Regulation and control element for regulating and controlling content of resistant starch in rice and application of regulation and control element

Abstract

The invention relates to a regulating element for regulating the content of resistant starch in rice and application thereof in the technical field of plant genetic engineering. The regulatory element provided by the invention contains a nucleotide sequence as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2. A CRISPR / Cas9 gene editing technology is used for carrying out fixed-point editing on the regulatory element in rice, and the expression level of the SBEIIb gene can be reduced after the regulatory element is mutated, so that the content of resistant starch in the rice is improved, and a gene resource and a technical thought are provided for improving the quality of the rice.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to a CRE regulatory element for regulating the expression of SBEIIb gene of rice resistant starch content and application thereof. BACKGROUND

[0002] Starch is usually directly converted into glucose to provide energy for the metabolic function of cells. If the energy required by cells decreases, the excess heat obtained from starch will be stored in the form of glycogen or fat for future use. Therefore, an unreasonable diet combined with a lack of exercise or a sedentary lifestyle can lead to obesity and type II diabetes and other health problems. Resistant starch (RS) is a dietary fiber, which is beneficial to health due to its difficulty in being digested by the gastrointestinal tract, thereby avoiding adverse health effects. Rice is the main food source for about half of the world's population, and the starch content of rice is more than 80%, but the content of resistant starch in most cooked rice is less than 2%. Therefore, cultivating high RS rice varieties can provide an effective method for improving public health.

[0003] The content of RS is controlled by a variety of genes involved in starch synthesis. It is reported that starch branching enzyme (SBE) affects the content of RS in rice, corn and wheat. Plants have two types of SBE, which have been historically named SBEI and SBEII in cereal crops. In rice, SBEIIb (also known as SBE3) is a key gene that controls the formation of RS, while neither SBEIIa nor SBEI can perform this function. A number of studies have shown that the content of RS and amylose in the endosperm of rice sbeIIb mutants is significantly increased. For example, sbe3-rs is a SBEIIb mutant with an amino acid mutation in SBEIIb, and a domestic first high RS japonica rice new strain ‘Diabetes Rice No. 1’ is cultivated by using the mutant. In addition, direct knockout or reduction of the expression of SBEIIb gene by using CRISPR / Cas gene editing and RNAi technology can significantly improve the content of RS in rice. However, previous studies have shown that high RS mutants or transgenic plants often have serious adverse defects such as seed shriveling, extremely low single grain weight, and high chalkiness, which do not meet the modern crop breeding concept of ‘improving nutrition on the premise of maintaining or improving yield’, and seriously affect the utilization rate of high RS crops.

[0004] Therefore, it is necessary to develop other methods to increase the content of RS while minimizing the impact on other important traits. By fine-tuning the expression of SBEIIb in the endosperm, it is explored to cultivate excellent varieties with high RS content, excellent quality and stable agronomic traits.

[0005] Cis-regulatory elements (CREs) are sequences that control the expression of genes at the transcriptional level in all developmental stages. Variations in CREs play a crucial role in the fine regulation of gene expression. Targeting the coding region of genes and CREs through the CRISPR / Cas editing system has been proven to be achievable. Compared with traditional rice variety improvement methods, the CRISPR / Cas9 gene editing technology has the advantages of short time consumption, no linkage drag, etc., and can achieve rapid and accurate targeted improvement.

[0006] Therefore, the mining of the CRE elements of the rice SBEIIb gene can further analyze the expression mechanism of the SBEIIb gene, enrich the expression regulation mode of the SBEIIb gene, provide gene resources for the fine regulation of the resistant starch content of rice, and have important application significance for cultivating functional rice that meets the needs of special groups such as diabetic patients. SUMMARY

[0007] The present application aims to meet the breeding needs of high RS content functional rice in the prior art, and provides a regulatory element for regulating the resistant starch content of rice and its application, which is used for regulating the expression of the SBEIIb gene to improve the RS content of rice and has excellent and stable agronomic traits.

[0008] To achieve the above-mentioned purpose, the present application first provides a regulatory element for regulating the resistant starch content of rice, characterized in that the regulatory element is CRE1 and / or CRE2, which is used for regulating the expression of the SBEIIb gene, and the nucleotide sequences of the regulatory elements CRE1 and CRE2 are respectively as follows: SEQ ID NO. 1 (5'-ACACGAAACAAA-3') and SEQ ID NO. 2 (5'-GGAGCACGTGAG-3').

[0009] Further, the regulatory elements CRE1 and CRE2 are CRE regulatory elements upstream of the SBEIIb gene.

