Method for improving resistant starch content of wheat through genome editing and technical system thereof
阅读说明：本技术 一种通过基因组编辑提高小麦抗性淀粉含量的方法及其技术体系 (Method for improving resistant starch content of wheat through genome editing and technical system thereof ) 是由 夏兰琴 李晶莹 马有志 孙永伟 陈隽 于 2019-11-01 设计创作，主要内容包括：本发明公开了一种通过基因组编辑提高小麦抗性淀粉含量的方法及其技术体系。本发明提供了一种通过基因编辑技术提高小麦种子中的抗性淀粉含量和/或直链淀粉含量和/或总戊聚糖含量的方法,包括如下步骤：降低小麦中SBEIIa蛋白的丰度。所述“降低小麦中SBEIIa蛋白的丰度”具体可通过对SBEIIa基因进行基因编辑实现。本发明的发明人利用CRISPR/Cas9技术,定点编辑小麦SBEIIa基因,通过造成移码突变,敲除了小麦SBEIIa基因,获得了直链淀粉、抗性淀粉以及总戊聚糖(健康纤维)含量明显提高的新一代小麦新种质。本发明对于小麦育种具有重大的应用推广价值。(The invention discloses a method for improving the resistant starch content of wheat by genome editing and a technical system thereof. The invention provides a method for improving resistant starch content and/or amylose content and/or total pentosan content in wheat seeds by a gene editing technology, which comprises the following steps: reducing the abundance of SBEIIa protein in wheat. The "reduction of the abundance of the SBEIIa protein in wheat" can be specifically realized by gene editing of the SBEIIa gene. The invention utilizes CRISPR/Cas9 technology to edit wheat SBEIIa gene at fixed point, knocks out the wheat SBEIIa gene by causing frameshift mutation, and obtains a new generation of wheat new germplasm with obviously improved amylose, resistant starch and total pentosan (healthy fiber) content. The invention has great application and popularization value for wheat breeding.)
1. A method of increasing the resistant starch content and/or amylose content and/or total pentosan content in wheat seeds comprising the steps of: reducing the abundance of SBEIIa protein in wheat.
2. A method of increasing the resistant starch content and/or amylose content and/or total pentosan content in wheat seeds comprising the steps of: and (4) carrying out gene editing on the SBEIIa gene.
3. The method of claim 2, wherein: the gene editing is realized by means of a CRISPR/Cas9 system.
4. The method of claim 3, wherein: in the CRISPR/Cas9 system, the target sequences of sgrnas are as follows: tcctgagccgcgcggcctct are provided.
5. The method of claim 3, wherein: in the CRISPR/Cas9 system, the target sequences of sgrnas are as follows: gggaaggtcctggtgcctga are provided.
6. A specific sgRNA or a specific recombinant plasmid;
the specific sgRNA is sgRNA1 or sgRNA 2;
the target sequences of sgRNA1 are as follows: tcctgagccgcgcggcctct, respectively;
the target sequences of sgRNA2 are as follows: gggaaggtcctggtgcctga, respectively;
the specific recombinant plasmid is pCXUN-Cas9-gRNA1 or pCXUN-Cas9-gRNA 2;
pCXUN-Cas9-gRNA1 contains a gene encoding a Cas9 protein and a gene encoding the sgRNA 1;
pCXUN-Cas9-gRNA2 contains the gene encoding the Cas9 protein and the gene encoding the sgRNA 2.
7. A method of producing transgenic wheat comprising the steps of: the coding gene of the specific sgRNA of claim 6 and the coding gene of the Cas9 protein are introduced into receptor wheat to obtain transgenic wheat with the resistant starch content and/or the amylose content and/or the total pentosan content higher than that of the receptor wheat in seeds.
8. A method of preparing gene-edited wheat comprising the steps of: introducing the coding gene of the specific sgRNA of claim 6 and the coding gene of the Cas9 protein into recipient wheat to obtain transgenic wheat; then selfing the transgenic wheat to obtain selfed progeny; then screening the genetic editing wheat from the selfing progeny; the content of resistant starch and/or amylose and/or total pentosan in the wheat seeds edited by the gene is higher than that of the receptor wheat.
