CN117646025B - A method for preparing a gene-edited plant with altered starch component content in barley grains - Google Patents
A method for preparing a gene-edited plant with altered starch component content in barley grains Download PDFInfo
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- CN117646025B CN117646025B CN202311567246.2A CN202311567246A CN117646025B CN 117646025 B CN117646025 B CN 117646025B CN 202311567246 A CN202311567246 A CN 202311567246A CN 117646025 B CN117646025 B CN 117646025B
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Abstract
本发明提供了一种大麦籽粒中淀粉组分含量改变的基因编辑植株的制备方法，该方法包括利用基因编辑技术，编辑大麦淀粉分支酶基因的上游启动子元件，获得大麦籽粒中淀粉组分含量改变的基因编辑植株。本发明利用基因编辑技术在大麦淀粉分支酶基因的上游启动子元件中找到了会影响大麦籽粒中淀粉组分含量的关键位点，并由此提供了一套能够制备大麦籽粒中淀粉组分含量改变的基因编辑植株的方法，为调控大麦籽粒中淀粉组分提供了新手段。
The present invention provides a method for preparing a gene-edited plant with a changed starch component content in barley grains, the method comprising using gene editing technology to edit the upstream promoter element of the barley starch branching enzyme gene to obtain the gene-edited plant with a changed starch component content in the barley grains. The present invention uses gene editing technology to find key sites that affect the starch component content in barley grains in the upstream promoter element of the barley starch branching enzyme gene, and thus provides a set of methods for preparing gene-edited plants with a changed starch component content in barley grains, providing a new means for regulating the starch components in barley grains.
Description
Technical Field
The invention relates to the technical field of biological gene engineering, in particular to a preparation method of a gene editing plant with the content of starch components in barley grains changed.
Background
Barley (Hordeum vulgare l.) is the fourth largest cereal crop worldwide next to wheat, rice and maize. Starch is the largest chemical component of barley kernels. Starch in barley kernels can be classified into amylose and amylopectin according to their structure, and cereal crops of different uses have different requirements on the quantity and proportion of amylose and amylopectin. Therefore, crops with different starch components can meet different living demands.
Currently, genetically edited starch synthesis genes are commonly used to alter crop starch components. For example, gene editing SSSIIa (sex 6 mutant) reduced amylopectin content, whereas significant increase in amylose synthesis (Morell,M.K.,Kosar-Hashemi,B.,Cmiel,M.,Samuel,M.S.,Chandler,P.,Rahman,S.,Buleon,A.,Batey,I.L.,&Li,Z.(2003).Barley sex6 mutants lack starch synthase IIa activity and contain a starch with novel properties.The Plant Journal,34,173-185.Yang,Q.,Ding,J.,Feng,X.,Zhong,X.,Lan,J.,Tang,H.,Harwood,W.,Li,Z.,Guzmán,C.,Xu,Q.,Zhang,Y.,Jiang,Y.,Qi,P.,Deng,M.,Ma,J.,Wang,J.,Chen,G.,Lan,X.,Wei,Y.,Zheng,Y.,&Jiang,Q.(2022).Editing of the starch synthase IIa gene led to transcriptomic and metabolomic changes and high amylose starch in barley.Carbohydrate Polymers,285,119238.). in maize, grain amylose content of SBEIIb deletion mutant (amylose extender, ae-) increased (Kim,K.N.,Fisher,D.K.,Gao,M.,&Guiltinan,M.J.(1998).Molecular cloning and characterization of the amylose-extender gene encoding starch branching enzyme IIb in maize.Plant Molecular Biology,38,945-956.). but gene editing a gene only achieved unidirectional control of phenotype.
The technique of fine-controlling gene expression has been applied (Zeng,D.,Liu,T.,Ma,X.,Wang,B.,Zheng,Z.,Zhang,Y.,Xie,X.,Yang,B.,Zhao,Z.,Zhu,Q.,&Liu,Y.G.(2020).Quantitative regulation of Waxy expression by CRISPR/Cas9 based promoter and 5'UTR-intron editing improves grain quality in rice.Plant Biotechnology Journal,18,2385-2387.Huang,L.,Li,Q.,Zhang,C.,Chu,R.,Gu,Z.,Tan,H.,Zhao,D.,Fan,X.,&Liu,Q.(2020).Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system.Plant Biotechnology Journal,18,2164-2166.), to the rice wall gene, which does not directly edit the gene coding region, but rather finely controls gene expression by knocking out the promoter element of the gene. The gene editing promoter region makes the gene editing result more possible.
The SBEIIb gene plays an important role in the biosynthesis of amylopectin and is the only enzyme that can be used to generate branches. The promoter region comprises a plurality of cis-acting elements which play an important role, and the expression and the function of the elements on the SBEIIb gene are still unknown.
In accordance with the above requirements, the present invention provides a method for altering the starch component of barley kernels using a gene editing promoter.
Disclosure of Invention
The invention provides a preparation method of a gene editing plant with changed starch component content in barley grains, which can effectively change the starch component content in barley grains and provides a new means for regulating and controlling the starch component in barley grains.
The specific technical scheme is as follows:
the invention provides a preparation method of a gene editing plant with changed starch component content in barley grains, which comprises the step of editing an upstream promoter element of a barley starch branching enzyme gene by utilizing a gene editing technology to obtain the gene editing plant with changed starch component content in barley grains.
Further, the barley starch branching enzyme Gene is SBEIIb Gene, and the Gene number of the SBEIIb Gene in BARLEX Morex v Gene Models is HORVU Hr1G077120.
Further, the original nucleotide sequence of the upstream promoter is shown as SEQ ID NO.1, the upstream promoter is positioned in front of the CDS region of the SBEIIb gene and has a length of 2.0-kb, and the nucleotide sequence of the upstream promoter after gene editing is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.
