CN116024232B - Application of HvBGlu3 gene in regulation and control of barley grain beta-glucan content - Google Patents
Application of HvBGlu3 gene in regulation and control of barley grain beta-glucan content Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses an application of HvBGlu3 gene in regulating and controlling the content of barley grain beta-glucan, wherein the nucleotide sequence of an open reading frame of the HvBGlu3 gene is shown as SEQ ID NO. 1; the regulation mode is to silence HvBGlu3 gene to raise the beta-glucan content of barley grain. According to the invention, the HvBGlu3 gene on the barley 3H chromosome is knocked out by using the CRISPR/Cas9 technology, the content of beta-glucan in grains of the obtained three homozygous mutant strains is obviously or extremely obviously increased, and the content of beta-glucan can be respectively increased by 5.56%,7.69% and 9.44% on the basis of wild type.
Description
Technical Field
The invention relates to the technical field of biological genetic engineering, in particular to application of HvBGlu3 gene in regulating and controlling the content of barley grain beta-glucan.
Background
Barley (Hordeum vulgare L.) is the fourth cereal crop in the world today, its planting area and total yield are inferior to rice, wheat, corn, have early maturing, adaptability is wide, nutrition is abundant etc. characteristics, wherein it is rich in beta-glucan that is one of its main characteristics. Beta-glucan is a non-cellulosic polysaccharide, mainly present in the aleurone layer of gramineae grains and the endosperm cell wall, which plays a different role in different uses of barley, which is a very important quality trait in barley. When barley is used for malting and beer brewing, beta-glucan is a bad property that increases wort viscosity, causes filtration difficulties (Mcclear and glenieholmes, 1985), and is susceptible to gel precipitation and deterioration of beer quality (Morgan and gotuard, 2013). When barley is used as a feed material, beta-glucan increases the viscosity of intestinal fluids of non-ruminant livestock and poultry animals, affecting digestion and absorption of the foodstuff, resulting in a decrease in the growth rate of the animal, and is therefore considered an animal dystrophy factor (Hessclman et al, 1982; wang et al, 1992). In contrast, when barley is used as a human diet, beta-glucan has a very good health-care function, and is known to have the functions of lowering blood sugar, lowering cholesterol, clearing the intestinal tract, improving immunity, and the like (Wang et al, 1992; martinez et al, 1992). Thus, the different uses of barley have quite different requirements for beta-glucan content, lower levels being required when barley is used in feed, malting and brewing, and higher levels being required as human diet (Houston et al, 2014).
Barley beta-glucan content is synthesized by the common regulation of cellulose synthase family members (CslF and CslH) (Burton et al, 2006; doblin et al, 2009), the CslF6 gene is the main gene known to synthesize beta-glucan content, however, the site of CslF6 gene is not identified in some QTL and whole genome association analysis (GWAS) studies about barley beta-glucan content, so we hypothesize that the regulation of some other genes eventually causes the difference of barley grain beta-glucan content, and so far the research about the gene regulating barley grain beta-glucan content is still very limited. The whole genome correlation analysis of the content of the barley grain beta-glucan is carried out to identify a candidate gene HvBGlu3 for regulating and controlling the content of the barley grain beta-glucan, the gene is positioned on a barley 3H chromosome (shown in figure 1), the function of the candidate gene is verified by further knocking out CRISPR/Cas9, and the content of the barley grain beta-glucan is obviously improved after the gene is knocked out. The research result provides a new view for elucidating the genetic regulation mechanism of the beta-glucan and also lays a certain theoretical foundation for genetic improvement of the barley grain beta-glucan. Therefore, the analysis and manipulation of the genes related to the beta-glucan content of the barley seeds are enhanced, and the method has wide application prospect in the field of barley genetic breeding.
Reference is made to:
Burton,R.A.,S.M.Wilson,H.Maria,A.J.Harvey,N.J.Shirley,M.Anne,B.A.Stone,E.J.Newbigin,B.Antony and G.B.Fincher(2006).Cellulose synthase-like CslF genes mediate the synthesis of cell wall(1,3；1,4)-beta-D-glucans.Science 311(5769):1940-1942.
Doblin,M.S.,F.A.Pettolino,S.M.Wilson,C.Rebecca,R.A.Burton,G.B.Fincher,N.Ed and B.Antony(2009).A barley cellulose synthase-like CSLH gene mediates
(1,3；1,4)-beta-D-glucan synthesis in transgenic Arabidopsis.Proceedings of the National Academy of Sciences of the United States of America 106(14):5996-6001.
Hesselman,K.,K.Elwinger and S.Thomke(1982).Influence of increasing levels ofβ-glucanase on the productive value of barley diets for broiler chickens.Animal Feed Science and Technology 7(4):351-358.
