A new allele regulating low amylose content in rice and its breeding application

Abstract

The present invention discloses a new allele Wxy57 for regulating the amylose content of rice, belonging to the technical field of molecular genetics. The guanine deoxynucleotide (G) at position 61 of the 3rd exon of the rice Wx gene is replaced by an adenine deoxynucleotide (A), and the gene contains a genome sequence as shown in SEQ ID No.1 and a coding region sequence as shown in SEQ ID No.2 and SEQ ID No.3. The present invention also discloses an encoded protein, which contains an amino acid sequence as shown in SEQ ID No.4. The new allele for regulating the amylose content of the present invention can also be applied to the breeding of new low-amylose rice varieties.

Description

Technical Field

[0001] The invention relates to a new allele Wxy57 for regulating low amylose content in rice and application thereof, and belongs to the field of molecular genetics. Background Art

[0002] The content of amylose is the main determinant of the taste quality of rice. Generally speaking, rice varieties with an amylose content of more than 20% have a poor taste, while rice varieties with an amylose content of less than 15% to 20% have a better taste. Low-amylose soft rice is a new type of rice with an amylose content between glutinous rice and sticky rice, with an amylose content between 5% and 12%. Due to its soft and elastic texture, no cold regeneration, good palatability and puffing properties, it is deeply loved by people and has great market demand and market value (Zhu Dawei et al., Journal of Yangzhou University, 2015, (1): 47-52; Zhang Changquan et al., Chinese Agricultural Science, 2016, 49 (22): 4267-4283).

[0003] The cultivation of soft rice in my country is mainly distributed in the southwest, especially in Yunnan. Yunnan has a long history of soft rice cultivation. The low amylose trait of the soft rice varieties is the result of natural variation of related genes in wild rice, and is preserved due to the eating habits of local people. Among them, Haopi and Haomuxi are typical representatives, listed as national high-quality and superior rice, and often used to entertain guests. In Yunnan, a series of high-quality soft rice varieties have been cultivated, including Yunhui 290, Diantun 502, Yinguang, Yunjing 20, Yunjing 29, Yunjing 37, Yunjing 41, Chujing 39, and Chujing 48. According to statistics, in recent years, my country's soft rice is mainly distributed in the southwest, accounting for 53% of the total soft rice in my country, followed by Central China and South China, and the planting area in East China, Northeast China, North China, Northwest China and other places is relatively small. With the increasing attention paid to the breeding and development of soft rice in Jiangsu and Northeast China, it is expected that the area in East China and Northeast China will increase significantly, and the proportion will also increase. As people's living standards improve, the demand for high-quality soft rice will increase rapidly.

[0004] Reducing the amylose content in rice is one of the effective ways to improve the taste quality of rice. However, the low-amylose gene resources currently available in production are extremely scarce, mainly including Wxmq used in the japonica soft rice Nanjing series and Wxop / hp used in Yunnan Province's high-quality indica soft rice varieties such as Haob, Yunhui 290, and Diantun 502. There is an urgent need to clone and utilize new excellent low-amylose soft rice genes or alleles.

[0005] Yunjing 57 is a soft rice material selected by us. This variety is going through the variety approval procedure and will soon be promoted and applied in Yunnan production. The rice appearance of Yunjing 57 is turbid and opaque. Through the research of this invention, it was found that the Wx gene of this variety had a single base substitution, but the amylose content was about 9%, and the rice quality was between glutinous rice and sticky rice, which belongs to the soft rice type. Using new soft rice resources and exploring new low amylose content regulatory genes have important theoretical and practical significance for enriching the soft rice regulatory gene library and analyzing the formation mechanism of soft rice. Summary of the invention

[0006] Technical issues:

[0007] The present invention aims to provide a new allele for regulating low amylose content in rice and its application, which can be used in breeding for controlling low amylose content in rice.

[0008] Technical solution:

[0009] The present invention provides a new allele Wxy57 for regulating the amylose content of rice, wherein the guanine deoxynucleotide (G) at position 61 of the third exon of the rice Waxy gene with gene accession number EU770319 is replaced by an adenine deoxynucleotide (A), and the allele contains a genome sequence as shown in SEQ ID No. 1 and coding region sequences as shown in SEQ ID No. 2 and SEQ ID No. 3.

