregulating a UXE gene according to any of claims 1-6 in biomass conversion, pulp and paper industry.
Technical Field
The invention belongs to the field of biomass energy, and particularly relates to a method for reducing the activity of uridine diphosphate Xylose Epimerase (UDP-Xylose Epimerase, UXE) in a plant body by utilizing a biotechnology, wherein the plant subjected to UXE reduction has higher saccharification efficiency; in particular to a method for improving plant saccharification efficiency by down-regulating a UXE gene and application thereof.
Background
The biomass energy combines the advantages of fossil energy and new energy, and is one of energy with great reserves and reproducibility. The fertilizer is also environment-friendly due to the fact that the fertilizer is derived from plants or agricultural and forestry industrial wastes. The main use in the world is still traditional energy, the storage is limited, the regeneration is not realized, the environmental hazard is serious, and the improvement and the popularization of biological energy are urgently needed. How to develop biomass energy efficiently and reasonably has important significance for sustainable development of economy and society, and has become a hot spot of great tendency.
Lignocellulose is an important biomass energy source and is a main raw material for producing bioethanol. Cellulose and hemicellulose in lignocellulose are hydrolyzed and saccharified into simple sugars and finally fermented into ethanol, the saccharification efficiency is an important factor influencing the biomass yield, and the composition and the crosslinking mode of the lignocellulose directly influence the saccharification efficiency. Hemicellulose of gramineous plants is mainly Glucuronic acid arabinoxylan (GAX) and Arabinoxylan (AX) having Xylan (Xylan) as a main chain and Arabinose (arabinosine) and Glucuronic acid (Glucuronic acid) as side chains. Xylan is crosslinked with partial structures of cellulose and lignin in a plant secondary wall through arabinose side chains to improve the toughness, stress resistance and the like of the plant, but the existence of the xylan side chains also has great influence on the industrial application of lignocellulose. The cross-linked network structure formed by xylan around cellulose prevents the entry of cellulolytic enzymes, and not only does the degree of side chain branching affect saccharification efficiency. Uridine diphosphate Xylose (UDP-Xylose, UDP-Xyl) as a substrate synthesized by the xylan side chain is catalyzed by uridine diphosphate Xylose epimerase (UXE) to generate uridine diphosphate arabinose (UDP-arabinase, UDP-Ara), and the regulation of the UXE directly influences the branching degree of an arabinose side chain on a xylan main chain, further influences the connection of arabinose with lignin through a ferulic acid ester bond, influences the crosslinking of hemicellulose and cellulose, and finally changes the saccharification efficiency of plants.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for improving the plant saccharification efficiency by using CRISPR/Cas9 to down-regulate a UXE gene.
The invention improves the plant saccharification efficiency by changing the side chain content and the branching degree of the plant xylan. Based on the working experience of the inventors and the current need for biomass material improvement, the inventors believe that down-regulation of the key enzyme UXE for arabinose synthesis may directly lead to a reduction in arabinose content, thereby altering the composition and structure of lignocellulose. Analysis of all UXEs in the rice genome shows that 3 UXEs are in total in the rice genome, and online prediction of TMHMM shows that OsUXEs all have transmembrane regions and are positioned in Golgi bodies. The inventor designs a plurality of targets for three UXEs in rice respectively by using a CRISPR/CAS9 gene editing technology and knocks out genes of the targets. Finally, plants UXE1UXE2 and UXE1UXE3 with three genes mutated in pairs are obtained, and after the sequencing identification reliability, the research work of the inventor is carried out aiming at the two mutants.
Under the same growth environment, the UXE1UXE2 mutant and the UXE1UXE3 mutant both show the same growth phenotype as a normal rice plant used as a control, and the agronomic characters of the two mutants are statistically analyzed, so that the UXE1UXE2 mutant and the UXE1UXE3 mutant have no difference in plant height, leaf shape and spike number with the normal rice. Stalk sections from both mutants showed unchanged cell wall. Monosaccharide analysis of arabinose in the cell walls of the two mutants revealed that arabinose content in the mutants was significantly reduced, while glucose and xylose were not significantly changed. Moreover, the ratio of mutant lignin monomers is also obviously changed, S-type lignin is increased and is easier to be cracked, and the method is also one of the obvious reasons for influencing saccharification efficiency. The mutant material was analyzed for saccharification efficiency, both of which showed higher saccharification efficiency than the wild-type material. The experimental results show that the Golgi-positioned UXE mutation does not bring great influence on the growth and development of plants, but can reduce the content of xylan side chain-arabinose in the plants so as to improve the saccharification efficiency.
Another object of the present invention is to provide the use of the UXE down-regulated plants obtained by the above method.
The purpose of the invention is realized by the following technical scheme:
a method for improving plant saccharification efficiency by down-regulating a UXE gene, wherein the plant saccharification efficiency is improved by deleting or down-regulating the UXE gene function of the plant.
The UXE gene function down-regulation is preferably to down-regulate the UXE activity in the plant body by a genome editing technology (CRISPR-CAS 9) so as to achieve the purpose of improving the saccharification efficiency.
The plant is preferably rice (Oryza sativa), Arabidopsis thaliana (Arabidopsis thaliana), poplar (Populus), Eucalyptus (Eucalyptus), pine (Pinus), or Jatropha (Jatropha carcas L.) or the like; more preferably rice. But can be derived from other plants, and the UXE activity in the plants is down-regulated by a genome editing technology (CRISPR-CAS 9), so that the aim of improving the saccharification efficiency is fulfilled.