SEQ ID NO.1：GATTGTTTTTCAACCCATGTGAGAGAGATGTGTGTGATTCATATAAGAG AGAAAATGGTATAACACCAAAGAAGACACGACTATTACAAAAGCCACATAACATGGCCCAACTAAAGAAAACAAACCATTGATGATCCGCATTGGCAATTAATAAACCGACAAAACCAACCTAACTAGAGCAAGGGCACCACATAGGACACCAAAGTAAGGGAACATTTTTACTGCATAAGACTACAATAGACGATAACACCATTCAAACACGACCCACATCGATTAAAACCCAAACAATACTTGAAGAATGCATGACATCACTGAGCAACGAGGCCACAAACCCAACAACGCTGGGCCAGCTGTCACCGAGGCCATAGAAGAAGGACTGAGGGGTGCATACGCATAAACCGAAAGGGAGTAGGAGGCACGAGAGCACCAAAGACAACGTCCACCACGACCGACAAAGATGACATCAACCATGATCAAGTACCGCAAGCAAGGGAGGATCGAGGAAAAGGCCGTTGGATACTCGAAATGACATTGGAGTATTTTTCTTGGATGACTCCAATTGGATGGCAATTGGTGGTGATGTCCATTTGGTCTTTAGTGATTCGCCCGAGATGCCAAGTTTCATAACCTCACACATCACACACACTCGCTAACACACTGACCCACTCGTCACTACTCTACCGTCCTACGTGGCACCTACTGCAAGGCGACGCCGTCCGACAATGTACCCCGACCCCTCCTGCTAAGGTTCCTTCCCCTCTCCCCGACCGCCACCGTCGGTCGCACTCACCCTGCTCCCCGCCCCCTCCCCCACCCCTCCCCCGTTGTCGCCCCTCGCCGCGTCCCCTGGAGGTGGCCGGGGCGGCAGCCCCCCTACTCCTCGTTGGCCTCCTCCCCCCTCCCTCCTGCTATTGCGTCCGAACGGCTACCGTCGTTGACAATGGAGGTTGAGCCCTCCCGTGTGCCAACGGTGCTGCTGTCCCTACTCCCGGCTCCTATTCGCCGCTATGCAGTCCTCATTTCGCGATGTCGTGGTGAGTCAACGTGGGGGCTTCAAGACCGTGTTGGCGCAGGGTGGTGATCGGTTTGGTGGCAGGCTCCGGAGCGTCCAAGGTGCTGGCAGGGGCGTAGCGAGGATTATCAACCATTAGGTTCAGCTCTACTGAAGAGTGGGTTCAGTTCTTTGCTTAGTCTTAAGCTAGTGTAAATCTATAAGGGACATGTCAAATTTCATTGGGTTCACTTGAACCCAATGCTAATGAGCTCGCTCCGCCCCTGGGTGCTGGTTGGGATCGGGGGAACCCCTATCGGCATGGCTGACGCTGACGCGGTGGCGCCTGTCGGTGCTGTCGGGCATTCCCGGAGGACGTCAGGGGCTACCCTTCCGCGTACTGGGGGAAATCCTTGGCAACAAGGTTGTCATTGTCGTGATCCTTCTTGAAGGCGTTGATTGGTACCGACGCTTTGGAATCTTGAAGCTTGGTGGAAGATGTTCGGTGGACGCAGCGATCGCGAGGCTTCTTAGTTTTTGTCAATTCGTCATTGTCAGCATTTATTTCTTTTGTACTTCATGTGTTGTTTTTTTTAGGCGTTGCTGTTTTCTTCACCACCTATCGTTAAATGTATTGCTTTGTAATAAAAAAGGGGAAACCCTTTTTCAACAAGGCGACGCCGTCCTGCAGCTACGCCACCGCAAAACCCCAAACCTCTTCGCCCCCATGCTACCATCCATGCAGCCGTCCGCCCGCGCGCGCGCGTTGGCCACGCCACCCGGTGGTGGCCAGGCCACCACGCCGCCACTCTCGCGTGAAGGCTCCGTCCGCTTCCTCCTAGTCCCACTCTCTGCCCGTGCTATATAGCATCCGCCCTCCGCCCCCTACCAATCTTAGCACACCCCCCTGCGCCTCCTCATTTCGCTCGCGTGGGTTTAAGCAGGAGACGAGGCGGGGTCAGTTGGGCGGTTGGGTTGGATCCGATCCGGCTGCGGCGGCAGCGACGAG.
SEQ ID NO.2：GATTGTTTTTCAACCCATGTGAGAGAGATGTGTGTGATTCATATAAGAG AGAAAATGGTATAACACCAAAGAAGACACGACTATTACAAAAGCCACATAACATGGCCCAACTAAAGAAAACAAACCATTGATGATCCGCATTGGCAATTAATAAACCGACAAAACCAACCTAACTAGAGCAAGGGCACCACATAGGACACCAAAGTAAGGGAACATTTTTACTGCATAAGACTACAATAGACGATAACACCATTCAAACACGACCCACATCGATTAAAACCC
AAACAATACTTGAAGAATGCATGACATCACTGAGCAACGAGGCCACAAACCCAACAAC
GCTGGGCCAGCTGTCACCGAGGCCATAGAAGAAGGACTGAGGGGTGCATACGCATAAA
CCGAAAGGGAGTAGGAGGCACGAGAGCACCAAAGACAACGTCCACCACGACCGACA
AAGATGACATCAACCATGATCAAGTACCGCAAGCAAGGGAGGATCGAGGAAAAGGCC
GTTGGATACTCGAAATGACATTGGAGTATTTTTCTTGGATGACTCCAATTGGATGGCAAT
TGGTGGTGATGTCCATTTGGTCTTTAGTGATTCGCCCGAGATGCCAAGTTTCATAACCTC
ACACATCACACACACTCGCTAACACACTGACCCACTCGTCACTACTCTACCGTCCTACG
TGGCACCTACTGCAAGGCGACGCCGTCCGACAATGTACCCCGACCCCTCCTGCTAAGG
TTCCTTCCCCTCTCCCCGACCGCCACCGTCGGTCGCACTCACCCTGCTCCCCGCCCCCT
CCCCCACCCCTCCCCCGTTGTCGCCCCTCGCCGCGTCCCCTGGAGGTGGCCGGGGCGG
CAGCCCCCCTACTCCTCGTTGGCCTCCTCCCCCCTCCCTCCTGCTATTGCGTCCGAACG
GCTACCGTCGTTGACAATGGAGGTTGAGCCCTCCCGTGTGCCAACGGTGCTGCTGTCC
CTACTCCCGGCTCCTATTCGCCGCTATGCAGTCCTCATTTCGCGATGTCGTGGTGAGTCA
ACGTGGGGGCTTCAAGACCGTGTTGGCGCAGGGTGGTGATCGGTTTGGTGGCAGGCTC
CGGAGCGTCCAAGGTGCTGGCAGGGGCGTAGCGAGGATTATCAACCATTAGGTTCAGC
TCTACTGAAGAGTGGGTTCAGTTCTTTGCTTAGTCTTAAGCTAGTGTAAATCTATAAGGG
ACATGTCAAATTTCATTGGGTTCACTTGAACCCAATGCTAATGAGCTCGCTCCGCCCCT
GGGTGCTGGTTGGGATCGGGGGAACCCCTATCGGCATGGCTGACGCTGACGCGGTGGC
