CN111139246A

CN111139246A - Application of qPLA6 gene in regulating or screening content of oil and fat components in rice

Info

Publication number: CN111139246A
Application number: CN202010051209.6A
Authority: CN
Inventors: 何予卿; 周浩; 夏朵
Original assignee: Hubei Shuangshui Shuanglu Biological Technology Co ltd; Huazhong Agricultural University
Current assignee: Hubei Shuangshui Shuanglu Biological Technology Co ltd; Huazhong Agricultural University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-05-12
Anticipated expiration: 2040-01-17
Also published as: CN111139246B

Abstract

The invention relates to an application of qPLA6 gene in regulating the content of oil and fat components in rice and an application of qPLA6 gene as a screening marker of the content of the oil and fat components in rice breeding, and also relates to a method for regulating the content of the oil and fat components in rice by carrying out genetic operation on qPLA6 gene, and a rice cross breeding method using the qPLA6 gene or a mutant thereof as the screening marker.

Description

Application of qPLA6 gene in regulating or screening content of oil and fat components in rice

Technical Field

The invention relates to the field of rice molecular breeding, in particular to application of qPLA6 gene in adjusting and screening the content of oil and fat components in rice.

Background

Lipids are stored in plant seeds mainly in the form of triglycerides. The oil in the rice is very important for the storage, the eating quality and the health of consumers, and the oil content of the rice is generally 2 to 4 percent. The eating quality and palatability of rice are affected by the fatty acid composition and content of rice. Currently, little is known in the art about the lipid synthesis pathway of rice, Liu et al demonstrate 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 researchers have also preliminarily identified several Quantitative Trait Loci (QTLs).

However, the genes and QTLs identified at present are too few, and the heredity and molecular basis of rice oil synthesis still cannot be known, so that main genes influencing oil components and contents in rice need to be found and used as screening marks or targets of genetic operation.

Disclosure of Invention

The inventor of the invention finds out through research that 23 sites which are significantly related to oil content and oil composition are identified, and 11 candidate genes which are possibly involved in a pathway related to rice oil metabolism in rice are obtained through verification of linkage analysis. After further investigation, we found that 4 of these sites contributed greatly to the natural variation in lipid composition and were differentiated in subpopulations. One of the genes is qPLA6 gene, and our research shows that qPLA6 is a main effective gene for positively regulating C16:0 content. If the mutation of the gene causes the reduction or disappearance of the function of the protein, the content of C16:0 in rice is reduced, and the content of C18:1 and C18:2 is increased.

Based on the research, the invention provides the application of qPLA6 gene in regulating the content of oil and fat components in rice, and the sequence of the qPLA6 gene is shown as SEQ ID NO. 1.

In a preferred embodiment, the grease component is one or more combinations of C16:0, C18:1, and C18: 2.

The invention also provides a method for regulating the content of oil and fat components in rice, which comprises the steps of introducing qPLA6 gene, improving the expression level of qPLA6 gene, reducing the expression level of qPLA6 gene or mutating qPLA6 gene to improve, reduce or eliminate the function of protein expressed by qPLA6 gene, wherein the sequence of the qPLA6 gene is shown as SEQ ID NO. 1.

For example, if the rice is excellent in other traits but the C16:0 content in rice is too high and the rice contains a qPLA6 gene with normal function, the C16:0 content in rice can be reduced by knocking out or mutating the qPLA6 gene in the rice to prevent the qPLA6 gene from being expressed, or by reducing or not expressing the acyl carrier protein thioesterase function of the expressed protein.

On the contrary, if the target rice species has excellent other properties but the content of C16:0 in rice is too low and the rice species does not contain the qPLA6 gene with normal function, the content of C16:0 in rice can be increased by introducing the qPLA6 gene or mutating the abnormal qPLA6 gene in the target rice species to restore and mutate the abnormal qPLA6 gene to the qPLA6 gene with normal function.

The invention also provides application of the qPLA6 gene as a screening marker of the content of the rice oil component in rice breeding, wherein the sequence of the qPLA6 gene is shown as SEQ ID NO: 1.

The invention also provides a rice cross breeding method, which comprises the step of selecting a target genotype by using the qPLA6 gene or a mutant thereof as a screening marker.