[0010] To further achieve the purpose of the present application, the present application also provides a method for regulating RS using the above-mentioned regulatory element, specifically: editing the CRE1 and / or CRE2 elements so that the sequence of the target CRE element changes, thereby changing the expression level of the SBEIIb gene in the rice variety.

[0011] Further, the method for editing the CRE1 and / or CRE2 elements includes adding, substituting, inserting or deleting one or more nucleotides to generate mutants, alleles or derivatives thereof.

[0012] Further, by editing the above-mentioned CRE element, the nucleotide sequence of the regulatory element CRE1 and / or CRE2 of the SBEIIb gene is changed, so that the resistant starch content of rice is increased, thereby improving the rice variety.

[0013] The application also provides a recombinant expression vector of a CRE regulatory element for regulating the expression of a rice SBEIIb gene, and a construction method of the recombinant vector: screening a sequence with NGG as a PAM site near the CRE element for a designed editing target site, the target site sequence for CRE1 is 5'-TTTGTTTCGTGTAAGCCTCC-3', and the target site sequence for CRE2 is 5'-AAACGTTGAGGAGCACGTGA-3', the target site sequence is designed into an inverted complementary primer and connected with an intermediate vector SK-gRNA, the enzyme digestion product after connection is connected with a final vector pC1300-Cas9, and a recombinant expression vector is obtained through Agrobacterium transformation.

[0014] Compared with the prior art, the above technical scheme of the application has the following beneficial effects: a CRE element upstream of the SBEIIb gene is found, and the sequence of the CRE element is destroyed through gene editing technology, so that the expression amount of the SBEIIb gene in rice is down-regulated, and the resistant starch content of rice is moderately increased. Compared with the existing sbeIIb functional deletion mutant, the mutation of the CRE sequence of the SBEIIb gene can increase the resistant starch content of rice without affecting other agronomic traits, and has important application value in the field of cultivating special functional rice varieties. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the mining of the CRE element upstream of the SBEIIb gene in rice in embodiment 1 of the application; Figure 2 is the regulatory effect of the mined CRE element identified by using a tobacco transient expression system in embodiment 1 of the application; Figure 3 is the wild type sequence and the mutant sequence of the CRE element upstream of the SBEIIb gene in rice in embodiment 2 of the application; Figure 4 is the influence of the mutation of the cre element upstream of the SBEIIb gene on the expression amount of the gene in embodiment 3 of the application; Figure 5 is the influence of the mutation of the cre element upstream of the SBEIIb gene on the quality traits such as the resistant starch content of rice in embodiment 4 of the application.

[0016] Figure 6 is the influence of the mutation of the cre element upstream of the SBEIIb gene on the yield-related traits of rice in embodiment 4 of the application. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present application, the following examples are used to illustrate the present application, but do not limit the scope of the present application.

[0018] The experimental methods not specified in the following examples are carried out according to the conventional technical procedures, and the materials and reagents used, unless otherwise specified, are commercially available conventional biochemical reagents.

[0019] Example 1: Mining and identification of SBEIIb gene CRE element in rice Nipponbare is a japonica rice variety, which has completed whole genome sequencing, is an important variety for rice breeding research, and is a completely publicly available variety. Taking the high-abundance expression tissue (developing seeds 10 days after flowering) of SBEIIb (LOC_Os02g32660) gene of rice japonica variety Nipponbare as the object, open chromatin accessibility (ATAC-seq) analysis and transcriptome sequencing (RNA-seq) analysis were carried out, and the genomic open chromatin data (DNase-seq) of rice leaf and callus obtained from the plant open chromatin database PlantDHS (http: / / plantdhs.org / ) were referenced, combined with bioinformatics analysis method, the chromatin accessibility sites related to the expression regulation of SBEIIb gene were screened, and two chromatin accessibility sites were mined, which are potential CRE elements for regulating the expression of SBEIIb gene, and the sequences are SEQ ID NO. 1 and 2, as shown in Figure 1 .

[0020] The effect of CRE on the expression of downstream target genes was detected in tobacco leaves by using a dual luciferase in vitro transient expression system. The sequences containing the unmutated CRE element, the sequence not containing the CRE1 element, the sequence not containing the CRE2 element, and the sequence not containing the CRE1 and CRE2 elements were respectively connected into the expression vector pGreenII0800-35Smini-LUC containing the dual luciferase reporter gene, to obtain recombinant expression vectors (Figure Figure 2 The correct recombinant vectors were transformed into Agrobacterium strain.