GCCTGTCGGTGCTGTCGGGCATTCCCGGAGGACGTCAGGGGCTACCCTTCCGCGTACT
GGGGGAAATCCTTGGCAACAAGGTTGTCATTGTCGTGATCCTTCTTGAAGGCGTTGATT
GGTACCGACGCTTTGGAATCTTGAAGCTTGGTGGAAGATGTTCGGTGGACGCAGCGAT
CGCGAGGCTTCTTAGTTTTTGTCAATTCGTCATTGTCAGCATTTATTTCTTTTGTACTTCA
TGTGTTGTTTTTTTTAGGCGTTGCTGTTTTCTTCACCACCTATCGTTAAATGTATTGCTTT
GTAATAAAAAAGGGGAAACCCTTTTTCAACAAGGCGACGCCGTCCTGCAGCTACGCCA
CCGCAAAACCCCAAACCTCTTCGCCCCCATGCTACCATCCATGCAGCCGTCCGCCCGCG
CGCGCGCGTTGGCCACGCCACCCGGTGGTGGCCAGGCCACCACGCCGCCACTCTCGCT
GAAGGCTCCGTCCGCTTCCTCCTAGTCCCACTCTCTGCCCGTGCTATATAGCATCCGCCC
TCCGCCCCCTACCAATCTTAGCACACCCCCCTGCGCCTCCTCATTTCGCTCGCGTGGGTT
TAAGCAGGAGACGAGGCGGGGTCAGTTGGGCGGTTGGGTTGGATCCGATCCGGCTGCG
GCGGCAGCGACGAG
SEQ ID NO.3:GATTGTTTTTCAACCCATGTGAGAGAGATGTGTGTGATTCATATAAGAG
AGAAAATGGTATAACACCAAAGAAGACACGACTATTACAAAAGCCACATAACATGGCC
CAACTAAAGAAAACAAACCATTGATGATCCGCATTGGCAATTAATAAACCGACAAAAC
CAACCTAACTAGAGCAAGGGCACCACATAGGACACCAAAGTAAGGGAACATTTTTACT
GCATAAGACTACAATAGACGATAACACCATTCAAACACGACCCACATCGATTAAAACCC
AAACAATACTTGAAGAATGCATGACATCACTGAGCAACGAGGCCACAAACCCAACAAC
GCTGGGCCAGCTGTCACCGAGGCCATAGAAGAAGGACTGAGGGGTGCATACGCATAAA
CCGAAAGGGAGTAGGAGGCACGAGAGCACCAAAGACAACGTCCACCACGACCGACA
AAGATGACATCAACCATGATCAAGTACCGCAAGCAAGGGAGGATCGAGGAAAAGGCC
GTTGGATACTCGAAATGACATTGGAGTATTTTTCTTGGATGACTCCAATTGGATGGCAAT
TGGTGGTGATGTCCATTTGGTCTTTAGTGATTCGCCCGAGATGCCAAGTTTCATAACCTC
ACACATCACACACACTCGCTAACACACTGACCCACTCGTCACTACTCTACCGTCCTACG
TGGCACCTACTGCAAGGCGACGCCGTCCGACAATGTACCCCGACCCCTCCTGCTAAGG
TTCCTTCCCCTCTCCCCGACCGCCACCGTCGGTCGCACTCACCCTGCTCCCCGCCCCCT
CCCCCACCCCTCCCCCGTTGTCGCCCCTCGCCGCGTCCCCTGGAGGTGGCCGGGGCGG
CAGCCCCCCTACTCCTCGTTGGCCTCCTCCCCCCTCCCTCCTGCTATTGCGTCCGAACG
GCTACCGTCGTTGACAATGGAGGTTGAGCCCTCCCGTGTGCCAACGGTGCTGCTGTCC
CTACTCCCGGCTCCTATTCGCCGCTATGCAGTCCTCATTTCGCGATGTCGTGGTGAGTCA
ACGTGGGGGCTTCAAGACCGTGTTGGCGCAGGGTGGTGATCGGTTTGGTGGCAGGCTC
CGGAGCGTCCAAGGTGCTGGCAGGGGCGTAGCGAGGATTATCAACCATTAGGTTCAGC
TCTACTGAAGAGTGGGTTCAGTTCTTTGCTTAGTCTTAAGCTAGTGTAAATCTATAAGGG
ACATGTCAAATTTCATTGGGTTCACTTGAACCCAATGCTAATGAGCTCGCTCCGCCCCT
GGGTGCTGGTTGGGATCGGGGGAACCCCTATCGGCATGGCTGACGCTGACGCGGTGGC
GCCTGTCGGTGCTGTCGGGCATTCCCGGAGGACGTCAGGGGCTACCCTTCCGCGTACT
GGGGGAAATCCTTGGCAACAAGGTTGTCATTGTCGTGATCCTTCTTGAAGGCGTTGATT
GGTACCGACGCTTTGGAATCTTGAAGCTTGGTGGAAGATGTTCGGTGGACGCAGCGAT
CGCGAGGCTTCTTAGTTTTTGTCAATTCGTCATTGTCAGCATTTATTTCTTTTGTACTTCA
TGTGTTGTTTTTTTTAGGCGTTGCTGTTTTCTTCACCACCTATCGTTAAATGTATTGCTTT
GTAATAAAAAAGGGGAAACCCTTTTTCAACAAGGCGACGCCGTCCTGCAGCTACGCCA
CCGCAAAACCCCAAACCTCTTCGCCCCCATGCTACCATCCATGCAGCCGTCCGCCCGCG
CGCGCGCGTTGGCCACGCCACCCGGTGGTGGCCAGGCCACCACGCCGCCACTCTCTGA
AGGCTCCGTCCGCTTCCTCCTAGTCCCACTCTCTGCCCGTGCTATATAGCATCCGCCCTC
CGCCCCCTACCAATCTTAGCACACCCCCCTGCGCCTCCTCATTTCGCTCGCGTGGGTTTA
AGCAGGAGACGAGGCGGGGTCAGTTGGGCGGTTGGGTTGGATCCGATCCGGCTGCGG
CGGCAGCGACGAG
SEQ ID NO.4:GATTGTTTTTCAACCCATGTGAGAGAGATGTGTGTGATTCATATAAGAG
AGAAAATGGTATAACACCAAAGAAGACACGACTATTACAAAAGCCACATAACATGGCC
CAACTAAAGAAAACAAACCATTGATGATCCGCATTGGCAATTAATAAACCGACAAAAC
CAACCTAACTAGAGCAAGGGCACCACATAGGACACCAAAGTAAGGGAACATTTTTACT
GCATAAGACTACAATAGACGATAACACCATTCAAACACGACCCACATCGATTAAAACCC
AAACAATACTTGAAGAATGCATGACATCACTGAGCAACGAGGCCACAAACCCAACAAC
GCTGGGCCAGCTGTCACCGAGGCCATAGAAGAAGGACTGAGGGGTGCATACGCATAAA
CCGAAAGGGAGTAGGAGGCACGAGAGCACCAAAGACAACGTCCACCACGACCGACA
AAGATGACATCAACCATGATCAAGTACCGCAAGCAAGGGAGGATCGAGGAAAAGGCC
GTTGGATACTCGAAATGACATTGGAGTATTTTTCTTGGATGACTCCAATTGGATGGCAAT
TGGTGGTGATGTCCATTTGGTCTTTAGTGATTCGCCCGAGATGCCAAGTTTCATAACCTC