Martinez,V.M.,R.K.Newman and C.W.Newman(1992).Barley diets with different fat sources have hypocholesterolemic effects in chicks.Journal of Nutrition 122(5):1070-1076.
Mcclear,B.V.and M.Glennieholmes(1985).Enzymic quantification of(1→3)
(1→4)-beta-D-glucan in barley and malt.Journal of the Institute of Brewing 91(5):285-295.
Morgan,A.H.and P.G.Gothard(2013).A rapid,simple viscometric technique for indirect estimation of solubleβ-glucan content of raw barley.Journal of the Institute of Brewing 83(1):37-38.
Wang,L.,R.K.Newman,C.W.Newman and P.J.Hofer(1992).Barley beta-glucans alter intestinal viscosity and reduce plasma cholesterol concentrations in chicks.Journal of Nutrition122(11):2292.
disclosure of Invention
The invention provides a new application of HvBGlu3 gene in regulating and controlling the content of beta-glucan in barley seeds, and provides basis and theoretical guidance for cultivating barley varieties with different beta-glucan contents in the seeds.
The specific technical scheme is as follows:
the invention provides application of HvBGlu3 gene in regulating and controlling the content of barley grain beta-glucan, wherein the nucleotide sequence of an open reading frame of the HvBGlu3 gene is shown as SEQ ID NO. 1; the regulation mode is to increase the beta-glucan content of barley grains by silencing HvBGlu3 gene.
The silencing mode is as follows: base insertions or deletions of different fragment sizes at both target positions ultimately result in premature termination of amino acid encoding.
According to the invention, the correlation analysis is carried out on the content of the barley grain beta-glucan to identify a candidate gene (HvBGlu 3) for regulating and controlling the content of the barley grain beta-glucan, and the gene is positioned on a barley 3H chromosome (shown in figure 1); the function of the candidate gene is verified through CRISPR/Cas9 knockout, and the content of the barley grain beta-glucan is obviously improved after the gene is knocked out.
Further, the application comprises the following steps:
(1) Designing a target sequence sgRNA1 of the HvBGlu3 gene, and constructing a CRISPR/Cas9 vector;
(2) Constructing agrobacterium genetically engineered bacteria containing the CRISPR/Cas9 vector of step (1);
(3) And (3) transforming the agrobacterium engineering bacteria into young barley embryos, and culturing to obtain a homozygous mutant strain which does not contain the exogenous Cas9 protein and is stably inherited.
Further, the nucleotide sequence of the target sequence sgRNA1 is shown as SEQ ID NO.2 or SEQ ID NO. 3.
Still further, the CRISPR/Cas9 vector is derived from the target sequence sgRNA1 introduced into knockout vector pRGEB 32.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, the HvBGlu3 gene on the barley 3H chromosome is knocked out by using the CRISPR/Cas9 technology, the content of beta-glucan in grains of the obtained three homozygous mutant strains is obviously or extremely obviously increased, and the content of beta-glucan can be respectively increased by 5.56%,7.69% and 9.44% on the basis of wild type.
(2) The research result provides a new view for elucidating the genetic regulation mechanism of the beta-glucan and also lays a certain theoretical foundation for the genetic improvement of the barley grain beta-glucan; enhances analysis and manipulation of genes related to the content of the beta-glucan in the barley seeds, and has wide application prospect in the field of barley genetic breeding.
Drawings
FIG. 1 is a Manhattan plot (3H) of a whole genome correlation analysis of barley grain beta-glucan content.
FIG. 2 is a schematic diagram of HvBGlu3 gene editing transgene.
FIG. 3 is a comparison of grain β -glucan content of knockout plants versus wild type (GP) plants;
wherein CK represents a wild type (Golden Promise, GP) plant; 3-3-9,3-3-12 and 3-3-13 represent transgenic positive plants of different mutation types, respectively; * And shows that the control and transgenic plants differed significantly and very significantly, with a significant level of P <0.05.
FIG. 4 is a 7DPA grain transcription analysis of knock-out plants versus wild-type (GP) plants, with red pentagram showing a significantly enriched KEGG pathway.
FIG. 5 is a comparison of the grain sugar content of 7DPA of a knockout plant versus a wild type (GP) plant; * And shows that the control and transgenic plants differed significantly and very significantly, with a significant level of P <0.05.