[0021] The specific information of the above sequences is as follows: The sequence of the unmutated CRE element: 5'-CAGGAGGCTT ACACGAAACAA ATTCTAGTGA-3', 5'-TAAACGTTGA GGAGCACGTGAG GGAGAAGCAC-3', which is composed of CRE and flanking sequences, wherein the CRE sequence is marked with an underline; Sequence without CRE1 element: 5'-CAGGAGGCTTATTCTAGTGA-3', consisting of CRE1 flanking sequence, i.e. cre1 sequence deletion mutation; Sequence without CRE2 element: 5'-TAAACGTTGAGGAGAAGCAC-3', consisting of CRE2 flanking sequence, i.e. cre2 sequence deletion mutation; Sequence without CRE1 and CRE2 elements: 5'-CAGGAGGCTTATTCTAGTGA-3', 5'-TAAACGTTGAGGAGAAGCAC-3', consisting of CRE1 and CRE2 flanking sequences, i.e. cre1 and cre2 sequence deletion mutations.

[0022] Further, the Nicotiana benthamiana leaves were infected with Agrobacterium containing the recombinant vector, and cultured for 48 hours. The tobacco leaves at the infection site were taken for luciferase activity detection (reference method: Xiong et al., 2022, Brassinosteroids regulate rice seed germination through the BZR1-RAmy3D transcriptional module. Plant Physiology). The fluorescence signal of the reporter gene in the tobacco leaf cells containing the mutant cre1 and / or cre2 element was weakened (Fig. 2B, left lower corner). Figure 2 Further, the quantitative fluorescence signal (LUC / REN activity) was determined, and it was found that the fluorescence signal of the tobacco leaves containing the mutant cre1, mutant cre1 & 2 elements was extremely significantly decreased, and the fluorescence signal of the tobacco leaves containing the mutant cre1 element was significantly decreased (Fig. 2B, right). Figure 2 Right), i.e. the mutation of the cre1 and / or cre2 element led to the decrease of the expression level of the downstream protein. The results showed that the mined CRE element had the ability to regulate the expression of the downstream target gene / protein.

[0023] Example 2: Creation and identification of rice materials containing mutant cre regulatory elements of SBEIIb gene According to the existing CRISPR / Cas9 related experimental method, a specific sequence containing NGG as PAM site is selected as the knockout target site on the CRE1 element of SBEIIb gene, and the target site sequence is 5'-TTTGTTTCGTGTAAGCCTCC-3'. A specific sequence containing NGG as PAM site is selected as the knockout target site on the CRE2 element of SBEIIb gene, and the target site sequence is 5'-AAACGTTGAGGAGCACGTGA-3'. After the target site sequences of CRE1 and CRE2 are designed into reverse complementary primers, they are connected into the intermediate vector SK-gRNA; the intermediate vector is digested, and the gRNA fragment containing the target site is recovered and connected into the final vector pC1300-Cas9 to obtain three recombinant vectors: pC1300-Cas9-CRE1, pC1300-Cas9-CRE2 and pC1300-Cas9-CRE1&2. After sequencing, the recombinant vector is transformed into the Agrobacterium strain (recombinant vector construction method Wang et al., 2017, A Simple CRISPR / Cas9 System for Multiplex Genome Editing in Rice, JGG).

[0024] The above three recombinant vectors are respectively transformed into rice by using the Agrobacterium-mediated genetic transformation method with Nipponbare as the transformation receptor, and positive transgenic seedlings are obtained through tissue culture. After final detection and identification, they are transplanted to the field to obtain T0 generation rice plants, i.e. plants containing mutant cre elements, including SBEIIb -cre1, SBEIIb -cre1, SBEIIb -cre1&2.

[0025] The wild type Nipponbare containing wild type CRE elements is used as a control variety, and the plants containing mutant cre elements prepared by the present application (including SBEIIb -cre1, SBEIIb -cre1, SBEIIb-cre1&2) only sequence difference exists near the CRE element. After the genomic DNA is quickly extracted from the rice tillering stage leaf by CTAB method, then a pair of PCR sequencing detection primers (primer sequence is CREp-F: 5'-CTCCCTGCTACTTGCTACTCCTGTGC-3'; CREp-R: 5'-TCGCCAGTCTGCTAGAGGATGTG-3') are designed on the upstream and downstream of the CRE1 and CRE2 element editing target site sequence, which are used to detect the sequence variation near the target site. After the PCR amplification and sequencing analysis based on the target site sequence, the sequence of the mutant cre element is obtained. Compared with the CRE element in the wild type, multiple base deletions exist in the cre element in the mutant, which indicates that the characteristics of the cre element have been completely destroyed, as shown in Figure 3 .