ACACATCACACACACTCGCTAACACACTGACCCACTCGTCACTACTCTACCGTCCTACG
TGGCACCTACTGCAAGGCGACGCCGTCCGACAATGTACCCCGACCCCTCCTGCTAAGG
TTCCTTCCCCTCTCCCCGACCGCCACCGTCGGTCGCACTCACCCTGCTCCCCGCCCCCT
CCCCCACCCCTCCCCCGTTGTCGCCCCTCGCCGCGTCCCCTGGAGGTGGCCGGGGCGG
CAGCCCCCCTACTCCTCGTTGGCCTCCTCCCCCCTCCCTCCTGCTATTGCGTCCGAACG
GCTACCGTCGTTGACAATGGAGGTTGAGCCCTCCCGTGTGCCAACGGTGCTGCTGTCC
CTACTCCCGGCTCCTATTCGCCGCTATGCAGTCCTCATTTCGCGATGTCGTGGTGAGTCA
ACGTGGGGGCTTCAAGACCGTGTTGGCGCAGGGTGGTGATCGGTTTGGTGGCAGGCTC
CGGAGCGTCCAAGGTGCTGGCAGGGGCGTAGCGAGGATTATCAACCATTAGGTTCAGC
TCTACTGAAGAGTGGGTTCAGTTCTTTGCTTAGTCTTAAGCTAGTGTAAATCTATAAGGG
ACATGTCAAATTTCATTGGGTTCACTTGAACCCAATGCTAATGAGCTCGCTCCGCCCCT
GGGTGCTGGTTGGGATCGGGGGAACCCCTATCGGCATGGCTGACGCTGACGCGGTGGC
GCCTGTCGGTGCTGTCGGGCATTCCCGGAGGACGTCAGGGGCTACCCTTCCGCGTACT
GGGGGAAATCCTTGGCAACAAGGTTGTCATTGTCGTGATCCTTCTTGAAGGCGTTGATT
GGTACCGACGCTTTGGAATCTTGAAGCTTGGTGGAAGATGTTCGGTGGACGCAGCGAT
CGCGAGGCTTCTTAGTTTTTGTCAATTCGTCATTGTCAGCATTTATTTCTTTTGTACTTCA
TGTGTTGTTTTTTTTAGGCGTTGCTGTTTTCTTCACCACCTATCGTTAAATGTATTGCTTT
GTAATAAAAAAGGGGAAACCCTTTTTCAACAAGGCGACGCCGTCCTGCAGCTACGCCA
CCGCAAAACCCCAAACCTCTTCGCCCCCATGCTACCATCCATGCAGCCGTCCGCCCGCG
CGCGCGCGTTGGCCACGCCACCCGGTGGTGGCCAGGCCACCACGCCGCCACTCTCGCG
TGAAGGCTCCGTCCGCTTCCTCCTAGTCCCACTCTCTGCCCGTGCTATATAGCATCCGCC
CTCCGCCCCCTACAATCTTAGCACACCCCCCTGCGCCTCCTCATTTCGCTCGCGTGGGTT
TAAGCAGGAGACGAGGCGGGGTCAGTTGGGCGGTTGGGTTGGATCCGATCCGGCTGCG
GCGGCAGCGACGAG
SEQ ID NO.5：GATTGTTTTTCAACCCATGTGAGAGAGATGTGTGTGATTCATATAAGAG AGAAAATGGTATAACACCAAAGAAGACACGACTATTACAAAAGCCACATAACATGGCCCAACTAAAGAAAACAAACCATTGATGATCCGCATTGGCAATTAATAAACCGACAAAACCAACCTAACTAGAGCAAGGGCACCACATAGGACACCAAAGTAAGGGAACATTTTTACTGCATAAGACTACAATAGACGATAACACCATTCAAACACGACCCACATCGATTAAAACCCAAACAATACTTGAAGAATGCATGACATCACTGAGCAACGAGGCCACAAACCCAACAACGCTGGGCCAGCTGTCACCGAGGCCATAGAAGAAGGACTGAGGGGTGCATACGCATAAACCGAAAGGGAGTAGGAGGCACGAGAGCACCAAAGACAACGTCCACCACGACCGACAAAGATGACATCAACCATGATCAAGTACCGCAAGCAAGGGAGGATCGAGGAAAAGGCCGTTGGATACTCGAAATGACATTGGAGTATTTTTCTTGGATGACTCCAATTGGATGGCAATTGGTGGTGATGTCCATTTGGTCTTTAGTGATTCGCCCGAGATGCCAAGTTTCATAACCTCACACATCACACACACTCGCTAACACACTGACCCACTCGTCACTACTCTACCGTCCTACGTGGCACCTACTGCAAGGCGACGCCGTCCGACAATGTACCCCGACCCCTCCTGCTAAGGTTCCTTCCCCTCTCCCCGACCGCCACCGTCGGTCGCACTCACCCTGCTCCCCGCCCCCTCCCCCACCCCTCCCCCGTTGTCGCCCCTCGCCGCGTCCCCTGGAGGTGGCCGGGGCGGCAGCCCCCCTACTCCTCGTTGGCCTCCTCCCCCCTCCCTCCTGCTATTGCGTCCGAACGGCTACCGTCGTTGACAATGGAGGTTGAGCCCTCCCGTGTGCCAACGGTGCTGCTGTCCCTACTCCCGGCTCCTATTCGCCGCTATGCAGTCCTCATTTCGCGATGTCGTGGTGAGTCAACGTGGGGGCTTCAAGACCGTGTTGGCGCAGGGTGGTGATCGGTTTGGTGGCAGGCTCCGGAGCGTCCAAGGTGCTGGCAGGGGCGTAGCGAGGATTATCAACCATTAGGTTCAGCTCTACTGAAGAGTGGGTTCAGTTCTTTGCTTAGTCTTAAGCTAGTGTAAATCTATAAGGGACATGTCAAATTTCATTGGGTTCACTTGAACCCAATGCTAATGAGCTCGCTCCGCCCCTGGGTGCTGGTTGGGATCGGGGGAACCCCTATCGGCATGGCTGACGCTGACGCGGTGGCGCCTGTCGGTGCTGTCGGGCATTCCCGGAGGACGTCAGGGGCTACCCTTCCGCGTACTGGGGGAAATCCTTGGCAACAAGGTTGTCATTGTCGTGATCCTTCTTGAAGGCGTTGATTGGTACCGACGCTTTGGAATCTTGAAGCTTGGTGGAAGATGTTCGGTGGACGCAGCGATCGCGAGGCTTCTTAGTTTTTGTCAATTCGTCATTGTCAGCATTTATTTCTTTTGTACTTCATGTGTTGTTTTTTTTAGGCGTTGCTGTTTTCTTCACCACCTATCGTTAAATGTATTGCTTTGTAATAAAAAAGGGGAAACCCTTTTTCAACAAGGCGACGCCGTCCTGCAGCTACGCCACCGCAAAACCCCAAACCTCTTCGCCCCCATGCTACCATCCATGCAGCCGTCCGCCCGCGCGCGCGCGTTGGCCACGCCACCCGGTGGTGGCCAGGCCACCACGCCGCCACTCTCGCGTGAAGGCTCCGTCCGCTTCCTCCTAGTCCCACTCTCTGCCCGTGCTATATAGCATCCGCCCTCCGCCCCCTACACACCCCCCTGCGCCTCCTCATTTCGCTCGCGTGGGTTTAAGCAGGAGACGAGGCGGGGTCAGTTGGGCGGTTGGGTTGGATCCGATCCGGCTGCGGCGGCAGCGACGAG.