9. Use of the specific sgRNA of claim 6 or the specific recombinant plasmid of claim 6 in wheat breeding; the aim of the wheat breeding is to increase the resistant starch content and/or the amylose content and/or the total pentosan content in wheat seeds.
10. A method of increasing the resistant starch content and/or amylose content and/or total pentosan content in wheat seeds comprising the steps of: inhibiting the activity of SBEIIa protein in wheat.
Technical Field
The invention belongs to the technical field of biology, and relates to a method for improving the content of resistant starch in wheat by genome editing and a technical system thereof.
Background
Diabetes, obesity and colon cancer are chronic non-infectious diseases that seriously harm human health. Diabetes Mellitus (DM) is a disease caused by partial or complete insulin loss, or decreased cellular insulin receptors, or decreased receptor sensitivity, and is a chronic, systemic metabolic disease caused by the combined action of genetic and environmental factors. Diabetes and its complications have become a worldwide public health problem seriously harming human health, and have attracted high attention from countries in the world. According to 2004 reports of the national institute of statistics, the number of people suffering from diabetes in China is over 2000 ten thousand. According to the prediction of the world health organization, the number of diabetic patients in China is doubled by 2030, and the number of diabetic patients reaches 4230 ten thousand. Diabetes has been classified as a3 rd chronic non-infectious disease threatening human health following cardiovascular disease, a tumor. Abnormal metabolism of blood sugar caused by diabetes often causes metabolic disorder of blood fat, and hyperlipidemia occurs. These two factors can cause the increase of blood viscosity and slow blood flow, and are easy to form thrombus and arteriosclerosis, thus causing vascular diseases and causing a plurality of serious chronic complications. Epidemiological evidence strongly suggests a correlation between blood glucose levels, atherogenesis, occurrence of cardiovascular events, and increased morbidity and mortality.
Resistant starch (also known as resistant starch and indigestible starch) is mainly present in high amylose, low amylopectin kernels or tubers, and its content is directly and positively correlated with the high amylose content. Research shows that the resistant starch cannot be digested and absorbed in the small intestine and provide glucose, and can directly enter the large intestine of a human body and be fermented by physiological bacteria to generate a plurality of short-chain fatty acids (butyric acid and the like) and gases. In addition, the resistant starch has the functions of stimulating the growth of beneficial flora, reducing human body caloric intake, controlling body weight and the like. In the research of mice and human beings, the resistant starch can prevent colorectal cancer and improve the content of short-chain fatty acid in the large intestine. High amylose starch (resistant starch) helps prevent the development of irreversible insulin resistance and reduces the concentration of plasma total lipid, cholesterol and triglycerides. Mantis et al have shown that resistant starch can promote lipid oxidation after meal, and reduce fat accumulation after long-term consumption, and help to control body weight. The high-resistance starch food is ingested by a human body, has less insulin response, can delay the rise of blood sugar after meal, and effectively controls the state of diabetes. The relevance of the blood sugar and blood fat level of the type II diabetic rat and the resistant starch is researched by the tylophora fimbriata and the like, and the resistant starch can reduce the blood sugar and blood fat level and urea nitrogen of the type II diabetic rat, so that the resistant starch is prompted to have the effect of relieving the symptoms of diabetes and possibly protecting the kidney function. The metabolism of resistant starch and the regulation effect on blood sugar researched by king bamboo and the like prove that the resistant starch has the metabolic characteristic of slow absorption, has certain effects on regulating the blood sugar steady state, reducing postprandial insulin secretion and enhancing insulin sensitivity, preliminarily discusses the influence of the resistant starch on glucose transport in a postprandial body, synthesizes other research results, predicts that the resistant starch is possibly beneficial to preventing chronic diseases and reducing postprandial tissue load. In 1992, the world Food and Agriculture Organization (FAO) defined resistant starch as: starch and its degradation products, which are not absorbed in the small intestine of healthy people. In addition, the resistant starch has processing characteristics such as low water holding capacity, and can be used for improving the processing technology of food, increasing the crispness and the expansibility of the food and improving the texture of the final product. Therefore, the functional dietary fiber can be used as a functional component of dietary fiber of food, and can be added into food in a proper amount to prepare flavor food and functional food with different characteristics. At present, resistant starch is applied to flour foods, such as bread, steamed stuffed bun, macaroni, biscuits and the like, as a food raw ingredient or a dietary fiber enhancer abroad. Among them, the use of resistant starch in bread is worth mentioning. The bread added with the resistant starch not only strengthens the dietary fiber components, but also is better than the nutrition-strengthened bread added with other traditional dietary fibers in the aspects of the sensory quality such as pore structure, uniformity, volume, color and the like. The addition of the resistant starch into macaroni and noodles can increase the boiling resistance, facilitate the maintenance of a tough structure and avoid the adhesion phenomenon after boiling. Therefore, the cultivation of the high-resistance starch wheat has important significance for human health and rich dietary diversity.
