阅读说明：本技术 一种调控作物种子直链淀粉含量和胶稠度的方法及应用 (Method for regulating and controlling amylose content and gel consistency of crop seeds and application ) 是由 刘耀光 祝钦泷 曾栋昌 于 2020-04-09 设计创作，主要内容包括：本发明公开了一种调控作物种子直链淀粉含量和胶稠度的方法及应用。所述方法为利用序列特异核酸酶基因组编辑技术对作物Wx基因的起始密码子上游部分进行位点特异性突变,创建作物Wx基因表达水平改变的突变系。利用本发明方法不同程度地降低了水稻突变系胚乳的OsWx基因表达量,快速有效地获得了胚乳直链淀粉含量适度下降、胚乳胶稠度适度增加的蒸煮和食味特性更好的新品系。利用该方法获得不同直链淀粉含量和胶稠度的作物突变系,大大丰富了作物的种质资源,快速培育优质新品种；因此,该方法在调控作物种子直链淀粉含量和/或胶稠度、以及培育优质水稻品系或品种中具有广泛的应用前景。(The invention discloses a method for regulating and controlling amylose content and gel consistency of crop seeds and application thereof. The method is characterized in that the upstream part of the initiation codon of the crop Wx gene is subjected to site-specific mutation by using a sequence-specific nuclease genome editing technology, and a mutation system with the changed expression level of the crop Wx gene is created. The method of the invention reduces the OsWx gene expression quantity of the rice mutant endosperm to different degrees, and quickly and effectively obtains a new strain with properly reduced content of endosperm amylose, properly increased endosperm gum consistency and better cooking and taste characteristics. The method is utilized to obtain the crop mutation lines with different amylose contents and gel consistencies, so that the germplasm resources of the crops are greatly enriched, and high-quality new varieties are quickly cultivated; therefore, the method has wide application prospect in regulating and controlling the amylose content and/or the gel consistency of crop seeds and cultivating high-quality rice strains or varieties.)

1. A method for regulating and controlling the amylose content and the gel consistency of crop seeds is characterized in that a site-specific mutation is carried out on the upstream part of an initiation codon of a crop Wx gene by using a sequence-specific nuclease genome editing technology to create a mutation line with changed expression level of the crop Wx gene.

2. The method of claim 1, wherein the site-specific mutagenesis is performed by: selecting one or more target points in the upstream part of the initiation codon of the Wx gene of the crop, constructing a knockout carrier containing guide RNAs of different target points, and transforming the knockout carrier into the crop.

3. The method of claim 1, wherein the portion upstream of the initiation codon is a promoter regulatory region or a 5' UTR regulatory region.

4. The method of claim 1, wherein the sequence-specific nuclease genome editing technology is a CRISPR/Cas genome editing technology.

5. The method of any one of claims 1 to 4, wherein the crop Wx gene is a rice OsWx gene.

6. The method as claimed in claim 5, wherein the upstream part of the initiation codon of the rice OsWx gene is targeted to any one or a combination of T1, T2, T3, T4, T5, T6, T7, T8, T9, T10 or T11; the sequences of the target points T1-T11 are respectively shown in SEQ ID NO. 2-12 in sequence.

7. The method of claim 6, wherein the target is a double target T1/T2, T3/T4, T5/T6, T7/T8, T9/T10, or a single target T11.

8. Use of the method of any one of claims 1 to 7 for controlling the amylose content and/or gel consistency of crop seeds.

9. Use of the method of any one of claims 1 to 7 for breeding a high quality rice line or variety.

10. A method for regulating and controlling the expression level of crop Wx genes is characterized in that one or more target points in the upstream part of the initiation codon of the crop Wx genes are selected, and the crop Wx genes are subjected to gene editing by using a sequence specific nuclease genome editing technology.

Technical Field

The invention belongs to the technical field of plant biology. More particularly, relates to a method for regulating and controlling amylose content and gel consistency of crop seeds and application thereof.

Background

The main component of endosperm of seeds of crops such as rice, wheat, corn and the like is starch, which is a main source for human and animal to take carbohydrate food; the endosperm starch mainly comprises amylose and amylopectin, the proportion change of the amylose and the amylopectin can affect the physicochemical properties of the starch product such as hardness, viscosity, gelatinization temperature, gum consistency and the like, and the higher the content of the amylose is, the harder the amylose is, the smaller the viscosity is and the lower the gum consistency is, so that the edible taste and the food processing characteristics of the starch product are affected. On the other hand, starches for industrial use also require starches with a high amylose content. Therefore, the development of starch products with different amylose contents can meet the consumption requirements of different populations and the needs of the food industry.