In a preferred embodiment, the rice cross-breeding method comprises the steps of:

s1: identifying the qpa 6 gene and its mutation status in the parent;

s2: selecting qPLA6 gene or mutant thereof as a screening marker;

s3: aggregating the screening markers with other dominant traits.

For example, although rice has excellent other properties, rice contains a qPLA6 gene with a high C16:0 content and a normal function, in order to obtain rice with higher unsaturated fatty acid by a cross breeding method, a parent with a mutation which causes the function of acyl carrier protein thioesterase to be reduced or disappeared in the qPLA6 gene is selected to be crossed with the rice, and the mutant gene is aggregated with other advantageous properties by using the qPLA6 gene mutant as a molecular marker.

On the contrary, the existing rice has excellent other properties, but the rice has too low content of C16:0 and does not contain qPLA6 gene with normal function, and in order to improve the content of C16:0 of the rice by a crossbreeding method, a parent with the function of the normal qPLA6 gene can be firstly selected to be crossbred with the rice, and the normal qPLA6 gene is used as a molecular marker to enable the mutant gene to be aggregated with other advantageous properties.

Drawings

FIG. 1 shows the percentage contents of 10 oil components in 533 rice varieties detected by GC-MS;

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

FIG. 3 is a Manhattan plot (Manhattan plot) of C16:0 content correlation analysis in indica subpopulations, with dashed lines representing significance threshold (-log)₁₀(P)＝7.06)

FIG. 4 is a Manhattan plot and Linkage Disequilibrium (LD) heatmap of the region around a significant peak on chromosome 6, with arrows indicating nucleotide variation sites of the candidate gene and vertical dashed lines indicating the candidate region around the peak;

FIG. 5 shows the gene structure of qPLA6 and the polymorphism of the gene;

FIG. 6 is a representation of the identification of qPLA6 as C16 in a Recombinant Inbred (RIL) population of ZS97/MH63 by linkage analysis: a statistical map of QTL scan results for 0-content major loci;

FIG. 7 is a C16:0 content statistical plot of different qPLA6 haplotypes, where test subjects of a are 305 indica rice varieties and test subjects of b are RIL populations derived from ZS97/MH 63;

figure 8 is a statistical plot of 10 fatty acid content in wild-type and knockout mutant families, P <0.05, P < 0.01; p < 0.001.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1. Sample source

The rice material used in the present invention includes natural population of 533 cultivars of cultivated rice and three self-bred recombinant populations. The natural population is used for whole genome association analysis, and the three recombinant inbred populations are used for linkage analysis.

Among them, 533 cultivars of rice included 50 cultivars of Australian rice (aus), 305 cultivars of indica (indica) and 178 cultivars of japonica (japonica).

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

All four groups are planted and grown in a test field of the university of Chinese agriculture in Wuhan, the four groups are sown in the middle of May, the four groups are transplanted to a field in the middle of June, the harvested paddy is aired and stored at room temperature for at least three months, and then the paddy is used for experiments.

2. Sample processing

Husking rice to obtain brown rice, grinding, sieving with 80 mesh sieve, drying at 80 deg.C for 24 hr, and extracting lipid. 0.2g of rice flour was put into a10 ml glass tube, and 4.5ml of a sulfuric acid-methanol solution (1:19) and 100. mu.l of a heptadecanoic acid standard solution in chloroform were added and mixed well. The preparation of the heptadecanoic acid standard solution was 1.07g of a heptadecanoic acid standard in 10ml of chromatographically pure chloroform. The mixture was incubated in a water bath at 85 ℃ for 2 h. After cooling to room temperature, fatty acid methyl esters were extracted using n-hexane and analyzed by gas chromatography-mass spectrometer (GC-MS). The sum of all identified fatty acid contents was taken as the oil content. Two tests were performed for each sample and the average was used for subsequent analysis.

3. GC-MS analysis results

The results of GC-MS analysis are shown in FIG. 1, and of the 10 fatty acids identified, palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2) accounted for more than 90% of the total fatty acid content. We found that there were wide variations in the oil-and-fat-related traits, 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 is in the range of 22.99-40.96 mg/g.

Of these, Australian rice (aus) had the highest C16:0 content, followed by indica (Ind) and japonica (Jap), and the opposite C18:1 and C18:2 contents (FIG. 2).