Further, the editing target of the upstream promoter is a sequence at a position 2bp-25bp away from the 5' -end of the A-box element, or a sequence containing the CAAT-box element.
Further, the preparation method specifically comprises the following steps:
(1) Designing an sgRNA target site aiming at a promoter region upstream of a CDS region of a barley starch branching enzyme gene SBEIIb;
(2) Preparing a CRISPR/Cas9 gene editing vector according to the sgRNA target site designed in the step (1);
(3) Constructing a genetic engineering bacterium containing a CRISPR/Cas9 gene editing vector, converting the genetic engineering bacterium into a barley genetic transformation material, and screening by a vector marker gene to obtain a gene editing plant with the starch component content in barley grains free of exogenous genes changed.
In the method, the barley starch branching enzyme gene SBEIIb can be obtained through Geneious software assembly, the promoter region upstream of the CDS region can be obtained through PLANTCARE (http:// bioinformation information. Psb. Ugent. Be/webtools/plantcare/html) online software prediction, the sgRNA target site can be obtained through TARGETDESIGN (http:// skl. Scau. Edu. Cn/TARGETDESIGN /) website design, and the target miss rate and the like of the target point can be detected in crispor (http:// crispor. Tefor. Net/crispor. Py).
The genetic transformation process comprises embryo stripping, infection and differentiation culture, after genetic transformation, transgenic positive seedlings can be obtained, the transgenic positive seedlings need to be continuously planted to T2 generation, plants which do not contain carrier marker genes are obtained through carrier marker gene screening, and the transgenic positive seedlings which do not contain exogenous genes are obtained through target sequence verification and screening. The obtained transgenic positive seedlings can obtain the amylose and amylopectin content of the seeds through the determination of quality characters.
Further, the nucleotide sequence of the sgRNA is shown as SEQ ID NO.6, specifically ACGCCGCCACTCTCGCGTGAAGG, or as SEQ ID NO.7, specifically CCCTACCAATCTTAGCACACCCC.
The sequence shown as SEQ ID NO.6 is adopted as sgRNA, the amylose content of the finally obtained barley gene editing plant grain is obviously improved, and the amylopectin content is obviously reduced, while the sequence shown as SEQ ID NO.7 is adopted as sgRNA, the amylopectin content of the finally obtained barley gene editing plant grain is obviously improved, and the amylose content is obviously reduced.
The invention also provides a gene editing plant with the starch component content in the barley grains changed, which is prepared by the preparation method.
The invention also provides an upstream promoter sequence of the starch branching enzyme gene SBEIIb, and the nucleotide sequence of the promoter element is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.
The invention also provides a combined Gene, which comprises a barley starch branching enzyme Gene SBEIIb and an upstream promoter element thereof, wherein the Gene number of the barley starch branching enzyme Gene SBEIIb is HORVU Hr1G077120 at BARLEX Morex v Gene Models, the upstream promoter is positioned in front of the CDS region of the SBEIIb Gene and has the length of 2.0-kb, and the nucleotide sequence of the upstream promoter is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.
The invention also provides a genetically engineered bacterium, which comprises a CRISPR/Cas9 gene editing vector, wherein the sgRNA sequence in the gene editing vector is shown as SEQ ID NO.6 or SEQ ID NO.7, and the host cell of the genetically engineered bacterium is agrobacterium.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention utilizes the gene editing technology to find out the key site which can influence the starch component content in the barley grain in the upstream promoter region of the barley starch branching enzyme gene, and thus provides a set of method for preparing the gene editing plant with changed starch component content in the barley grain, and provides a new means for regulating and controlling the starch component in the barley grain.
(2) The invention also provides a corresponding upstream promoter element, a combined gene and a genetic engineering bacterium according to key sites influencing the starch component content in the barley grain, and provides more means and tools for realizing the starch component change of the barley grain.
Drawings
FIG. 1 shows the target site of the SBEIIb gene promoter region and the gene editing of the mutant.
FIG. 2 is a measurement of the starch trait of the gene editing barley SBEIIb promoter mutant.
FIG. 3 shows the expression of SBEIIb in control GP and mutant.
FIG. 4 shows the expression levels of different starch synthesis genes in the control and mutant during grain development.
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 construction of knockout vector and acquisition of Gene editing plants (SBEIIb promoter B3 target)
1) Prediction of SBEIIb Gene promoter element
The gene sequence of SBEIIb (HORVU Hr1G 077120) was assembled by Geneious software, and the first 2.0-kb of CDS region was selected as the promoter region of the gene, PLANTCARE was used
(Http:// bioinformation. Psb. Ugent. Be/webtools/plantcare/html) on-line software predicts the promoter elements of the gene.
2) Design of promoter knockout sgRNA
The sgRNA target site of the target SBEIIb gene was designed using the site at wire mesh station TARGETDESIGN (http:// skl. Scau. Edu. Cn/TARGETDESIGN /), and then the off-target rate was checked on site crispor (http:// crispor. Tefor. Net/crispor. Py), and the B3-sgRNA sequence was determined by the integrated knockout element and off-target detection, i.e.:
CCTCATTTCGCTCGCGTGGGTTT。
3) Construction of promoter knockout vector
① Linearizing U6 vectors with BsaIHF-V2 restriction enzymes (R3733L, NEB, U.S.), rapid CIP dephosphorylation of the digested products (M0525V, NEB, U.S.) to prevent vector self-ligation;
② The design target primer is B3-F CTTGAACCCACGCGAGCGAAATG and B3-R AAACCATTTCGC
TCGCGTGGGTT;
③ The target primers are respectively dissolved in TE buffer solution, the final concentration is 100 mu M/L, and the PCR instrument is used for annealing at 90 ℃ for 1-2min;
④ Adding T4 PNK (M0201S, NEB, U.S.) into the target primer annealing product to carry out phosphorylation treatment;
⑤ U6 linearized vectors and annealed phosphorylation products were ligated with T4 ligase (M0202L, NEB, USA) to obtain Cas9-U6-B3 knockout vectors.
