CN111808865B - Application of WCR1 gene in regulating rice heart white rate or taste quality - Google Patents

Abstract

The invention relates to a novel pleiotropic heart white related gene WCR1, and the construction of transgenic materials is verified, and finally, functional mutation sites are found and corresponding molecular markers are developed, thereby providing effective theoretical basis and technical support for the improvement of rice chalkiness. The WCR1 gene is used, and genetic manipulation is carried out, so that the heart white rate can be reduced by about 20%, the polished rice rate can be improved by about 10%, and certain positive influence is also exerted on the yield and the taste value of a single plant, which is rare in production application. The invention provides a molecular marker with low heart white rate. The detection of the molecular marker can quickly identify the homozygous genotype single plant with low heart-white rate in the seedling stage, and eliminate the single plant with high heart-white rate in time, thereby not only saving the production cost, but also greatly improving the selection efficiency and greatly shortening the breeding period of rice varieties.

Description

Application of WCR1 gene in regulating rice heart white rate or taste quality

Technical Field

The invention relates to the field of rice breeding, in particular to application of a WCR1 gene in adjusting the rice heart white rate or taste quality.

Background

Rice (Oryza sativa L.) as one of the three major food crops in the world provides a food source for half of the population on the earth, and up to 76% of the caloric intake for people in more southeast Asia (Sasaki et al, 2000; Wing et al, 2018). With the improvement of living standard, people pay more and more attention to the quality of rice. Scientists introduced the concept of "green super rice" in the beginning of the 21 st century, where high quality was an important goal in rice breeding. Rice chalkiness refers to a white, opaque portion of the endosperm, which is an undesirable appearance quality attribute for consumers and the market, and also has some negative impact on process quality and cooked food quality. The 'high-quality rice standard' GB/T17891-2017 issued by China also takes chalkiness as an important index of high-quality rice. At present, the quality of rice of most examined varieties in China is relatively common, the requirement of the nation on the rice with excellent quality cannot be met, the phenomenon of strong rice and weak rice is deepened to a certain extent, and the import quantity of the rice in China is increased. Therefore, breeding rice varieties with improved appearance and taste quality of rice and reducing chalkiness content in rice are one of the concerns of breeders.

In traditional breeding, the appearance quality of rice is improved by crossing a high-yield line with a high-quality line and then continuously backcrossing the high-yield line to obtain lines with good yield and quality. However, chalkiness is a quantitative trait commonly influenced by multiple genes and environments, and needs to be selected in a larger population, so that trait investigation is time-consuming and labor-consuming, and the actual improvement process is difficult. Therefore, there is a need for molecular breeding methods that utilize the identified chalkiness-related molecular markers to screen for and retain favorable alleles. The method has the advantages that only genotype identification is needed, phenotype identification is not needed, manpower and material resources can be saved, and the method is very effective in improving the chalkiness character of rice and improving the quality.

Chalkiness is divided into abdominal, cardiac and dorsal whites according to where it occurs on the endosperm, and the trait is usually characterized by chalkiness grain rate (PGWC), chalkiness Area (ACE) and chalkiness Degree (DEC). In reported studies, although a number of chalkiness-associated Quantitative Trait Loci (QTLs) have been identified, involving multiple chromosomes in the rice genome, there are essentially no transgenes to validate the effects of these QTLs and lack functional molecular markers, and therefore cannot be effectively applied to production practice. Up to now, only one major gene, Chalk5, which controls the white abdomen, has been cloned (Li et al, Chalk5 encodes a vacuolar H + -translocating pyrophoric in fluidic in rice [ J ]. Nature Genetics,2014,46: 398).

Therefore, the isolation and cloning of the chalkiness-related genes and the development of functional molecular markers have become the primary task and key for chalkiness improvement.

Disclosure of Invention

A new pleiotropic cardiac related gene is identified by using natural population through a map-based cloning method, a transgenic material is constructed for verification, and finally, a functional mutation site is found and a corresponding molecular marker is developed, so that an effective theoretical basis and technical support are provided for chalkiness improvement of rice.

Based on the above, the invention provides the application of the WCR1 gene in regulating the rice heart white rate or taste quality.

