CN111235170A - Application of qMYR2 gene in adjusting or screening content of oil and fat components in rice - Google Patents

Abstract

The invention relates to the application of qMYR2 gene in regulating the content of oil and fat components in rice and the application of it as a screening marker for the content of oil and fat components in rice in rice breeding. content method, and rice cross-breeding method using qMYR2 gene or its mutants as selectable markers.

Description

Application of qMYR2 gene in regulating or screening the content of oil and fat components in rice

technical field

The present invention relates to the field of rice molecular breeding, more particularly, to the application of qMYR2 gene in regulating and screening the content of oil and fat components in rice.

Background technique

Lipids in plant seeds are mainly stored in the form of triglycerides. The oil in rice is very important to rice storage, eating quality and consumers' health, and its oil content is generally 2-4%. The edible quality and palatability of rice are affected by the composition and content of fatty acids in rice. Currently, little is known about the lipid synthesis pathway of rice in the field, and Liu et al. confirmed that OsFAD3 converts linoleic acid (C18:2) to linolenic acid (C18:2). Zaplin et al. demonstrated that OsFAD2-1 converts oleic acid to linoleic acid. Several investigators have also tentatively identified several quantitative trait loci (QTL).

However, there are too few identified genes and QTLs, and the genetic and molecular basis of oil synthesis in rice still cannot be known. Therefore, it is necessary to find the main genes that affect the oil components and contents in rice, which can be used as screening markers or genetic manipulations. target.

SUMMARY OF THE INVENTION

Through research, the inventors of the present invention have identified 23 loci that are significantly related to oil content and oil composition, and have obtained 11 loci that are likely to be involved in the pathways related to rice oil metabolism in rice through linkage analysis. candidate gene. After further study, we found that among these loci, 4 genes contributed greatly to the natural variation in lipid composition and were differentiated in subgroups. One of them is the qMYR2 gene. Our study shows that qMYR2 is a major gene that positively regulates the content of C14:0. If the mutation of this gene leads to the reduction or disappearance of protein function, the C14:0 content in rice will be reduced.

Based on the above research, the present invention provides the application of the qMYR2 gene in regulating the content of oil and fat components in rice, and the sequence of the qMYR2 gene is shown in SEQ ID NO: 1.

In a preferred embodiment, the oil component is C14:0.

The present invention also provides a method for regulating the content of oil and fat components in rice, including introducing qMYR2 gene, increasing the expression level of qMYR2 gene, reducing the expression level of qMYR2 gene, or mutating the qMYR2 gene to increase, decrease or disappear the protein function expressed by the qMYR2 gene. Step, the sequence of the qMYR2 gene is shown in SEQ ID NO:1.

For example, if the target rice seed has excellent other traits, but the C14:0 content in the rice is too high, and the rice seed contains a normal function qMYR2 gene, the qMYR2 gene in the target rice seed can be knocked out or mutated to make the qMYR2 gene not expressed , or the myristoyl-ACP thioesterase function of the expressed protein is reduced or has no myristoyl-ACP thioesterase function, the C14:0 content in rice can be reduced.

Conversely, if the target rice seed has excellent other traits, but the C14:0 content in the rice is too low, and the rice seed does not contain the normal function qMYR2 gene, the qMYR2 gene can be introduced by introducing the qMYR2 gene, or by mutating the abnormal qMYR2 gene in the target rice seed. The C14:0 content in rice can be increased by re-mutating it into the qMYR2 gene with normal myristoyl-ACP thioesterase function.

The present invention also provides the application of the qMYR2 gene as a screening marker for the content of oil and fat components in rice in rice breeding, and the sequence of the qMYR2 gene is shown in SEQ ID NO: 1.

The present invention also provides a method for cross-breeding of rice, including the step of using the qMYR2 gene or its mutant as a screening marker to select a target genotype.

In a preferred embodiment, the rice hybrid breeding method comprises the following steps:

S1: Identify the qMYR2 gene in the parent and its mutation status;

S2: select the qMYR2 gene or its mutant as a screening marker;

S3: Aggregate the selection marker with other dominant traits.

For example, the existing rice seeds have excellent other traits, but the C14:0 content in the rice is too high and contains the normal function qMYR2 gene. In order to cross the parent of the mutation resulting in reduced or abolished phosphatidylcholine:diglyceride phosphorylcholine 2 function with this rice species, the mutant gene was aggregated with other dominant traits by using the qMYR2 gene mutant as a molecular marker come together.

On the contrary, the existing rice seeds have excellent other traits, but the C14:0 content in the rice is too low and does not contain the normal function qMYR2 gene. The myristoyl-ACP thioesterase functional qMYR2 gene parent was crossed with this rice seed, and the mutant gene was aggregated with other dominant traits by using the normal qMYR2 gene as a molecular marker.