⑥ The knock-out vector was transformed into competent E.coli DH5a using primers
P2-5'-GCGATTAAGTTGGGTAACGC-3' and cas9-B3-R were subjected to monoclonal validation and sequencing.
After sequencing, the plasmid is extracted to obtain cas9:B3 promoter editing vector.
2) Obtaining of Gene-edited plants
The constructed knockdown vector positive plasmids were transformed with Agrobacterium by heat shock method using commercial Agrobacterium competent cells
AGL1 (AC 1020S, indigenous organism, china) was used as the recipient strain.
The nucleotide sequence of the upstream promoter after gene editing is shown in SEQ ID NO. 68.
SEQ ID NO.68:GATTGTTTTTCAACCCATGTGAGAGAGATGTGTGTGATTCATATA
AGAGAGAAAATGGTATAACACCAAAGAAGACACGACTATTACAAAAGCCACATAACAT
GGCCCAACTAAAGAAAACAAACCATTGATGATCCGCATTGGCAATTAATAAACCGACA
AAACCAACCTAACTAGAGCAAGGGCACCACATAGGACACCAAAGTAAGGGAACATTTT
TACTGCATAAGACTACAATAGACGATAACACCATTCAAACACGACCCACATCGATTAAA
ACCCAAACAATACTTGAAGAATGCATGACATCACTGAGCAACGAGGCCACAAACCCAA
CAACGCTGGGCCAGCTGTCACCGAGGCCATAGAAGAAGGACTGAGGGGTGCATACGC
ATAAACCGAAAGGGAGTAGGAGGCACGAGAGCACCAAAGACAACGTCCACCACGACC
GACAAAGATGACATCAACCATGATCAAGTACCGCAAGCAAGGGAGGATCGAGGAAAA
GGCCGTTGGATACTCGAAATGACATTGGAGTATTTTTCTTGGATGACTCCAATTGGATGG
CAATTGGTGGTGATGTCCATTTGGTCTTTAGTGATTCGCCCGAGATGCCAAGTTTCATAA
CCTCACACATCACACACACTCGCTAACACACTGACCCACTCGTCACTACTCTACCGTCC
TACGTGGCACCTACTGCAAGGCGACGCCGTCCGACAATGTACCCCGACCCCTCCTGCTA
AGGTTCCTTCCCCTCTCCCCGACCGCCACCGTCGGTCGCACTCACCCTGCTCCCCGCCC
CCTCCCCCACCCCTCCCCCGTTGTCGCCCCTCGCCGCGTCCCCTGGAGGTGGCCGGGG
CGGCAGCCCCCCTACTCCTCGTTGGCCTCCTCCCCCCTCCCTCCTGCTATTGCGTCCGA
ACGGCTACCGTCGTTGACAATGGAGGTTGAGCCCTCCCGTGTGCCAACGGTGCTGCTG
TCCCTACTCCCGGCTCCTATTCGCCGCTATGCAGTCCTCATTTCGCGATGTCGTGGTGAG
TCAACGTGGGGGCTTCAAGACCGTGTTGGCGCAGGGTGGTGATCGGTTTGGTGGCAGG
CTCCGGAGCGTCCAAGGTGCTGGCAGGGGCGTAGCGAGGATTATCAACCATTAGGTTC
AGCTCTACTGAAGAGTGGGTTCAGTTCTTTGCTTAGTCTTAAGCTAGTGTAAATCTATAA
GGGACATGTCAAATTTCATTGGGTTCACTTGAACCCAATGCTAATGAGCTCGCTCCGCC
CCTGGGTGCTGGTTGGGATCGGGGGAACCCCTATCGGCATGGCTGACGCTGACGCGGT
GGCGCCTGTCGGTGCTGTCGGGCATTCCCGGAGGACGTCAGGGGCTACCCTTCCGCGT
ACTGGGGGAAATCCTTGGCAACAAGGTTGTCATTGTCGTGATCCTTCTTGAAGGCGTTG
ATTGGTACCGACGCTTTGGAATCTTGAAGCTTGGTGGAAGATGTTCGGTGGACGCAGC
GATCGCGAGGCTTCTTAGTTTTTGTCAATTCGTCATTGTCAGCATTTATTTCTTTTGTACT
TCATGTGTTGTTTTTTTTAGGCGTTGCTGTTTTCTTCACCACCTATCGTTAAATGTATTGC
TTTGTAATAAAAAAGGGGAAACCCTTTTTCAACAAGGCGACGCCGTCCTGCAGCTACG
CCACCGCAAAACCCCAAACCTCTTCGCCCCCATGCTACCATCCATGCAGCCGTCCGCCC
GCGCGCGCGCGTTGGCCACGCCACCCGGTGGTGGCCAGGCCACCACGCCGCCACTCTC
GCGTGAAGGCTCCGTCCGCTTCCTCCTAGTCCCACTCTCTGCCCGTGCTATATAGCATCC
GCCCTCCGCCCCCTACCAATCTTAGCACACCCCCCTGCGCCTCCTCATTCGCTCGCGTG
GGTTTAAGCAGGAGACGAGGCGGGGTCAGTTGGGCGGTTGGGTTGGATCCGATCCGGC
TGCGGCGGCAGCGACGAG
Genetic transformation is carried out on the isolated golden hope (GP) young embryo by using agrobacterium infection, and a gene editing plant is obtained after hygromycin screening culture. DNA of T 0 generation gene editing plant and wild leaf is extracted, target region is sequenced by using B357 primer, mutation type is detected, amplification length is 575bp, and the primer is B357-F5' -AGGCGTTGCTGTTTTCTT
CAC-3’;B357-R:5’-ACGAGAGAATGCGAGTGAGC-3’;
Continuously planting gene editing plants, extracting DNA from T2 generation plants, simultaneously screening by using HYG and Cas9 screening markers to obtain plants which do not contain the two marker genes, simultaneously detecting mutation sites by using a B357 primer, and finally screening to obtain the gene editing plants which do not contain exogenous genes. The primer is as follows, HYG-F is 5'-TAAATAGCTGCGCCGATGGT-3', and HYG-R:
5’-GGCGACCTCGTATTGGGAAT-3’;Cas9-F:5’-CCTGGCCCACATGATCAAGT-3’;
cas 9-R5'-TGTACTTCTCAGGCAGCTGC-3', and the T2 generation seeds can be used for the subsequent steps after positive identification. Example 2 construction of knockout vector and acquisition of Gene editing plants (SBEIIb promoter B5 target)
1) Prediction of 35SBEIIb Gene promoter element
The gene sequence of SBEIIb (HORVU Hr1G 077120) was assembled by Geneious software, and the first 2.0-kb of CDS region was selected as the promoter region of the gene, PLANTCARE was used
(Http:// bioinformation. Psb. Ugent. Be/webtools/plantcare/html) on-line software predicts the promoter elements of the gene.