Amylose of endosperm of different crops is controlled by a highly conserved orthologous gene, namely wax gene (Wx for short, also called Waxy gene), which encodes a key enzyme of plant Starch synthesis with function conservation, namely Granule-Bound Starch Synthase I (GBSSI), and different allelic variation of the Wx gene can cause variation of amylose content and consistency of the endosperm. In rice (Oryza sativa L.), the amylose content of different subspecies (indica and japonica) and rice of different varieties (endosperm) is obviously different; wherein, the amylose content of a typical indica rice variety (O.sativa L.sspindica) is about 25 percent, and rice is hard; the amylose content of japonica rice varieties (O.sativa L.ssp japonica) and part of indica rice introduced into the bloody margin of japonica rice is about 15-18%, and the rice is moderate in hardness and softness, so that the taste preference of most people is met; the amylose content of a small amount of soft rice originally produced in Yunnan is about 10-11%, and the rice is soft. The amylose content of glutinous rice varieties (indica glutinous rice and japonica glutinous rice) is about 2 percent, and the rice is sticky and soft and is used as a material for rice dumplings, cakes and the like. The above rice endosperm amylose content difference is mainly due to the control of different variants of OsWaxy gene (OsWx for short, gene number: Os06g0133000) located in chromosome 6, typically OsWx of indica rice^aType, evolutionarily belonging to a strongly functional primitive type; function weakened variant OsWx of japonica rice^bMolding; modified OsWx of soft rice with weakened function^mpMolding; oswx mutant of glutinous rice with complete loss of function; i.e., the stronger the OsWx function, the rice speciesThe higher the amylose content in the endosperm of the seed.

Because the taste of the rice variety with high amylose content is poor, in order to cultivate the high-quality indica rice variety with moderate amylose content and good edible taste, the traditional breeding method comprises the following steps: carrying out hybridization and backcross breeding on new varieties by using Wx alleles generated by natural mutation existing in crop germplasm resources; for example, OsWx of japonica rice is obtained by crossing and backcrossing^bAllele transfer into indica background varieties to replace the original OsWx^a. However, this method has the following limitations: (1) the available Wx alleles are of a few species, such as in rice, which is predominantly indica-type OsWx^a(amylose content about 25%), japonica OsWx^b(amylose content about 15% to 18%) and waxy oswx (amylose content about 2%); (2) needs multiple-generation backcross and has long breeding time. In recent years, sequence-specific nuclease genome editing technologies, such as TALENs technology, CRISPR/Cas (including multiple Cas enzymes such as Cas9, Cas12a, etc.) technology, have developed to maturity and have been successfully applied in various organisms.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing method for regulating and controlling the content of amylose in crop seeds and provides a method for regulating and controlling the content and the consistency of the amylose in the crop seeds and application thereof.

The invention aims to provide a method for regulating and controlling the amylose content and the gel consistency of crop seeds.

Another object of the present invention is to provide the use of said method for regulating the amylose content and/or the gel consistency of crop seeds.

The invention further aims to provide application of the method in breeding high-quality rice lines or varieties.

It is still another object of the present invention to provide a method for regulating the expression level of Wx gene in crops.

The above purpose of the invention is realized by the following technical scheme:

the invention firstly provides a method for regulating and controlling the amylose content and the gel consistency of crop seeds, which utilizes a sequence specific nuclease genome editing technology to carry out site-specific mutation on the upstream part of an initiation codon of a crop Wx gene and create a mutation system with changed expression level of the crop Wx gene.

The site-specific mutation can change the 5 'non-coding region (5' UTR) of the Wx gene and the precursor RNA splice point and the adjacent sequence of the first intron region thereof, so that the splicing efficiency is changed, and the promoter region sequence is changed to influence the transcription activity, thereby effectively carrying out expression regulation on the crop Wx gene and obtaining the mutant system with the changed expression level of the crop Wx gene.

Preferably, the site-specific mutation method is: selecting one or more target points in the upstream part of the initiation codon of the Wx gene of the crop, constructing a knockout carrier containing guide RNAs of different target points, and transforming the knockout carrier into the crop.

More preferably, the guide RNA containing different targets is a sgRNA expression cassette.

Preferably, the upstream part of the initiation codon is a promoter regulatory region or a 5' UTR regulatory region.

Preferably, the sequence-specific nuclease genome editing technology is CRISPR/Cas genome editing technology.

More preferably, the CRISPR/Cas genome editing technique is a CRISPR/Cas9 genome editing technique.

Preferably, the crop Wx gene is a rice OsWx gene. The sequence of the upstream part of the initiation codon of the rice OsWx gene is shown as SEQ ID NO. 1.

Preferably, the target point of the upstream part of the initiation codon of the rice OsWx gene is any one or a combination of more of T1, T2, T3, T4, T5, T6, T7, T8, T9, T10 or T11; the sequences of the target points T1-T11 are sequentially shown as SEQID NO. 2-12 respectively.

More preferably, the target point is a double target point T1/T2, T3/T4, T5/T6, T7/T8, T9/T10 or a single target point T11.