Detailed Description
The invention will be further described with reference to the following examples, which are given by way of illustration only, but the scope of the invention is not limited thereto. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
EXAMPLE 1 cloning of HvBGlu3 Gene and coding region thereof
1. Cloning of HvBGlu3 Gene
After barley golden hope (GP) sprouted for one week, cutting about 2cm leaves for DNA extraction. Extracting DNA by using a CTAB method to obtain GP genome DNA for standby; obtaining the HvBGlu3 gene sequence from a plant genome database (https:// plants. Ensembl. Org/index. Html); GP genomic DNA was used as a templateMax Master Mix (Dye Plus) high-fidelity DNA polymerase (P525, vazyme, china) was used for gene amplification.
The amplification primers are as follows:
HvBGlu3-F：5’-AAGCACATCTCATCAGCACA-3’；
HvBGlu3-R：5’-CACCTAAACAAAAAGCATAA-3’；
PCR reaction System (50. Mu.L): 25. Mu.L of 2X Phanta Max Master Mix (Dye Plus), 2. Mu.L of each of the upstream and downstream primers (10. Mu.M), 2. Mu.L of cDNA (100 ng/. Mu.L), 19. Mu.L of sterilized double distilled water.
PCR procedure: pre-denaturation at 95℃for 3min, (denaturation at 95℃for 15s, tm-3℃for 15s, extension at 72℃for 60 s/Kb) 35 cycles, complete extension at 72℃for 5min. The amplified product was purified and sequenced, leaving a portion for use.
2. Cloning of HvBGlu3 Gene coding region
After the barley GP germinates for one week, the whole seedling is frozen by liquid nitrogen for standby. Extracting total RNA of barley by using a MiniBest plant RNA extraction kit (9769, takara, japan), wherein the extraction steps are shown in reference specifications, and RNase pollution is prevented in the operation process; the extracted RNA was assayed for purity and concentration using NanoDrop 2000 (Thermo Fisher Scientific inc., USA) and ready for use. First strand reverse transcription DNA synthesis was performed using the RNA reverse transcription kit PrimeScript II 1st Strand cDNASynthesis Kit (RR 037A, takara, japan) with total RNA as a template. Primers were designed based on the HvBGlu3 database reference sequence and gene cDNAs were amplified using high fidelity DNA polymerase. The amplification primer is the same as the gene cloning primer, and the amplification enzyme and the PCR reaction system are used in the same way as the procedure.
Example 2 construction and functional verification of knockout vector
1) Design of sgRNA
The gRNA target site of the HvBGlu3 gene is designed by using an online website (http:// skl. Scau. Edu. Cn/targetdiesign /), then off-target detection is carried out on the website (http:// www.rgenome.net/cas-offinder /), and finally two sgRNA sequences, namely the gRNA1, are determined by combining the target knockout position and the off-target detection of the sites: ACACATATTGCAATCCTTCC GGG; gRNA2: CCTATCTATGTGCAAGAGAA TGG.
2) Knock-out vector construction
Cloning and introducing the two sgRNAs in the steps into a linearized pRGEB32 expression vector, then transforming the recombinant vector into escherichia coli competent, plating the escherichia coli competent to LB culture medium containing 50 mug/ml kanamycin, and picking up a monoclonal after overnight culture to perform bacterial liquid PCR verification and sequencing verification (namely nucleotide sequences shown as SEQ ID No.2 and SEQ ID No. 3) by using primers F (5'-GACCATGATTACGCCAAGCTTAAGGAATCTTTAAACATACG-3') and R (5'-GGACCTGCAGGCATGCACGCGCTAAAAACGGACTAGC-3').
Positive clones with correct sequences were subjected to overnight culture to extract plasmids. Plasmid extraction kit was purchased from beijing Tiangen biochemical company and the extraction method was as described in the specification. Transforming the knocked-out recombinant vector with correct sequencing into agrobacterium tumefaciens AGL1 by a thermal activation method, and screening positive recombinants to obtain the AGL1 agrobacterium tumefaciens strain containing the knocked-out recombinant vector.
3) Acquisition of transgenic plants
The knocked-out recombinant vector containing SEQ ID No.2 and SEQ ID No.3 is transformed into young embryo of barley Golden Promise by agrobacterium mediation method, and tens of transgenic candidate plants are obtained after hygromycin screening. And sequentially extracting DNA of the transgenic candidate plant and the wild GP, and obtaining a transgenic positive plant by a PCR amplification and sequencing verification method, wherein the verification primers are F (5'-ACCCCAGGAAGTAGCAAGGT-3') and R (5'-TTCGCTCCATTCCTGCAGAG-3'), the amplified fragment size is 625bp, and the verified mutant plant is respectively propagated for two generations to obtain a stable strain.
4) Transgenic plant analysis
Three different mutation types of transgene materials, namely 3-3-9,3-3-12 and 3-3-13 (figure 2), are obtained through gene knockout, and have base insertions or deletions with different fragment sizes at two target positions, so that the premature termination of amino acid coding is finally caused. And then, carrying out measurement analysis on the beta-glucan content of the mature grains of the transgenic positive plants and the CK of the control plants, and determining by adopting a Megazyme kit.