Description of drawings

Fig. 1 is the percentage content of 10 kinds of oil components in 533 kinds of rice varieties detected by GC-MS;

Figure 2 shows the phenotypic distribution of oil-related traits of Australian rice (Aus), indica rice (Ind), and japonica rice (Jap) among 533 rice varieties;

Figure 3 is the Manhattan plot of the correlation analysis of C14:0 content in indica rice subpopulations, the dotted line represents the significance threshold (-log ₁₀ (P)=7.06)

Figure 4 is a Manhattan plot and linkage disequilibrium (LD) heat map of the region around the significant peak on chromosome 2, arrows indicate nucleotide variation sites of candidate genes, and vertical dashed lines indicate candidate regions around the peak;

Fig. 5 is the gene structure of qMYR2 and the polymorphism of this gene;

Figure 6 is a statistical graph of the QTL scan results of the identification of qMYR2 as the major site of C14:0 content in the recombinant inbred (RIL) population of 88B2/HD9802S by linkage analysis;

Fig. 7 is the C18:2 content statistics chart of different qMYR2 haplotypes, wherein the test object of a is the 305 indica rice varieties in the obtained population, and the test object of b is the RIL population derived from 88B2/HD9802S;

Figure 8 is a statistical graph of the content of 10 fatty acids in wild-type and knockout mutant families, *P<0.05, **P<0.01; ***P<0.001.

Detailed ways

The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

1. Sample source

The rice material used in the present invention includes a natural population composed of 533 cultivated rice varieties and three self-cultivated recombinant inbred populations. Natural populations were used for genome-wide association analysis, and three recombinant inbred populations were used for linkage analysis.

Among them, 533 cultivated rice varieties included 50 aus varieties (aus), 305 indica varieties (indica), and 178 japonica varieties (japonica).

The three recombinant inbred varieties were derived from the following hybrid lines: Zhenshan 97/Minghui 63 (ZS97/MH63), 88B-2/HD9802S and B805D/H6S, respectively.

2. Sample processing

The rice was dehulled into brown rice, then milled, passed through an 80-mesh sieve, dried at 80°C for 24 hours, and then subjected to lipid extraction. Add 0.2g rice flour into a 10ml glass tube, add 4.5ml sulfuric acid:methanol solution (1:19) and 100μl standard solution of heptadecanoic acid dissolved in chloroform, and mix well. The configuration of the heptadecanoic acid standard solution is that 1.07g of the heptadecanoic acid standard solution is dissolved in 10ml of chromatographically pure chloroform. The mixture was incubated in an 85°C water bath for 2h. After cooling to room temperature, fatty acid methyl esters were extracted with n-hexane and analyzed by gas chromatography-mass spectrometry (GC-MS). The sum of all identified fatty acid contents was taken as the oil content. Each sample was tested twice and the average was used for subsequent analysis.

3. GC-MS analysis results

The GC-MS analysis results are shown in Figure 1. Among the 10 fatty acids identified, palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2) accounted for 90% of the total fatty acid content. %above. We found wide variation in a number of lipid-related traits in our study, ranging from a 1.5-fold difference in C18:2 content to a 7.4-fold difference in myristic acid (C14:0) content. The total fatty acid content was in the range of 22.99-40.96 mg/g.

Among them, Australian rice (aus) had the highest content of C16:0, followed by indica rice (Ind) and japonica rice (Jap), and the contents of C18:1 and C18:2 were opposite (Fig. 2).

4. Association analysis and linkage analysis

Genome-wide association study (GWAS): GWAS was performed separately for indica, japonica, and all samples. In each analysis group, only SNPs with minor locus frequency (MA) F > 5% and deletion rate < 15% were selected for association analysis.

Linkage Analysis: Linkage analysis was performed on three RIL populations - ZS97/MH63 (96 lines), 88B-2/HD9802S (108 lines) and B805D/H6S (135 lines). These lines were all subjected to next-generation sequencing, and then the 50bp paired-end reads were aligned to the rice reference genome. Sequences with alignment quality ≥ 40 and bases with average quality ≥ 10 were used to identify SNPs. The genotypes of the parental lines were inferred by genotyping using the MPR algorithm, and a genetic Bin map was constructed for QTL identification.

Based on the above work, we finally obtained 5.2 million high-quality SNPs for evaluating population structure and affinities, and identified 23 significant association loci among them. Among 23 loci, we found qMYR2 to be the major QTL associated with C14:02 content (Fig. 3).

As shown in Figure 4, qMYR2 is located on the long arm of chromosome 2. We analyzed the sequence in the 25.86-26.02Mb (162kb) region based on the point-paired linkage disequilibrium (LD) correlation coefficient (r ² <0.6), and finally determined The candidate gene corresponding to this QTL is LOC_Os02g43090 (SEQ ID NO: 1), which encodes myristoyl-ACP thioesterase and its homologous gene in Arabidopsis is FATB (ester acyl-ACP thioesterase B) gene .

The G-A mutation at position 169 on exon 1 of the LOC_Os02g43090 gene (Ex1-G/A), which resulted in an amino acid change, had a large sub-allelic frequency (Figure 5).

The genotypes of the Ex1-G/A locus of qMYR2 differed between the indica varieties 88B-2 and HD9802S. Furthermore, QTL analysis using the RIL population derived from the 88B-2/HD9802S hybrid line confirmed that the contrastive effects of qMYR2 haplotypes on C14:0 content qMYR2 (Figure 6, Table 1).