[0010] The present invention also discloses a protein encoded by Wxy57, which contains an amino acid sequence as shown in SEQ ID No.4.

[0011] The gene of the present invention can be used in improving the quality of rice.

[0012] Beneficial effects:

[0013] There is no report of this allele variation of Waxy gene. The present invention found that the guanine deoxynucleotide (G) at position 61 of exon 3 of Waxy (Wx) gene of Yunjing 57 with gene accession number EU770319 was replaced by adenine deoxynucleotide (A), and the sequence is shown in SEQ ID NO.1. This mutation caused the corresponding aspartic acid (D) at position 134 to be replaced by asparagine (N). Therefore, the Wx gene of Yunjing 57 is a new allele variation occurring on the basis of Wxb type, named Wxy57.

[0014] The present invention provides a protein related to the amylose content of rice and a gene Wxy57 encoding the protein. This gene encodes granule-bound starch synthase I (GBSS I), which is directly responsible for the synthesis of amylose in rice endosperm and plays a key role in the cooking and eating quality of rice. The rice grains carrying the homozygous genotype of Wxy57 have an amylose content of about 9%, belonging to the typical soft rice type.

[0015] Through the research of the present invention, it is found that a single-base substitution occurs in the Wx gene of this variety, with an amylose content of about 9%. The rice quality is between glutinous rice and sticky rice, belonging to the soft rice type. Utilizing the new soft rice resources and exploring new genes regulating low amylose content have important theoretical and practical significance for enriching the soft rice regulatory gene pool and analyzing the formation mechanism of soft rice. By using conventional technical means such as hybridization and backcrossing, this gene can be introgressed into other rice varieties to cultivate new soft rice materials. Brief Description of the Drawings

[0016] Figure 1 Schematic diagram of the appearance of rice grains;

[0017] Figure 2 Cross-sectional I2-KI color reaction of rice grains (A is the cross-sectional view before staining, B is the cross-sectional view after I2-KI staining);

[0018] Figure 3 Amylose content of rice grains;

[0019] Figure 4 Sequence alignment of the 3rd exon of the Wx gene (Nipponbare is Nipponbare, Yunjing57 is Yunjing 57);

[0020] Figure 5 Amino acid sequence alignment of the Wx gene encoding (Nipponbare is Nipponbare, Yunjing57 is Yunjing 57);

[0021] Figure 6 Expression of the Wxy57 allele at different times;

[0022] Figure 7 Activity of GBSSI encoded by Wxy57 at different times. Specific Embodiment

[0023] The embodiments of the present invention will be described in detail below in conjunction with specific implementation steps. For those not specified in the implementation steps, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents and instruments not specified for the manufacturer, they are all conventional products that can be obtained through market purchase. The specific implementation steps are as follows:

[0024] 1. Test Materials

[0025] Nipponbare, Shengnuo 18 and 9311 are all public known and public materials, which can be provided free of charge by the China Agricultural Germplasm Resource Bank. Yunjing 57 is a soft rice material that we are currently going through the variety approval process.

[0026] 2. Rice appearance and amylose content determination

[0027] The seeds were dried until the moisture content was about 14%, and then ground into polished rice. It was found that the rice of Yunjing 57 was turbid and opaque, the rice of Shengnuo 18 was milky white and opaque, and the rice of Nipponbare and 9311 was translucent ( Figure 1 , Figure 2 .A). The results of the I2-KI staining reaction of rice showed that the glutinous rice variety Shengnuo 18 was yellow-brown after staining; at the same staining time, Nipponbare and 9311 showed a darker blue-purple color, while Yunjing 57 showed a lighter blue-purple color ( Figure 2 .B). Further determination of the amylose content revealed that the amylose content of Yunjing 57 in Chuxiong was 9.35±0.23%, which was higher than that of the glutinous rice variety Shengnuo 18 (amylose content was 1.58±0.13%), and lower than that of the japonica rice variety Nipponbare (amylose content was 15.64±0.47%) and the indica rice variety 9311 (amylose content was 14.97±0.36%) ( Figure 3 ). Therefore, Yunjing 57 is a typical low-amylose variety, and the amylose content of the variety grown in Yunnan is about 9%.