In a specific embodiment, the application is the application of the promoter region sequence of the WCR1 gene as a rice low heart white rate marker, and the promoter region sequence of the WCR1 gene is shown as SEQ ID NO. 3. We have identified that the promoter region of WCR1 gene contains characteristic sequence, which can increase the expression of its coding sequence and show the character of reducing heart white rate phenotypically.

In a specific embodiment, the use of the coding region which is the WCR1 gene for modulating rice heart rate or taste quality. The overexpression box containing the coding region of the WCR1 gene is introduced into rice or the WCR1 gene is knocked out by a transgenic method, and the results show that the upregulation expression of the WCR1 gene caused by genetic manipulation can cause the reduction of the rice heart whiteness rate, the deletion or downregulation of the WCR1 gene caused by genetic manipulation can cause the increase of the rice heart whiteness rate, and the change of the expression level of the WCR1 gene can cause the change of the rice substance composition, so that the taste quality is changed. For example, the increase of the expression level of the WCR1 gene leads to the increase of the content of total starch, amylose and total fat, and simultaneously leads to the reduction of the content of gluten, albumin and total protein, thereby leading to certain improvement of the taste index, namely the taste value, such as viscosity, hardness, appearance and the like of the cooked rice. Of course, the WCR1 gene could be down-regulated to achieve the reverse effect and thereby match the taste of other people.

The invention also provides a method for regulating the heart-white rate of rice, which comprises the step of increasing or reducing the expression of the WCR1 gene in rice producing the rice or the step of knocking out the WCR1 gene.

In a specific embodiment, WCR1 gene expression in said rice is increased by introducing into said rice a overexpression cassette for the WCR1 gene.

In a specific embodiment, the overexpression cassette of the WCR1 gene is composed of a strong promoter and a WCR1 gene coding region under the control of the strong promoter, and the protein sequence coded by the WCR1 gene coding region is shown as SEQ ID NO. 1 or 2. Constitutive promoters with high expression activity in rice can be used for driving the expression of the WCR1 gene, such as the promoter of the BL130 WCR1 gene, the Maize ubiquitin promoter, other promoters capable of strong expression in rice and the like.

In a specific embodiment, the expression of the WCR1 gene in the rice is decreased by knock-down, or the expression of the WCR1 gene in the rice is removed by knock-out.

In one embodiment, the high efficiency WCR1 gene is introduced into rice containing the low efficiency WCR1 gene by crossing.

In a preferred embodiment, a molecular marker having the sequence shown in SEQ ID NO. 3 is used as a selection marker to select progeny containing the high efficiency WCR1 gene during hybridization.

In one embodiment, the method of reducing heart rate by hybridization comprises the steps of:

s1: taking a parent containing a high-efficiency WCR1 gene as a donor, taking a parent containing a low-efficiency WCR1 gene as a receptor, and carrying out hybridization to obtain a progeny F1;

s2: backcrossing F1 with the parent containing the low-efficiency WCR1 gene, using the screening marker to screen the offspring containing the high-efficiency WCR1 gene, and establishing a near-allelic gene line of the WCR 1;

s3: selfing is carried out by using the near allele line of the WCR1, and screening homozygous offspring containing high-efficiency WCR1 genes by using the screening marker, so that the material with low-heart-and-white-rate backbone is obtained.

The above method may be used to improve the taste quality.

The invention identifies a pleiotropic heart-white related gene WCR1, can reduce the heart-white rate by about 20 percent and improve the whole polished rice rate by about 10 percent through genetic operation, and has certain positive influence on the yield and the taste value of a single plant, which is rare in production application. The invention provides a molecular marker with low heart white rate. The detection of the molecular marker can quickly identify the homozygous genotype single plant with low heart-white rate in the seedling stage, and eliminate the single plant with high heart-white rate in time, thereby not only saving the production cost, but also greatly improving the selection efficiency and greatly shortening the breeding period of rice varieties.