The research of the invention finds that: and (3) carrying out single knockout or combined knockout on the target spots to obtain mutant lines with different Wx gene expression levels, further obtaining a plurality of mutant lines with different amylose contents, and realizing artificial regulation and control on the amylose content of crops. For example, OsWx is the homozygous mutant plant of T2 generation edited by CRISPR/Cas genome editing technology^aDouble-target editing of gene promoter regulatory regions (T1/T2, T3/T4, T5/T6, T7/T8, T9/T10), and OsWx^aSingle target point (T11) editing of gene 5' UTR control region can effectively generate various deletion and insertion mutants, and amylose content of a plurality of mutant systems is lower than 25% (9%; E.E.)20%) and increased endosperm consistency (50-85 mm).

The method can be used for rapidly cultivating the mutant line with the changed Wx gene expression level of the crops to obtain the mutant line with the changed amylose content and/or gel consistency; therefore, the application of the method in the regulation of the amylose content and/or the gel consistency of crop seeds and the cultivation of high-quality rice lines or varieties is within the protection scope of the invention.

In addition, the invention also provides a method for regulating the expression level of the crop Wx gene, which selects one or more target points in the upstream part of the initiation codon of the crop Wx gene and carries out gene editing on the crop Wx gene by using a sequence specific nuclease genome editing technology.

The invention has the following beneficial effects:

the invention provides a method for regulating and controlling the amylose content and the gel consistency of crop seeds and application thereof. The invention utilizes CRISPR/Cas genome editing technology to perform expression regulation on rice OsWx genes, and reduces endosperm OsWx gene relative expression quantity of rice mutation lines to different degrees. Compared with wild indica rice variety Tianfeng B (the amylose content of seeds is about 25 percent), the new rice variety with moderately reduced endosperm amylose content and moderately increased endosperm gum consistency can be quickly and effectively obtained; can quickly cultivate new crop varieties with better cooking and taste characteristics and adaptive to industrial requirements, and greatly meet the consumption requirements of different crowds and the industrial requirements.

The method obtains various mutant lines with different amylose contents and gel consistencies, greatly enriches the germplasm resources of crops, and the obtained mutant lines can be directly popularized and applied as crop varieties and can also be used as breeding parents, so that the breeding time is short; therefore, the method has wide application prospect in regulating and controlling the amylose content and/or the gel consistency of crop seeds and cultivating high-quality rice strains or varieties.

Drawings

FIG. 1 shows OsWx of rice^aA gene structure, a promoter and a regulatory region sequence thereof, and a CRISPR/Cas9 target point design schematic diagram; (A) the figure is rice OsWx^aGene structure (partial Exon/intron are omitted), wherein "promoter" represents promoter, "5 'UTR" represents 5' regulatory region, "3 'UTR" represents 3' regulatory region, and "Exon" represents Exon; (B) the figure is a schematic diagram of the design of a promoter and a regulatory region sequence thereof as well as a CRISPR/Cas9 target point, wherein underlined characters are T1-T11 (T11 is positioned in a 5 'UTR regulatory region), italic characters (a sense strand is NGG, and an antisense complementary strand is CCN) are a pre-spacer sequence adjacent motif (PAM) required by the target point, and a lower case character is a first intron positioned in the 5' UTR regulatory region.

FIG. 2 shows OsWx expression of rice^aA CRISPR/Cas9 genome editing vector schematic diagram for carrying out double-target combination and single-target editing on a promoter regulatory region and a 5' UTR regulatory region of a gene; wherein, "OsU 6 a" and "OsU 6 b" represent the rice micronucleus RNA promoter; "sgRNA" represents a single guide RNA; "P_ubi"represents a strong promoter of maize ubiquitin; "NLS" represents a nuclear localization signal; "Cas 9 p" represents a rice codon optimized Cas9 protein; "Tnos" represents nos terminator; "2 xP_35s"represents a 2-fold 35S promoter; "HPT" represents a hygromycin selectable marker gene; "T_35S"represents a 35S terminator; "LB" represents the left sequence; "RB" represents the right border sequence; "Kan^R"represents a screening marker for clarithromycin; "pBR 322 ori" represents a bacterial replicon; "pVS 1 replicon" stands for Agrobacterium replicon.

FIG. 3 shows OsWx of rice^aThe target point sequence variation of partial mutant line edited by promoter regulatory region and 5' UTR regulatory region of gene; wherein "Ref" represents a reference sequence of wild-type transformed receptor indica (tianfeng B), "-" represents a base deletion, "del" represents a base deletion, "in" represents a base insertion, and "sub" represents a base substitution.

FIG. 4 shows the results of endosperm gum consistency measurements of rice mutant lines; wherein, the graph (A) is the result of measuring the content of the amylose in the endosperm of the rice mutant line; (B) the graph shows the results of endosperm gum consistency measurements of rice mutant lines.

FIG. 5 shows the results of the measurement of relative expression level of OsWx gene in endosperm of rice mutant lines.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.