[0028] 3. Genetic analysis

[0029] A hybrid combination was prepared with Yunjing 57 as the male parent and Nipponbare as the female parent, and the F0 hybrid was harvested; the F0 was planted, and the main ear F1 seeds of one F0 plant were randomly taken, dried and ground into polished rice, and a total of 155 polished rice grains were obtained, and genetic analysis was performed based on appearance and I2-KI display. 42 turbid rice grains with I2-KI coloring of light blue-purple and 113 translucent rice grains with I2-KI coloring of dark blue-purple were detected, and the chi-square test (2=0.26<20.05=3.84) showed that the segregation ratio of turbid rice grains with I2-KI coloring of light blue-purple and transparent rice grains with I2-KI coloring of dark blue-purple met the ratio of 1:3, indicating that the low amylose trait of Yunjing 57 is controlled by a pair of recessive nuclear genes.

[0030] 4. Gene cloning

[0031] The Wx gene is the master gene regulating the content of amylose in rice. We first sequenced the Wx gene of Yunjing 57. The genomic DNA of Yunjing 57 leaves was extracted by CTAB method, and PCR amplification was performed using the extracted genomic DNA as a template. The upstream primer used for amplification: 5'-CTCAAGACACAAATAACTGC-3', downstream primer: 5'-GTACCTGTCTGCAACCTTGA-3'. PCR system: DNA template (50ngμL-1) 1μL, 10×PCRbuffer 2.5μL, dNTP (2mmol L-1) 2μL, upstream and downstream primers (10μmol L-1) 1μL each, Taq DNA polymerase (5UμL-1) 0.2μL, ddH2O 17.3μL, a total of 25uL. Amplification conditions: 94℃5min; 94℃30s, 56℃30s, 72℃2min, 35 cycles; 72℃ extension 5min, end the reaction. PCR reaction was carried out in an EppendorfMastercycle thermal cycler. 5uL of amplified product was separated and identified by agarose gel electrophoresis, and the remaining PCR products that met the band size were sent to Kunming Qingke Biotechnology Co., Ltd. for sequencing. Vector NTI 11.5 software was used to align the sequence of Yunjing 57 with the Wx gene of Nipponbare with the gene accession number EU770319 in the database. It was found that the first base of the first intron of the Wx gene of Yunjing 57 was T, which belongs to the Wxb type. In addition, the guanine deoxynucleotide (G) at the 61st position of the 3rd exon of the Wx gene of Yunjing 57 was replaced by an adenine deoxynucleotide (A) ( Figure 4 ), the sequence is shown in SEQ ID NO.1. This mutation causes the corresponding aspartic acid (D) at position 134 to be replaced by asparagine (N) ( Figure 5 ). So far, there has been no report of this allele variation of the Wx gene. Therefore, the Wx gene of Yunjing 57 is a new allele variation based on the Wxb type and is named Wxy57.

[0032] 5. Analysis of Wxy57 gene transcripts in Yunjing 57

[0033] RNA was extracted from the endosperm of Yunjing 57 and reverse transcribed into cDNA. PCR amplification was performed using cDNA as a template. The upstream primer used for amplification was 5'-AGCCACCCACACCACCACC-3', and the downstream primer was 5'-TGGTATAATATGGAACAGGGG-3'. PCR system: cDNA template (50ngμL-1) 1μL, 10×PCR buffer 2.5μL, dNTP (2mmolL-1) 2μL, upstream and downstream primers (10μmol L-1) 1μL each, Taq DNA polymerase (5UμL-1) 0.2μL, ddH2O 17.3μL. Amplification conditions: 94℃5min; 94℃30s, 55℃30s, 72℃3.7min, 35 cycles; 72℃ extension 5min, end the reaction. PCR was performed in an EppendorfMastercycle thermal cycler. The amplified products were separated by agarose gel electrophoresis, the target bands were recovered by gel cutting, and sent to Kunming Qingke Biotechnology Co., Ltd. for sequencing. The cDNA sequence of Yunjing 57 was compared with the gene sequence of Yunjing 57 using Vector NTI 11.5 software, and it was found that the Wx gene of Yunjing 57 can produce two transcription products, as shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. The full length of the SEQ ID NO.2 transcript is 3129 bp, and its first intron has not been cut off. The full length of the SEQ ID NO.3 transcript is 2006 bp, and its first intron has been cut off. The splicing pattern of Wxy57 is the same as that of the reported Nipponbare Wx gene (Wxb) (Wang et al., Plant Journal, 1995, 7(4): 613-622). The difference in the splicing products is that the guanine deoxynucleotide (G) at position 61 of exon 3 is replaced by adenine deoxynucleotide (A), which results in the replacement of the corresponding aspartic acid (D) at position 134 by asparagine (N). Figure 5 ) whose amino acid sequence is shown in SEQ ID NO.4.