Drawings

FIG. 1 is a graph of the results of the phenotype and predicted candidate genes of an important recombinant singleton set during the comparison of the map-based cloning of the cardiac gene WCR1 and agronomic traits of a near isogenic line;

FIG. 2 shows WCR1 ^B Near isogenic lines and WCR1 ^J Photo comparison of rice of near isogenic lines;

FIG. 3 shows the nucleotide polymorphism comparison of the promoter and coding region of the 5 WCR1 haplotypes between parents, and their line composition and average heart rate;

FIG. 4 shows WCR1 ^B Near isogenic lines and WCR1 ^J A rice agronomic character statistical chart of a near isogenic line;

FIG. 5 is a photograph comparison of rice of WCR1 gene complementation strain, overexpression strain and knockout strain;

FIG. 6 shows WCR1 ^B Near isogenic lines and WCR1 ^J A statistical graph of the expression quantity of the WCR1 gene in different tissues at different periods in an approximately isogenic line;

FIG. 7 is a photograph of GUS staining of various tissues of transgenic positive material driving GUS by WCR1 promoter;

FIG. 8 is a fluorescent micrograph of rice protoplasts expressing WCR1-YFP and Ghd 7-CFP;

FIG. 9 shows a WCR1 ^B Near isogenic lines and WCR1 ^J Scanning electron microscope photographs of endosperm cross sections of rice of near isogenic lines;

FIG. 10 shows WCR1 ^B Near isogenic lines and WCR1 ^J Total starch content, Amylose Content (AC), Total protein content of Rice of near isogenic lines, complementary strains (Positive and negative), over-expressed strains (Positive and negative), and knock-out strains (Positive and negative)Albumin content, gluten content, globulin content, prolamine content and taste value, wherein the taste value is the comprehensive score of viscosity, hardness and appearance of the cooked polished rice;

FIG. 11 is a histogram showing the statistics of WCR1 mRNA level and heart rate in group A oryza sativa and group B oryza sativa;

FIG. 12 is a photograph of rice of a WCR1 near isogenic line constructed with BL130 as a donor and 9311 as a recurrent parent.

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. Material selection and establishment of Recombinant Inbred Line (RIL) populations

And (3) selecting the high-chalk white rice variety Bei Lu 130(BL130) and the low-chalk white rice variety Jin 23B (J23B) to hybridize to obtain F1, continuously selfing progeny, and obtaining 184 strains of F6 as RIL population according to a single seed transmission method.

2. Phenotypic identification and genetic map construction

And (3) identifying the genotype by using an SSR method, screening polymorphic SSR primers, and obtaining 190 pairs of SSR markers with polymorphism and 2 pairs of InDel markers. The RM series primers were used to construct the genetic linkage map, and the gap was filled in with the InDel marker. Genetic linkage maps were generated using the "Kosambi" mapping function of the MAPMAKER/Exp3.0 software. The genetic map obtained finally had a total length of 1339.5cM and the average distance between the markers was 7.0 cM.

3. Preliminary location heart white rate QTL

A population of RIL containing 184 lines was planted for two consecutive years in the breeding base of this team in Hainan and test fields located at the university school of agriculture in Wuhan Huazhong, respectively. Each line was planted in 2 rows of 10 plants. The row spacing of the plants is 16.5cm multiplied by 26.4 cm. The heart white rate of the intermediate 8 strains was examined for each strain, and the mean of the 8 strains was taken as the heart white rate phenotype of the strain.

The identification of the QTL is based on genotype, phenotype and genetic linkage map, a composite interval mapping method of WinQTLCart2.5 software is utilized, and 2cM is taken as a step length to detect the QTL related to the heart white rate in the whole genome. The threshold value of QTL detection is LOD >2.5, namely that QTL influencing the heart white rate exists in the interval. Eventually 5 cardiac QTLs were detected, with qWCR1 of chromosome 1 located between RM315 and RM14, accounting for 6.4% of phenotypic variation. It can be seen that qWCR1 is likely to contain genes that affect heart white rate.