Table 1 Phenotypic analysis of lipid-related traits

We divided qMYR2 into haplotype A (SEQ ID NO: 1) and haplotype B (SEQ ID NO: 2) based on the A and T bases at the mutated site. The C14:0 content of haplotype A lines was higher than that of haplotype B lines in both the 305 indica cultivar populations and in the RIL population (Fig. 7).

5. Transgenic analysis

To further determine the function of qMYR2, we knocked out qMYR2 in ZS97 using the CRISPR-cas9 system. In preparing the CRISPR/Cas9 knockout construct, we carefully examined the gene coding region and designed a 23 bp target with a C-terminal NGG. After determining the target, we inserted the 20bp target into the intermediate vector pER8-Cas9-U6 or pER8-Cas9-U3, and cloned it into pCXUN-Cas9 to obtain a knockout vector, named KO-MYR2. The knockout construct was introduced into E. coli strainTrans 5α for sequencing, and after verification was correct, it was introduced into Agrobacterium tumefaciens strain EHA105, and then transformed into ZS97. After obtaining the transgenic plants, the genotype of the knockout line was verified by sequencing.

Compared with wild-type ZS97 (haplotype A), the ZS97 knockout line KO-qMYR2 had significantly higher C18:1 content and significantly lower C14:0 content (Figure 8). It was demonstrated that this site can be used to modulate the fatty acid composition in rice.

Based on the above experiments, it can be seen that qMYR2 is the main gene that positively regulates the content of C14:0. If the mutation of this gene leads to the reduction or disappearance of protein function, the C14:0 content in rice will be reduced.

Although only the knockout mutants and Ex1-G/A mutants obtained by the CRISPR-cas9 system are shown in the above examples, those skilled in the art will know after reading the contents disclosed in this application that only need to introduce the gene into, Knockout, knockdown or mutation to reduce or no related function can achieve the purpose of regulating C14:0 content in rice.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

<110> Huazhong Agricultural University

Hubei Shuangshui Shuanglu Biotechnology Co., Ltd.

Application of <120> qMYR2 gene in regulating or screening the content of oil and fat components in rice

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Claims (9)

1. Application of qMYR2 gene in regulating the content of oil and fat components in rice, the sequence of the qMYR2 gene is shown in SEQ ID NO: 1. 2. The application according to claim 1, wherein the oil component is C14:0. 3. a method for regulating oil and fat component content in rice, it is characterized in that, comprise to the rice seed of producing described rice and import qMYR2 gene, improve qMYR2 gene expression level, reduce qMYR2 gene expression level or mutate qMYR2 gene to make qMYR2 gene express The step of increasing, decreasing or disappearing the protein function of the qMYR2 gene, the sequence of the qMYR2 gene is shown in SEQ ID NO: 1. 4. The method according to claim 3, wherein the rice seed used for producing the rice contains the qMYR2 gene with normal myristoyl-ACP thioesterase function, and the qMYR2 gene is knocked out or down-regulated, or the qMYR2 gene is mutated. The qMYR2 gene is used so that the myristoyl-ACP thioesterase function of the protein expressed by the qMYR2 gene is reduced or has no myristoyl-ACP thioesterase function. 5. method according to claim 3, is characterized in that, the rice seed that produces described rice does not contain the qMYR2 gene with normal myristoyl-ACP thioesterase function, and is transferred into with normal myristoyl-ACP thioester Enzymatically functional qMYR2 gene, or backmutating said qMYR2 gene without normal myristoyl-ACP thioesterase function to a qMYR2 gene with normal myristoyl-ACP thioesterase. 6. The application of the qMYR2 gene as a screening marker for the content of oil and fat components in rice in rice breeding, the sequence of the qMYR2 gene is shown in SEQ ID NO: 1. 7. A method for cross-breeding of rice, comprising the step of selecting a target genotype by using the qMYR2 gene or a qMYR2 gene mutant as a screening marker. 8. according to the described rice hybrid breeding method of claim 7, it is characterized in that, existing rice seed contains the qMYR2 gene with normal myristoyl-ACP thioesterase function, S1: Identify rice seeds containing a mutation in the qMYR2 gene with reduced or absent myristoyl-ACP thioesterase function as a parent; S2: crossing the existing rice seed with a parent containing a mutation in the qMYR2 gene whose function of myristoyl-ACP thioesterase is reduced or eliminated; S3: Using the qMYR2 gene mutant as a selection marker, the qMYR2 gene mutant and the dominant traits of the existing rice seeds are aggregated together. 9. according to the described rice hybrid breeding method of claim 7, it is characterized in that, existing rice seed does not contain the qMYR2 gene of normal myristoyl-ACP thioesterase function, S1: Identify rice seeds containing the qMYR2 gene with normal myristoyl-ACP thioesterase function as parents; S2: crossing the existing rice seed with the parent containing the qMYR2 gene with normal myristoyl-ACP thioesterase function; S3: Using the qMYR2 gene with normal myristoyl-ACP thioesterase function as a selection marker, the qMYR2 gene with normal myristoyl-ACP thioesterase function and the dominant traits of the existing rice seeds are aggregated into Together.

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