Common wheat (Triticum aestivum l., AABBDD,2n ═ 6x ═ 42) is an important food crop, and wheat is taken as a staple food in over 40% of the population worldwide. It is the main source of protein intake for human, and is rich in vitamin B, vitamin E, fiber, magnesium, phosphorus, etc. Starch is the main component of wheat grains and accounts for more than 80% of the weight of wheat endosperm. Starch is a granule having crystalline and amorphous regions formed by the arrangement and stacking of amylose (amylose) and amylopectin (amylopectin), wherein amylose accounts for 15% -25% of endosperm starch, and amylopectin accounts for 75% -85%. Wheat starch can be classified into type A, type B and type C starches according to the form and size. Wherein, the A-type starch is generally round and has a particle diameter of 10-35 μm, the B-type starch is generally spherical or polygonal and has a particle diameter of 3-10 μm, and the C-type starch particle is generally smaller than 3 μm. The starch synthesis process is regulated by a series of enzymes.
Disclosure of Invention
The invention aims to provide a method for improving the resistant starch content of wheat by genome editing and a technical system thereof.
The invention provides a method for improving resistant starch content and/or amylose content and/or total pentosan content in wheat seeds, which comprises the following steps: reducing the abundance of SBEIIa protein in wheat.
The "reduction of the abundance of the SBEIIa protein in wheat" can be specifically achieved by inhibiting the expression of the SBEIIa gene. The "reduction of the abundance of the SBEIIa protein in wheat" can be specifically realized by knocking out the SBEIIa gene. The knockout includes the knockout of the entire gene, as well as the knockout of a partial segment of the gene.
The "reducing the abundance of the SBEIIa protein in wheat" can be specifically achieved by silencing the SBEIIa gene.
The "reduction of the abundance of the SBEIIa protein in wheat" can be specifically realized by gene editing of the SBEIIa gene.
The invention provides a method for improving resistant starch content and/or amylose content and/or total pentosan content in wheat seeds, which comprises the following steps: and (4) carrying out gene editing on the SBEIIa gene.
Any of the above gene editing is realized by means of a CRISPR/Cas9 system.
In the CRISPR/Cas9 system, the target sequence of sgRNA (sgRNA1) is as follows: tcctgagccgcgcggcctct are provided.
In the CRISPR/Cas9 system, the target sequence of sgRNA (sgRNA2) is as follows: gggaaggtcctggtgcctga are provided.
The gene editing is realized by introducing a specific DNA molecule containing a coding gene of Cas9 protein and a coding gene of sgRNA1 into recipient wheat. The gene editing is realized by introducing a recombinant plasmid containing the specific DNA molecule into receptor wheat.
The gene editing is realized by introducing a DNA molecule containing a coding gene of Cas9 protein and a DNA molecule containing a coding gene of sgRNA1 into recipient wheat respectively.