Example 3 functional verification
1. Determination of grain beta-glucan content
Pulverizing the harvested mature barley seeds by a pulverizer, sieving by a 40-mesh sieve, and storing the obtained powder sample under a sealed drying condition for later use. Beta-glucan assays were performed using beta-glucan kit (K-BGLU) from Megazyme, inc., 3 biological replicates per material assay.
As a result, as shown in FIG. 3, the grain beta-glucan content of the transgenic positive plants was significantly or extremely significantly increased, and the grain beta-glucan content (3-3-13) of barley was increased by 9.44% at the highest (FIG. 3) compared with the control.
Through the analysis, the content of the beta-glucan in the barley seeds can be increased by knocking out HvBGlu3 in the barley, and the gene negatively regulates the content of the beta-glucan in the barley seeds.
2. Starch and sucrose metabolic pathways
Seeds 7 days after flower extraction (7 DPA), total RNA of mutant and wild type materials was extracted using plant RNA extraction kit (Takara, japan) according to the specification. After quality detection and purification of total RNA, RNA samples were reverse transcribed into cDNA using reverse transcription kit (Takara, japan), after which the company was commissioned to transcriptome library construction and sequencing of mutant and wild type materials.
As shown in fig. 4 and 5, transcriptome analysis of the grain (7 DPA) 7 days after flowers showed that the deletion of HvBGlu3 gene promoted the starch and sucrose metabolism pathways in the developing grain (fig. 4), thereby affecting the sugar metabolism level of the developing grain (fig. 5), and the 6 sugar content in the mutant system was significantly or very significantly increased and the 2 sugar content was significantly or very significantly decreased compared to the control, so we speculated that HvBGlu3 was affecting the accumulation of barley grain β -glucan in the mature period by affecting the starch and sucrose metabolism of the developing grain.
Thus, the barley grain beta-glucan content can be increased by terminating expression of the gene in barley plants.
The above-described embodiment is only a preferred embodiment of the present invention, and is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims. All nucleotide sequences which relate to the coding of the gene and biological materials which contain any one of the genes belong to the protection scope of the invention.
Claims (4)
- The application of the HvBGlu3 gene in regulating and controlling the content of the barley grain beta-glucan is characterized in that the nucleotide sequence of an open reading frame of the HvBGlu3 gene is shown as SEQ ID NO. 1; the regulation mode is to increase the beta-glucan content of barley grains by knocking out the HvBGlu3 gene.
- 2. The use according to claim 1, comprising the steps of:(1) Designing sgRNA of HvBGlu3 gene, and constructing a CRISPR/Cas9 vector;(2) Constructing agrobacterium genetically engineered bacteria containing the CRISPR/Cas9 vector of step (1);(3) And (3) transforming the agrobacterium genetic engineering bacteria into young barley embryos, and culturing to obtain a homozygous mutant strain which does not contain the exogenous Cas9 protein and is stably inherited.
- 3. The use according to claim 2, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID No.2 or in SEQ ID No. 3.
- 4. The use of claim 2, wherein the CRISPR/Cas9 vector is derived from the introduction of sgRNA into knockout vector pRGEB 32.
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| WO1992005258A1 (en) * | 1990-09-20 | 1992-04-02 | La Trobe University | Gene encoding barley enzyme |
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| CN114990135A (en) * | 2022-06-23 | 2022-09-02 | 浙江大学中原研究院 | Preparation method of transgenic rice with improved grain beta-glucan content |
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| JP5557205B2 (en) * | 2009-02-27 | 2014-07-23 | 独立行政法人農業・食品産業技術総合研究機構 | Barley β-glucan deletion gene, synthetic gene and use thereof |
| US9681620B2 (en) * | 2014-04-10 | 2017-06-20 | The United States Of America, As Represented By The Secretary Of Agriculture | Barley mutant lines having grain with ultra-high beta glucan content |
| WO2018148738A1 (en) * | 2017-02-13 | 2018-08-16 | General Mills, Inc. | Wheat lines, plants, and grain with increased beta-glucan content |
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| WO1992005258A1 (en) * | 1990-09-20 | 1992-04-02 | La Trobe University | Gene encoding barley enzyme |
| CN108823215A (en) * | 2018-07-06 | 2018-11-16 | 南京大学 | Gene relevant to rice yield and its application |
| CN114990135A (en) * | 2022-06-23 | 2022-09-02 | 浙江大学中原研究院 | Preparation method of transgenic rice with improved grain beta-glucan content |
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