[0026] Example 3: Effect of mutation of the CRE element upstream of the SBEIIb gene on the expression amount of the gene in rice The grains (developed for 10 days) of the plant containing the mutant cre element prepared in Example 2 of the present application and the grains (developed for 10 days) of the control variety wild-type Nipponbare containing the wild-type CRE element are ground in liquid nitrogen, the cells are broken, and total RNA is extracted. The cDNA is obtained by using a reverse transcription kit, and then the expression amount of the SBEIIb gene mRNA in the control variety Nipponbare and the plant containing the mutant cre element is detected according to the SYBR Green fluorescent quantitative PCR method. The detection primers are as follows: SBEIIbq-F: 5'-CCGCAGGAGAAATCCCATAC-3', SBEIIbq-R: 5'-CCGAAACGACTACTTGGTGC-3'. The results show that, as shown in Figure 4 , the mRNA expression amount of the SBEIIb gene in the plant containing the mutant cre element is significantly reduced, and the expression abundance of the SBEIIb protein is also decreased, which is consistent with the characteristics that the typical CRE element can regulate the expression of the target gene downstream thereof.

[0027] Example 4: Determination of agronomic traits of rice containing mutant cre element In this embodiment, in order to clarify the biological function of the CRE element, the field agronomic traits of the plant containing the mutant cre element prepared in Example 2 of the present application are investigated. The plants are planted in the field environment according to the conventional cultivation management conditions, and when the rice is mature, compared with the control variety Nipponbare, the amylose content of the plant containing the mutant cre element is significantly increased, the amylopectin content is slightly increased, the total starch content is slightly decreased, and the grain chalkiness has a slight increase (not obvious) (as shown in Figure 5 ); at the same time, the plant height, effective tiller number, ear length, primary branch number, grain number per ear, seed setting rate, thousand-grain weight, grain shape, and other yield trait indexes have no obvious change.Figure 6 ), the overall trend of change is consistent with the phenotype effect caused by the specific down-regulation of SBEIIb gene expression. Therefore, the CRE in the present application can fine-tune the expression of SBEIIb gene at the transcription level, and after the knockout and destruction, the mRNA and protein levels of SBEIIb can be reduced, thereby increasing the resistant starch content of rice, and having important application value in the field of cultivating special functional rice varieties.

Claims

1. A regulatory element for regulating the content of resistant starch in rice, characterized in that, The regulatory elements are CRE1 and / or CRE2, which are used to regulate the expression of SBEIIb gene, and the nucleotide sequences of the regulatory elements CRE1 and CRE2 are respectively as follows: SEQ ID NO. 1 (5'-ACACGAAACAAA-3') and SEQ ID NO. 2 (5'-GGAGCACGTGAG-3').

2. The regulatory element for regulating amylose content of rice according to claim 1, wherein, The regulatory elements CRE1 and CRE2 are CRE regulatory elements upstream of the SBEIIb gene.

3. A method of modulating the content of resistant starch in rice, characterized in that, The editing of the CRE1 and / or CRE2 elements changes the sequence of the target CRE element, thereby changing the expression level of the SBEIIb gene in the rice variety.

4. The method for modulating the resistant starch content of rice according to claim 3, wherein, The method for editing the CRE1 and / or CRE2 elements includes adding, substituting, inserting or deleting one or more nucleotides to generate mutants, alleles or derivatives thereof.

5. The method for modulating amylose content of rice according to claim 3, wherein, By changing the nucleotide sequence of the regulatory elements CRE1 and / or CRE2 of the SBEIIb gene, the content of resistant starch in rice is increased, thereby improving the rice variety.

6. A recombinant expression vector of a CRE regulatory element regulating the expression of a rice SBEIIb gene, characterized in that, The construction method of the recombinant vector is as follows: sequences with NGG as PAM sites are screened near the CRE element for the design of editing target sites, the target site sequence for CRE1 is 5'-TTTGTTTCGTGTAAGCCTCC-3', and the target site sequence for CRE2 is 5'-AAACGTTGAGGAGCACGTGA-3', the target site sequence is designed into an inverted complementary primer and connected with an intermediate vector SK-gRNA, the cleavage product after connection is connected with a final vector pC1300-Cas9, and through Agrobacterium transformation, a recombinant expression vector is obtained.

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