The gene editing is realized by introducing a specific DNA molecule containing a coding gene of Cas9 protein and a coding gene of sgRNA2 into recipient wheat. The gene editing is realized by introducing a recombinant plasmid containing the specific DNA molecule into receptor wheat.
The gene editing is realized by introducing a DNA molecule containing a coding gene of Cas9 protein and a DNA molecule containing a coding gene of sgRNA2 into recipient wheat respectively.
The invention also protects specific sgrnas. The specific sgRNA is sgRNA1 or
The invention also protects the specific recombinant plasmid. The specific recombinant plasmid is pCXUN-Cas9-gRNA1 or pCXUN-Cas9-
The target sequences of sgRNA1 are as follows: tcctgagccgcgcggcctct are provided.
The target sequences of sgRNA2 are as follows: gggaaggtcctggtgcctga are provided.
pCXUN-Cas9-gRNA1 contains the gene encoding the Cas9 protein and the gene encoding the
pCXUN-Cas9-gRNA2 contains the gene encoding the Cas9 protein and the gene encoding the
The encoding gene of the Cas9 protein can be specifically a DNA molecule which is reversely complementary with the 392-4522 th nucleotide in the
The pCXUN-Cas9-gRNA1 can be specifically shown as a
The difference between pCXUN-Cas9-gRNA2 compared to pCXUN-Cas9-gRNA1 is only that "AGAGGCCGCGCGGCTCAGGA" is replaced with "GGGAAGGTCCTGGTGCCTGA".
The invention also provides a method for preparing transgenic wheat, which comprises the following steps: and (3) introducing the coding gene of the specific sgRNA and the coding gene of the Cas9 protein into receptor wheat to obtain transgenic wheat of which the resistant starch content and/or the amylose content and/or the total pentosan content in seeds are higher than those of the receptor wheat. The encoding gene of the specific sgRNA and the encoding gene of the Cas9 protein are specifically introduced into receptor wheat through the recombinant plasmid.
The invention also provides a method for preparing gene-edited wheat, which comprises the following steps: introducing the coding gene of the specific sgRNA and the coding gene of the Cas9 protein into receptor wheat to obtain transgenic wheat, and identifying the wheat with gene editing and the gene editing type thereof (screening the wheat with gene editing and SBEIIa gene expression inhibited from the transgenic wheat); then, the transgenic gene is edited and wheat is selfed to obtain selfed progeny; then screening the selfed progeny for a gene-edited wheat without transgene, wherein the resistant starch content and/or amylose content and/or total pentosan content of the wheat seeds without transgene is higher than that of the receptor wheat. The encoding gene of the specific sgRNA and the encoding gene of the Cas9 protein are specifically introduced into receptor wheat through the recombinant plasmid.
The gene editing wheat is wheat which meets the following conditions: the A genome, the B genome and the D genome are all mutated in target regions and are all homozygous mutants.
The gene editing wheat is wheat which meets the following conditions: the A genome, the B genome and the D genome are all mutated in a target region and are all homozygous mutant types; no vector sequence was carried.
The gene-edited wheat may specifically be: t obtained in example 30Inbreeding generation plant B041-S103 to obtain T1Generation plants from which T mutants based on the target sequence were selected of the type "homozygous mutant for a genome D13 and homozygous mutant for B genome D10 and homozygous mutant for D genome D411And (5) plant generation.
The gene-edited wheat may specifically be: t obtained in example 30Inbreeding generation plant B041-S103 to obtain T1Generation plants from which T with the type of mutation based on the target sequence of "homozygous mutant for a genome D13 and homozygous mutant for B genome D10 and homozygous mutant for D genome D41 and not carrying a vector sequence" were selected1And (5) plant generation.
And (3) selfing the gene editing wheat to obtain a progeny, namely the gene editing strain.
The invention also protects the application of the specific sgRNA or the specific recombinant plasmid in wheat breeding; the aim of the wheat breeding is to increase the resistant starch content and/or the amylose content and/or the total pentosan content in wheat seeds.