4. Fine localization and agronomic trait review of Gene WCR1

To fine-locate WCR1, we constructed BC with J23B as the background ₅ The generation is Near Isogenic Line (NIL). As shown in FIGS. 1 and 2, NIL-WCR1 ^B Has the performance which is significantly lower than NIL-WCR1 ^J Heart white rate of. At 3000 BC ₅ F ₂ Among the individuals, we identified 36 recombinant individuals that exchanged between the markers RM315 and RM 14. Using these recombinant individuals, we located WCR1 between W7 and W8, which is located at the promoter of LOC _ Os01g 69270. Therefore, we predicted LOC _ Os01g69270 to be a candidate gene for WCR1, which encodes an F-box protein. By sequencing, we identified two different alleles whose coding regions encode amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively (corresponding to WCR1, respectively) ^B And WCR1 ^J ). As a result of the alignment, an amino acid mutation (alanine) was caused in the coding region of the gene>Glycine), one deletion and one synonymous mutation of glycine; analyzing the 3' -UTR region to find an insertion/deletion; analysis of the upstream 2kb promoter region revealed 28 nucleotide polymorphisms that may cause differences in expression level (FIG. 3).

In addition, we examined the agronomic traits between NILs and the results showed that compared to NIL-WCR1 ^J ，NIL-WCR1 ^B The whole polished rice rate, the grain length, the grain width, the grain thickness, the thousand grain weight and the single plant yield are obviously increased (figure 4).

The prediction of the domain of the WCR1 protein was made by our conjecture that the three mutations in the coding region did not affect the function of the protein and thus were not due to the variation in the coding region, and the subsequent experiments also confirmed our conjecture.

5. Functional verification of WCR1

To further confirm the function of WCR1 gene, and to verify whether the low-heart-white phenotype is indeed caused by the mutation in the promoter region contained in BL130, we constructed complementation, overexpression and knock-out vectors for transfecting plants, respectively, and obtained transgenic positive plants. Wherein, the complementary strain (ZpZc) is obtained by transfecting an indica rice variety J23B with a promoter region 3.5kb upstream of a BL130 coding region and a coding region of J23B; the overexpression strain (OX) is a constitutive strong promoter (Maize ubiquitin promoter) and is obtained by transfecting a japonica rice variety middle flower 11 with a combination of a coding region of J23B; knock-out strains (KO) were obtained by deleting a partial sequence of exon 3 of the WCR1 coding region of flower 11 using the Cas9 editing system.

As a result, as shown in FIG. 5, the rice of both the complementation plants and the overexpression plants significantly decreased the heart-white rate, while the rice of the knockout plant significantly increased the heart-white rate, compared to the rice of the transgenic negative line. This indicates that the gene WCR1 is a gene that can affect the heart whitening rate of rice, and that the gene has a negative regulatory effect on the heart whitening rate of rice. Similar results were obtained by repeating the above experiment using the coding region of BL 130.

6. Spatio-temporal expression analysis of WCR1

We compared expression differences between NIL using fluorescent real-time quantitative PCR (RT-PCR). The results showed that WCR1 was expressed in root, stem, flag leaf, scion and endosperm, and the expression was most differentially expressed in endosperm, as indicated by NIL-WCR1 ^B Is significantly higher than NIL-WCR1 ^J (FIG. 6). We further analyzed the spatiotemporal expression characteristics of WCR1 using GUS staining. The GUS staining results were consistent with the RT-PCR results, showing that WCR1 was expressed in all roots, stems, stem nodes, flag leaves, leaf sheaths, leaf pillows, young ears, glumes, and endosperm of germinated seeds, with the deepest staining in young ears and seeds (fig. 7). Therefore, WCR1 is a gene constitutively expressed in rice, however, WCR1 ^B The expression activity of the polypeptide is obviously higher than that of WCR1 ^J This means that the difference between the two is likely to be due to the difference in expression regulatory sequences.