2) Design of 40 promoter knockout sgRNA
The B5-sgRNA sequence was determined using the integrated knockout element and off-target detection using the sgRNA target site of the target SBEIIb gene designed on site at wire mesh station TARGETDESIGN (http:// skl. Scau. Edu. Cn/TARGETDESIGN /) followed by detection of off-target rate on site crispor (http:// crispor. Tefor. Net/crispor. Py), i.e., B5-sgRNA: ACGCCGCCACTCTCGCGTGAAGG
3) Construction of promoter knockout vector
① Linearizing U6 vectors with BsaIHF-V2 restriction enzymes (R3733L, NEB, U.S.), rapid CIP dephosphorylation of the digested products (M0525V, NEB, U.S.) to prevent vector self-ligation;
② The design target primer is B5-F CTTGCGCCGCCACTCTCGCGTGA and B5-R aaacTCACGCGA GAGTGGCGGCG;
③ The target primers are respectively dissolved in TE buffer solution, the final concentration is 100 mu M/L, and the PCR instrument is used for annealing at 90 ℃ for 1-2min;
④ Adding T4 PNK (M0201S, NEB, U.S.) into the target primer annealing product to carry out phosphorylation treatment;
⑤ U6 linearized vectors and annealed phosphorylation products were ligated with T4 ligase (M0202L, NEB, USA) to obtain Cas9-U6-B5 knockout vectors.
⑥ The knock-out vector was transformed into competent E.coli DH5a using primers
P2-5'-GCGATTAAGTTGGGTAACGC-3' and cas9-B5-R were subjected to monoclonal validation and sequencing.
After sequencing, the plasmid is extracted to obtain cas9:B5 promoter editing vector.
4) Obtaining of Gene-edited plants
The knockdown vector positive plasmids were transformed with agrobacteria by heat shock method, using commercial agrobacteria competent cell AG L1 (AC 1020S, plasmodium, china) as recipient strain. Genetic transformation is carried out on the isolated golden hope (GP) young embryo by using agrobacterium infection, and a gene editing plant is obtained after hygromycin screening culture. Extracting DNA of T 0 generation gene editing plants and wild type leaves, sequencing target areas by using a B357 primer, detecting mutation types, and amplifying the length to 575bp, wherein the primers are as follows:
B357-F:5’-AGGCGTTGCTGTTTTCTTCAC-3’;B357-R:5’-ACGAGAGAATGCGAGTGAG C-3’;
Continuously planting gene editing plants, extracting DNA from T2 generation plants, simultaneously screening by using HYG and Cas9 screening markers to obtain plants which do not contain the two marker genes, simultaneously detecting mutation sites by using a B357 primer, and finally screening to obtain the gene editing plants which do not contain exogenous genes. The primer is as follows, HYG-F is 5'-TAAATAGCTGCGCCGATGGT-3', and HYG-R:
5’-GGCGACCTCGTATTGGGAAT-3’;Cas9-F:5’-CCTGGCCCACATGATCAAGT-3’;
Cas 9-R5'-TGTACTTCTCAGGCAGCTGC-3', and the T2 generation seeds can be used for the subsequent steps after positive identification.
Example 3 construction of knockout vector and acquisition of Gene editing plants (SBEIIb promoter B7 target)
1) Prediction of SBEIIb Gene promoter element
The gene sequence of SBEIIb (HORVU Hr1G 077120) was assembled by Geneious software, and the first 2.0-kb of CDS region was selected as the promoter region of the gene, PLANTCARE was used
(Http:// bioinformation. Psb. Ugent. Be/webtools/plantcare/html) on-line software predicts the promoter elements of the gene.
2) Design of promoter knockout sgRNA
The sgRNA target site of the target SBEIIb gene was designed using the site at wire mesh station TARGETDESIGN (http:// skl. Scau. Edu. Cn/TARGETDESIGN /), then the off-target rate was detected on site crispor (http:// crispor. Tefor. Net/crispor. Py), and the two sgRNA sequences were determined by the integrated knockout element and off-target detection, namely, B7-sgRNA: CCCTACCAATCTTAGCACAC CCC;
3) Construction of promoter knockout vector
① Linearizing U6 vectors with BsaIHF-V2 restriction enzymes (R3733L, NEB, U.S.), rapid CIP dephosphorylation of the digested products (M0525V, NEB, U.S.) to prevent vector self-ligation;
② The design target primer is B7-F CTTGGGGTGTGCTAAGATTGGTA, B7-R aaacTACCAATCT TAGCACACCC;
③ The target primers are respectively dissolved in TE buffer solution, the final concentration is 100 mu M/L, and the PCR instrument is used for annealing at 90 ℃ for 1-2min;
④ Adding T4PNK (M0201S, NEB, U.S.) into the target primer annealing product to carry out phosphorylation treatment;
⑤ U6 linearized vectors and annealed phosphorylation products were ligated with T4 ligase (M0202L, NEB, USA) to obtain Cas9-U6-B7 knockout vectors.
⑥ The knock-out vector was transformed into competent E.coli DH5a using primers
P2-5'-GCGATTAAGTTGGGTAACGC-3' and cas9-B7-R were subjected to monoclonal validation and sequencing.
After sequencing, the plasmid is extracted to obtain cas9:B7 gene editing vector.
4) Obtaining of Gene-edited plants
The constructed knockdown vector positive plasmids were transformed with Agrobacterium by heat shock method using commercial Agrobacterium competent cells
AGL1 (AC 1020S, indigenous organism, china) was used as the recipient strain. And (3) carrying out genetic transformation by using agrobacterium infection in-vitro GP immature embryo, and obtaining a gene editing plant after screening and culturing hygromycin. Extracting DNA of T 0 generation gene editing plants and wild type leaves, sequencing target areas by using a B357 primer, detecting mutation types, and amplifying the length of 575bp, wherein the primers are as follows:
5’-AGGCGTTGCTGTTTTCTTCAC-3’;B357-R:5’-ACGAGAGAATGCGAGTGAGC-3’;
continuously planting gene editing plants, extracting DNA from T2 generation plants, simultaneously screening by using HYG and Cas9 screening markers to obtain plants which do not contain the two marker genes at the same time, simultaneously detecting mutation sites by using a B357 primer, and finally screening to obtain the gene editing plants which do not contain exogenous genes. The primer is as follows, HYG-F is 5'-TAAATAGCTGCGCCGATGGT-3', and HYG-R:
5’-GGCGACCTCGTATTGGGAAT-3’;Cas9-F:5’-CCTGGCCCACATGATCAAGT-3’;
5'-TGTACTTCTCAGGCAGCTGC-3', and after seed reproduction, enough transgenic seeds are harvested, and the T2 generation seeds can be used for the subsequent steps after positive identification.