4. Association analysis and linkage analysis

Genome Wide Association Study (GWAS): GWAS was performed on indica, japonica and all samples, respectively. In each analysis group, only SNPs with a minor site frequency (MAF) > 5% and a deletion rate < 15% were selected for association analysis.

Linkage analysis: linkage analysis was performed on three RIL populations-ZS 97/MH63(96 strains), 88B-2/HD9802S (108 strains) and B805D/H6S (135 strains). These lines were all sequenced by second generation and then aligned to the rice reference genome with 50bp double-ended reads. Wherein, the sequences with the aligned mass more than or equal to 40 and the bases with the average mass more than or equal to 10 are used for identifying SNP. Genetic Bin maps were constructed for identifying Quantitative Trait Loci (QTLs) by using the MPR algorithm to infer the genotype of the parental lines.

Based on the above work, we finally obtained 520 ten thousand high quality SNPs for assessing population structure and affinity, and identified 23 significant association sites among them. In 23 loci, we found qPAL6 to be a major QTL associated with C16:0 content (fig. 3).

As shown in FIG. 4, qPAL6 is located at the end of the short arm of chromosome 6, and we are based on the point-to-point Linkage Disequilibrium (LD) index (r)²<0.6) the sequence in the region of 2.01Mb-2.28Mb (267kb) was analyzed, and it was finally determined that the candidate gene for the QTL was LOC _ Os06g05130 (sequence SEQ ID NO:1), encoding an acyl carrier protein thioesterase whose homologous gene in Arabidopsis was FATB (acyl-ACP thioesterase B) gene.

There are many base variations of LOC _ Os06g05130 gene, wherein the A-T mutation at 1096 th position on exon 6 (Ex6-A/T) has larger allele frequency, and the mutation causes the change from alanine (A) to threonine (T) (FIG. 5).

The genotype of the Ex6-a/T locus differed between indica rice varieties ZS97 and MH63, and QTL analysis of the ZS97/MH63 hybrid-derived RIL population demonstrated an effect of qPLA6 on C16:0 content (fig. 6, table 1).

TABLE 1 phenotypic analysis of lipid-related traits

Based on the A and T bases of the mutation sites, we divided qPla6 into haplotype A (SEQ ID NO:1) and haplotype B (SEQ ID NO: 2). The mean C16:0 content of haplotype B was significantly higher than haplotype a in 305 indica varieties; the mean C16:0 content of haplotype B was also significantly higher in the RIL population than haplotype A (FIG. 7).

5. Transgene analysis

In order to further determine the function of qPLA6, qPLA6 in ZS97 is knocked out by using a CRISPR-Cas9 system, in the preparation of a CRISPR/Cas9 knock-out construct, a gene coding region is carefully checked, and a 23bp target point with C-terminal NGG is designed, after the target point is determined, a 20bp target point is inserted into an intermediate vector pER8-Cas9-U6 or pER8-Cas9-U3 and is cloned to pCXUN-Cas9 to obtain a knock-out vector, namely KO-PLA6, the knock-out construct is introduced into an escherichia coli strainn 5 α competent cell for sequencing, after the correctness is verified, agrobacterium tumefaciens A EH105 is introduced, then the strain is transferred to ZS97, and after a transgenic plant is obtained, the genotype of the knock-out system is verified through sequencing.

Compared to the wild-type ZS97, the ZS97 knockout line KO-qPLA6 had significantly lower C16:0 content and significantly higher C18:1 and C18:2 content (fig. 8).

The above experiments show that qPLA6 is a main effective gene for positively regulating the content of C16: 0. The mutation of the gene can cause the reduction or disappearance of protein function, so that the content of C16:0 in rice is reduced, and the content of C18:1 and C18:2 is increased.

Although the above examples only show that the CRISPR-cas9 system is used to obtain the knockout mutant and the Ex6-A/T mutant, it will be understood by those skilled in the art after reading the disclosure of the present application that the gene is only needed to be transferred, knocked out, knocked down or mutated into related functions with reduced or no related functions, so as to achieve the purpose of regulating the content of C16:0, C18:1 and C18:2 in rice.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> university of agriculture in Huazhong

Hubei Shuangliu Shuanglv Biotech Co Ltd

Application of <120> qPLA6 gene in regulating or screening content of oil component in rice

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