7. Subcellular localization and scanning Electron microscopy of WCR1

The expression position of the gene in the cell has great influence on the function of the gene, and understanding the subcellular localization of the gene is helpful to the prediction of the gene functionThe function of the gene has good reference value. We analyzed the amino acid sequence of WCR1 and found two nuclear localization signal peptides KRKRKR and RRKR at the N-terminus. Therefore, we transiently co-expressed WCR1-YFP and the nuclear marker Ghd7-CFP in rice protoplasts. The results showed that WCR1-YFP and Ghd7-CFP were co-located in the nucleus (FIG. 8). In addition, we also co-transformed WCR1-GFP, a nuclear tag and a membrane tag into Nicotiana benthamiana, and the same nuclear localization results were obtained. The morphology structure of starch particles is examined by using a Scanning Electron Microscope (SEM), and the result shows that NIL-WCR1 ^B The arrangement of starch granules in the center and at the edges of the endosperm of the centreless white seed is very dense, regular and without gaps. However, from NIL-WCR1 ^J The starch granules in the central endosperm of the heart-white seed are small and round, and have larger space gap, while the starch granules in the periphery of the endosperm are mixed with NIL-WCR1 ^B Are the same. The results show that the formation of the white heart is closely related to the structure and spatial arrangement of the starch (fig. 9).

8. Effect of WCR1 on stored Material and taste values

To investigate the effect of WCR1 on storage materials, we measured grain starch, protein, fat content and taste values using field grown NIL and seeds of transgenic material. NIL-WCR1 ^B ZpZc (+) and OX (+) significantly increased the total starch, amylose and total fat content while decreasing the gluten, albumin and total protein content relative to NIL-WCR1, respectively ^J ZpZc (-) and OX (-). Whereas the result for KO (+) is the opposite. Furthermore, NIL-WCR1 compared to control ^B And OX (+) has certain improvement effect on the taste index, namely the taste value, such as viscosity, hardness, appearance and the like of the cooked rice. These results indicate that WCR1 can affect the accumulation of storage materials and further improve the cooked taste quality (fig. 10).

8. Analysis of natural variation of WCR1

We aligned the sequences of 492 cultivars in the WCR1 promoter (about 2kb) and coding region in the rice core germplasm in this laboratory, and examined the heart-whitening rate of 235 of them (with the floury and brown rice removed). The sequence of 492 varieties was divided into 5 haplotypes (H1-5) based on the nucleotide variation of the WCR1 gene between the two parents. As a result, as shown in FIG. 3, we found that the breed containing the H1 haplotype (ClassA) had a lower heart white rate (40.13%), whereas the breed containing the H2-5 haplotype (ClassB) had a higher heart white rate (58.62%). Further analysis of the sequence differences of H1-5 revealed that H1 has a signature sequence (SEQ ID NO:3) around the promoter region-1696 bp; the sequence of H2 at the corresponding position was similar to H1, but the A (adenosine) at-1696 bp was replaced by G (guanylic acid); the sequence of H3-5 at the corresponding position is completely different from H1 and H2.

We then further analyzed the WCR1 expression level and the heart-white ratio of mature seeds in the endosperm at day 10 for some of ClassA and ClassB, and as a result, as shown in FIG. 11, the WCR1 expression level of ClassA is significantly higher than that of ClassB, while the heart-white ratio phenotype of ClassA is significantly lower than that of ClassB.

The above results suggest that the signature sequence of SEQ ID NO 3 of H1 is likely to be responsible for the increased expression of the WCR1 gene and for the low-heart-rate phenotype.

We further analyzed the H1 and H2 promoter (about 2kb) sequences, and found that there was only one SNP (A/G) (at-1696), and also analyzed the cis-elements near the site of this SNP (A/G), and found that the second nucleotides of the Dof Transcription Factors (TFs) binding core sequence T/AAAAG were altered from AAAAG to AGAAG. This means that natural variation a/G may affect the binding of Dof to the WCR1 promoter, thereby further interfering with Dof regulation of WCR1 transcript levels. In addition, we carried out phylogenetic analysis of WCR1 using the sequencing data of 492 parts of oryza sativa and 99 parts of oryza sativa, and showed that the sequence corresponding to the signature sequence of H1 in H2 originated from oryza sativa, while the signature sequence of H1 originated from japonica rice subgroup. Therefore, we speculate that the signature sequence of H1 may be an acquired-function variation generated during long-term acclimation and retained during phenotypic selection.

In order to verify the role of the characteristic sequence of H1 in rice chalkiness improvement, an NIL is constructed by multi-generation hybridization with BL130 as a male parent and 9311 as a recurrent parent, and the characteristic sequence of H1 is selected by MAS in the process,at BC ₄ F ₂ In the segregating population of (2), we examined the heart white rate phenotype and genotype and showed NIL-9311 ^BL The heart-white rate of the hybrid rice is obviously lower than that of NIL-9311 (figure 12), which indicates that the characteristic sequence of H1 may have important function and potential application value in breeding high-quality rice in hybrid breeding.