Example 4 analysis of starch Properties of Gene-edited plants
T2 generation transgenic line subjected to gene knockout, B3 target knockout totally obtains 1 mutant B3M 1 (1 base deletion at target), and B5 target knockout totally obtains 2 mutants B5M 1 (1 base deletion at target) and B5M 2
(3 Base deletion at target point), B7 target point knockout to obtain 2 mutants, namely B7M 1 (1 base deletion at target point) and B7M 2 (10 base deletion at target point) (figure 1).
And then, measuring and analyzing the starch content of the CK mature grains of the mutant and the control plants, wherein the starch content comprises the total starch content, the amylose content and the amylopectin content, and the measuring method adopts Megazyme K-TSTA and K-AMYL kit.
As a result of analysis, compared with control CK, the contents of B3M 1 amylose and amylopectin are not significantly changed, the contents of B5M 1 and B5M 2 amylose are extremely significantly increased by 36.53% and 41.64%, respectively, and the contents of amylopectin are extremely significantly reduced by 42.84%
And 39.90%, the B7M 1 and B7M 2 amylose content was extremely significantly reduced by 33.27% and 24.97%, respectively, and the amylopectin content was extremely significantly increased by 40.17% and 44.95%, respectively (FIG. 2).
Example 5 functional verification of SBEIIb Gene
1) Planting and sampling of 30 barley materials
The control GP and the mutants B5M 1, B5M 2, B7M 1 and B7M 2 are simultaneously planted in a plant growth chamber, the temperature of the plant growth chamber is 22 ℃ in the daytime, 18 ℃ at night, the photoperiod is 14h in the daytime and 10h at night, the light intensity is 300 mu M-M -2s-1, and the planted soil is a mixture of peat and vermiculite, and the ratio is 9:1. Sampling barley seeds 7 days, 14 days, 21 days and 28 days after flowers, cutting off the snapping seeds, removing the awns, rapidly placing the seeds into a 15mL centrifuge tube after liquid nitrogen precooling, and storing at-80 ℃ after liquid nitrogen quick freezing. RNA contamination is avoided in the whole sampling process.
2) Extraction, reverse transcription and fluorescent quantitative PCR of RNA
Total RNA was extracted using Quick RNA extraction kit (Hua-Vietnam Biotechnology Co., ltd., beijing, china), cDNA was synthesized according to the reverse transcription kit (Takara, japan) instruction, and the housekeeping gene 3-phosphoglyceraldehyde dehydrogenase gene was used as an internal reference, and the amplification sequence was GAPDH-F:5'-GTGAGGCTGGTGCTGATTACG-3', GAPDH-R:
5’-TGGTGCAGCTAGCATTTGAGAC-3’;
The expression level of SBEIIb gene and other starch synthesis related genes including AGPS, AGPL, GBSSI, SSI, SSIIa, SSIIIa, SSIIIb, SSIVa, SBEI, SBEIIa, SBEIIIa, SBEIIIb, ISAI, ISAII, ISAIII, PUL were determined in controls and mutants. The primer list used is as shown in Table 1.
TABLE 1 list of primers required for gene expression level measurement.
QRT-PCR reactions were performed using a TB Green TMPreMix Ex TaqTM II kit (Takara, japan) and a LightCycler480 fluorescent quantitative PCR apparatus (Roche, switzerland) with a procedure of 95℃pre-denaturation 90s,95℃denaturation 5s,60℃annealing 20s,40 cycles. The response Ct value of each gene was determined from the amplification curve, and the relative gene expression level was calculated by the 2-DeltaCt method (Schefe et al, 2006). Each value was assayed for 4 biological replicates.
The analysis result shows that the expression quantity of SBEIIb is higher in the early stage of grain development, and the expression quantity is obviously reduced along with the development progress, which shows that the gene has functions in the early stage of grain development (figure 3 a). Compared with GP, the expression level of the B7M 1 and B7M 2 mutants was significantly higher and the expression level of the B5M 2 mutant was significantly lower 7 days after flowers, and the difference of the expression level of SBEIIb was substantially consistent with the difference of the final AP content (figure 2). The overall decrease in SBEIIb expression in the B5 mutant and the overall increase in the B7 mutant (fig. 3B) resulted in a significant decrease in AP content in the B5 mutant and a significant increase in the B7 mutant (fig. 2) throughout the whole grain development period (7 DAF-21 DAF).
Q-PCR was performed on 16 starch synthesis genes. The gene expression profile showed that the genes involved in starch synthesis in the mutant were significantly altered, indicating that starch synthesis was indeed co-regulated by many synthetic genes (FIG. 4). The determination analysis shows that the SBEIIb promoter B5 target spot is knocked out to reduce the gene expression of SBEIIb, thereby reducing the amylopectin content, and the SBEIIb promoter B7 target spot is knocked out to reduce the gene expression of SBEIIb, thereby improving the amylopectin content. Therefore, the B5 or B7 target spot can be knocked out to change the starch component of the barley grain.
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)
1. A preparation method of a gene editing plant with changed starch component content in barley grains is characterized by comprising the steps of editing an upstream promoter element of a barley starch branching enzyme gene by utilizing a gene editing technology to obtain the gene editing plant with changed starch component content in barley grains;
The barley starch branching enzyme Gene is SBEIIb Gene, and the Gene number of the SBEIIb Gene in BARLEX Morex v Gene Models is HORVU Hr1G077120;
The original nucleotide sequence of the upstream promoter region is shown as SEQ ID NO.1, and the length of the upstream promoter region is 2.0-kb before the CDS region of the SBEIIb gene;
the nucleotide sequence of the upstream promoter after gene editing is shown as SEQ ID NO.2 and SEQ ID NO.3, and the starch component content is changed into that the amylose content of barley gene editing plant grains is obviously improved and the amylopectin content is obviously reduced;
Or the nucleotide sequence of the upstream promoter after gene editing is shown as SEQ ID NO.4 or SEQ ID NO.5, and the starch component content is changed into that the amylopectin content of barley gene editing plant grains is obviously improved and the amylose content is obviously reduced.
2. The method for preparing a gene-edited plant with altered starch content in barley grain according to claim 1, wherein the editing target of the upstream promoter is a sequence at a position 2bp to 25bp away from the 5' -end of the a-box element or a sequence comprising the CAAT-box element.
3. The method for preparing a gene-edited plant with altered starch content in barley grain according to claim 1, comprising the steps of:
(1) Designing an sgRNA target site aiming at a promoter region upstream of a CDS region of a barley starch branching enzyme gene SBEIIb;
(2) Preparing a CRISPR/Cas9 gene editing vector according to the sgRNA target site designed in the step (1);
(3) Constructing a genetic engineering bacterium containing a CRISPR/Cas9 gene editing vector, converting the genetic engineering bacterium into a barley genetic transformation material, and screening by a vector marker gene to obtain a gene editing plant with the starch component content in barley grains free of exogenous genes changed.
4. The method for preparing a gene editing plant with altered starch content in barley grain according to claim 3, wherein the nucleotide sequence of sgRNA is shown as SEQ ID NO.6 or SEQ ID NO. 7.
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