The invention also provides a method for improving the resistant starch content and/or the amylose content and/or the total pentosan content in wheat seeds, which comprises the following steps: inhibiting the activity of SBEIIa protein in wheat.
The SBEIIa protein is (a1), or (a2) or (a 3):
(a1) a protein consisting of an amino acid sequence shown in a
(a2) a protein derived from the protein obtained by substituting and/or deleting and/or adding (a1) one or more amino acid residues and having the same function;
(a3) a protein derived from wheat, having 98% or more identity to (a1) and having the same function.
The SBEIIa gene is a nucleic acid molecule for coding the SBEIIa protein.
The SBEIIa gene is a DNA molecule of 1) or 2) or 3) or 4) or 5) or 6) or 7) or 8) as follows:
1) the coding region is a DNA molecule shown as a
2) the coding region is a DNA molecule shown as a
3) the coding region is a DNA molecule shown as a
4) DNA molecule shown in
5) DNA molecule shown in
6) DNA molecule shown in
7) a DNA molecule which hybridizes under stringent conditions with a DNA sequence as defined in any one of 1) to 6) and which encodes said SBEIIa protein;
8) a DNA molecule derived from wheat and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or more homology to a DNA sequence defined in any one of 1) to 6) and encoding said SBEIIa protein.
The encoding gene of the sgRNA1 is shown as the 6915-position 7017 nucleotide in the
The encoding gene for sgRNA2 differed from the encoding gene for sgRNA1 only in that "AGAGGCCGCGCGGCTCAGGA" was replaced with "GGGAAGGTCCTGGTGCCTGA".
The receptor wheat can be Zheng wheat 7698.
The invention utilizes CRISPR/Cas9 technology to edit wheat SBEIIa gene at fixed point, knocks out the wheat SBEIIa gene by causing frameshift mutation, and obtains a new generation of wheat new germplasm with obviously improved amylose, resistant starch and total pentosan (healthy fiber) content. Compared with the wild type control, the obtained editing line with three genomes of the SBEIIa gene knocked out at fixed points has the advantages that the content of amylose, resistant starch and total pentosan in seeds is obviously increased, the content of amylopectin with the polymerization degree of DP 9-12 is reduced, and the content of long-chain starch with the polymerization degree of DP >13 is increased. RVA measurement results show that: the starch peak viscosity value and the final viscosity value are both obviously reduced. The invention has great application and popularization value for wheat breeding.
Drawings
FIG. 1 is the electrophoresis chart of 5 regenerated plants obtained in example 2 after enzyme digestion
FIG. 2 shows the results of
FIG. 3 shows a part T in example 21The identification result of the step (4) is carried out on the generation plant
FIG. 4 is the electrophoresis chart of 2 regenerated plants obtained in example 3 after enzyme digestion
FIG. 5 shows the partial sequencing results of 2 regenerated plants in example 3
FIG. 6 shows a part T in example 31The identification result of the step (4) is carried out on the generation plant
FIG. 7 shows the results of the analysis of the properties of starch granules in example 4.
FIG. 8 shows the results of determination of amylose and resistant starch contents in example 4.
FIG. 9 shows the results of the starch RVA value determination in example 4.
FIG. 10 shows the results of analysis of the distribution of starch chain lengths in example 4.
FIG. 11 shows the results of the determination of the total pentosan content in example 4.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Zheng wheat 7698. Reference to the literature of "zheng mai 7698" (referred to in the literature as "Zhengmai 7698"): GuoG, Lei M, Wang Y, Song B, Yang J, et al, accumulation of As, Cd, and Pb in western heat accumulation in associated resources and associated thermal waste [ J ]. Journal of Environmental Research and Public Health,2018,15(11):2601. Zheng Mai7698 is denoted by WT, the corresponding sequence in the A genome is denoted by WT-A, the corresponding sequence in the B genome is denoted by WT-B, and the corresponding sequence in the D genome is denoted by WT-D.