8. Application of WCR1 in rice chalkiness improvement

According to the study of the WCR1 gene, we can use the WCR1 gene as the basis to improve breeding for chalkiness or taste quality by means of transgenosis or hybridization.

In the aspect of transgenosis, the WCR1 gene coding sequence and a promoter with higher expression level (such as a promoter of a BL130 WCR1 gene, a Maize ubiquitin promoter and other promoters capable of strongly expressing in rice) are combined to form a WCR1 gene expression cassette and are introduced into a target rice plant, so that the heart white rate of the target rice plant is improved.

In the aspect of cross breeding, the characteristic sequence of H1 is used as a molecular marker for auxiliary selection, so that the planting area can be reduced, the breeding process is simplified, the manpower and material resources are saved, and the breeding efficiency is improved. Therefore, the material with H1 haplotype in rice germplasm material (such as BL130 in the present experiment) is used for the improvement of rice chalkiness.

The applicant selects a backbone material with high chalkiness and without H1 haplotype (most of the 'backbone materials' are from indica rice varieties and comprise conventional rice varieties, sterile lines and restorer lines with excellent production properties and chalkiness to be improved), and a cultivated rice material with H1 haplotype (part of japonica rice varieties such as BL130) to be hybridized to obtain a hybrid F ₁ Generation; f ₁ Then continuously backcrossing with the backbone material, selecting true hybrid individual plants by using the characteristic sequence of H1 as a molecular marker (for example, the true hybrid individual plants can be selected by amplifying by using a primer pair containing the sequences shown in SEQ ID NO:4 and 5, or can be selected by means of sequencing, probe or other means, only the offspring containing the characteristic sequence of H1 can be selected), backcrossing for multiple generations to obtain a near isogenic line, then selfing for one generation, and taking the characteristic sequence of H1 as a branchThe sub-marker is selected from a single strain with homozygous H1 haplotype, i.e., the modified low chalk skeleton material.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Sequence listing

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Application of <120> WCR1 gene in adjusting rice heart white rate or taste quality

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Claims (4)

1. A method for regulating the rice heart white rate comprises the step of improving the expression of WCR1 gene in the rice, a promoter region sequence shown as SEQ ID NO. 3 and a WCR1 gene sequence shown as SEQ ID NO. 1 or 2 of a gene coding protein sequence are introduced into the rice by a hybridization or transgene method, and the sequence shown as SEQ ID NO. 3 is used as a screening marker to screen filial generations containing the WCR1 gene coding sequence in the hybridization or transgene process, so that the rice material with low heart white rate is obtained.

2. The method of adjusting rice heart rate according to claim 1, comprising the steps of:

s1: using a parent containing a promoter region sequence shown as SEQ ID NO. 3 and a WCR1 gene sequence shown as SEQ ID NO. 1 or 2 of a gene coding protein sequence thereof as a parent donor, using a parent containing a promoter region sequence shown as aaaaaaaagg ctaccgagaa tgtctttcta gggaagc and a WCR1 gene sequence shown as SEQ ID NO. 1 or 2 of a gene coding protein sequence thereof as a parent receptor, and hybridizing the parent donor and the receptor to obtain a filial generation F1;

s2: backcrossing the F1 progeny with the parent receptor in the S1 step, screening the progeny containing the promoter region sequence shown in SEQ ID NO. 3, and establishing a near allele line of the WCR 1; s3: selfing the WCR1 near allele line, and screening homozygous progeny containing the sequence shown in SEQ ID NO. 3 to obtain the rice material with low heart-white rate.

3. Use of the method of regulating a rice heart-white rate according to claim 1 for regulating a rice heart-white rate.

4. The use of the method for regulating rice heart white percentage according to claim 1 for regulating the quality of rice taste by regulating the expression level of the coding region of the WCR1 gene through the promoter region sequence shown in SEQ ID NO. 3.

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