[0034] 6. Tissue expression analysis of Wxy57 gene in Yunjing 57

[0035] The Wx gene encodes granule-bound starch synthase I (GBSSI), which is responsible for amylose synthesis in the endosperm and is mainly expressed in the immature embryo (Zhou et al., Journal of Integrative Plant Biology, 2021, 63(5), 878-888). RNA was extracted from the endosperm of Yunjing 57 and Nipponbare at six stages: flowering, 5 days after flowering, 10 days after flowering, 15 days after flowering, 20 days after flowering, and 25 days after flowering, and reverse transcribed into cDNA. OsActin-1 was used as the internal reference gene, and real-time fluorescence quantitative PCR was used to identify the expression level of the Wx gene. Figure 6 As shown in the figure, in Yunjing 57 and Nipponbare, the expression level of Wx gene was very low when it just bloomed, and the expression level increased sharply 5 days after flowering, reaching the highest expression level 10 days after flowering, and the expression level showed a slow downward trend 15, 20, and 25 days after flowering. Compared with Nipponbare, the expression difference of Wx gene in Yunjing 57 at different stages was not significant, indicating that the replacement of guanine deoxynucleotide (G) at position 61 of exon 3 by adenine deoxynucleotide (A) did not affect the expression level of Wx gene in endosperm.

[0036] 7. Activity determination of GBSSI encoded by Wxy57 gene

[0037] Granule-bound starch synthase ELISA kit (Shanghai ELISA Biotechnology Co., Ltd. 076667) was used to detect the activity of GBSSI encoded by the Wxy57 gene. Fresh grains of Yunjing 57 and Nipponbare were harvested 5, 10, 15, 20 and 25 days after flowering, and the endosperm, embryo skin and embryo were ground into powder in liquid nitrogen after removing the glumes. The powder was placed in a 1.5 ml centrifuge tube and the enzyme activity was determined according to the manufacturer's instructions. The results are shown in Figure 7 As shown in the figure, in both Yunjing 57 and Nipponbare, GBSSI has high enzyme activity 5 days after flowering, reaches the highest value 10 days after flowering, and shows a downward trend 15, 20, and 25 days after flowering. Compared with Nipponbare (Wxb), the activity of GBSSI in Yunjing 57 (Wxy57) was significantly reduced at each time point, indicating that the replacement of guanine deoxynucleotide (G) at position 61 of exon 3 by adenine deoxynucleotide (A) affected the enzyme activity of the corresponding encoded GBSSI.

Claims

1. A rice amylose content allele, characterized in that: The gene Wxy57 is a rice Waxy gene with gene accession number EU770319, in which the 61st guanine deoxynucleotide (G) of the 3rd exon is replaced by an adenine deoxynucleotide (A); the Wxy57 genomic DNA sequence is shown in SEQ ID No.

1.

2. The gene according to claim 1, characterized in that: The gene Wxy57 contains two transcription products, which are shown as SEQ ID No.2 and SEQ ID No.3 respectively.

3. The protein encoded by the gene according to claim 1 or 2, characterized in that: Its amino acid sequence is shown in SEQ ID No.

4.

4. Use of the gene according to claim 1 or 2 in improving rice quality, characterized in that: The application refers to the application in regulating the low amylose content of rice.

Images