Bioinformatics analysis was performed on 3 UXE proteins in rice. Through sequence homology alignment analysis, the amino acid sequence similarity of OsUXE1, OsUXE2 and OsUXE3 is as follows: 81.24 percent. Transmembrane region analysis of 3 UXE proteins by TMHMM software shows that the OsUXEs all have transmembrane regions and are positioned in a Golgi apparatus.
Preferably, the UXE gene is at least one of OsUXE1, OsUXE2 and OsUXE 3; further comprises at least two of OsUXE1, OsUXE2 and OsUXE 3.
Phenotypic analysis was performed on mutants UXE1UXE2 and UXE1UXE 3. All mutants grew normally, the panicle number trait of the UXE1UXE3 mutant alone was shown to be weak, and all mutant xylem vessels were found to be normal by sectioning the basal stem segments of both mutants.
The cell wall components of mutants UXE1UXE2 and UXE1UXE3 were analyzed. Collecting the stem material of the mutant, and analyzing monosaccharide composition of the mutant material (1)
The xylan structures of the mutants UXE1UXE2 and UXE1UXE3 were analyzed. Hemicellulose of the mutant material was extracted by the 0.5M KOH method (Zhao X, Ouyang K, Gan S, Zeng W, Song L, Zhao S, Li J, Doblin MS, Bacic A, Chen XY, Marchant A, Deng X, Wu AM (2014) Biochemical and molecular dynamics associated with a heterologous biologically synthesized in Neomaracaulia cadamba (Rubiaceae) during xylogenesis. front Plant Sci 5: 602). Structural analysis of hemicellulose was performed using 2D-HSQC and 1H-NMR as well as FT-IR. The results show that in the OsUXEs mutant, the methylation degree of glucuronic acid (GlcA) is not obviously reduced, and the acetylation degree is reduced. The results show that arabinose of the two mutants is reduced obviously, the methylation degree and acetylation degree of xylan side chains are reduced, and the structure of hemicellulose is changed greatly.
The mutants UXE1UXE2 and UXE1UXE3 were analyzed for saccharification efficiency. Arabinose is crosslinked to lignin through ferulic acid, so that the entrance of cellulolytic enzyme is prevented from influencing saccharification efficiency, and the structure of the side chain of the obtained mutant xylan is greatly changed, so that the saccharification efficiency of the mutant is also greatly changed. After the mutants are subjected to enzymolysis by cellulose mixed enzyme, the content of the obtained monosaccharide is measured to calculate the saccharification efficiency, and the saccharification efficiencies of the two mutants are greatly improved.
In addition, Golgi-localized UXE is also present in plants such as Arabidopsis, poplar, eucalyptus, pine, and Jatropha curcas, and multiple mutants thereof have been expected to result in improved saccharification efficiency.
A UXE down-regulated plant is prepared by the above preparation method.
The UXE down-regulated plants are useful in the biomass conversion, pulp and paper industries.
Compared with the prior art, the invention has the following advantages and effects:
the invention has the advantages that the utilization efficiency of the biomass energy depends on the quality of the raw materials to a great extent, the available components of the raw materials are improved, the treatment cost of the raw materials is reduced, and the utilization efficiency of the biomass energy can be improved. The prior method mostly focuses on changing the pretreatment method of the material, has limited improvement degree of the saccharification efficiency, fundamentally changes the biomass characteristic of the material, fundamentally improves the saccharification efficiency, can save energy and reduce the labor intensity.
Drawings
FIG. 1 is an alignment chart of amino acid sequences of 3 OsUXE (OsUXE1, OsUXE2 and OsUXE3) proteins in rice.
FIG. 2 is a graph showing the prediction of the transmembrane domains of OsUXE1 and OsUXE2 proteins in rice.
FIG. 3 is a graph showing the prediction of the transmembrane region of OsUXE3 protein in rice.
FIG. 4 is a map schematic of pYLCRISPR/Cas 9-MH.
FIG. 5 is a graph showing the results of detection of rice mutants; wherein (a): schematic diagram of sequence mutation site; left is Wildtype (WT), middle is UXE1UXE2 or
FIG. 6 is a rice mutant and rice stem section slice; wherein WT and wildtype refer to No. 11 of rice medium flower, UXE1UXE2 refers to
FIG. 7 is a graph showing the results of real-time fluorescent quantitative PCR.
FIG. 8 is an analysis chart of monosaccharide components of cell walls, wherein UXE1UXE2 refers to
FIG. 9 is a graph of saccharification efficiency analysis, wherein UXE1UXE2 refers to
FIG. 10 is a nuclear magnetic map of xylan side chain methylation and acetylation analysis; wherein (a): mutant hemicellulose 2D-HSQC; (b) the method comprises the following steps Mutant hemicellulose H-NMR; (c) the method comprises the following steps Modifying and locally amplifying a hemicellulose side chain; WT refers to No. 11 rice medium flower; UXE1UXE2 refers to
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The experimental procedures for specific experimental conditions not specified in the following examples are generally performed according to conventional experimental conditions or according to experimental conditions recommended by the manufacturers. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.