A Wx allele for regulating very low amylose content in rice and its application

Abstract

The present invention belongs to the field of molecular genetics, and discloses a rice WxC2701T allele for regulating very low amylose content. In this gene, the cytosine (C) at the 20th position in the 12th exon of the coding region of the Wx gene in wild-type japonica rice Wuyunjing 8 is replaced by thymine (T). Its nucleotide sequence is as shown in SEQ ID No.1, the coding region sequence is as shown in SEQ ID No.2, and the amino acid sequence of the GBSSI protein encoded by the rice WxC2701T allele is as shown in SEQ ID No.3. The above WxC2701T allele significantly reduces the amylose content to a very low level, moderately reduces the transparency of rice grains; can significantly change the viscosity and digestion characteristics of starch, increase the content of rapidly digestible starch, and has little impact on the agronomic traits and yield of rice, and has strong application in the breeding of very low amylose rice.

Description

Technical Field

[0001] The present invention relates to a Wx allele of rice for regulating very low amylose content and its application in breeding, belonging to the field of molecular genetics. C2701T Background Art

[0002] The amylose content in rice endosperm determines the rice quality and its final use. Rice can be classified into glutinous rice (<5%), very low amylose content rice (5 - 12%), low amylose content rice (12 - 20%), medium amylose content rice (20 - 25%) and high amylose content rice (>25%) according to the amylose content. Among them, very low amylose content rice can be used as raw materials for soft rice breeding or rice wine brewing. The granule-bound starch synthase I (GBSSI) encoded by the Waxy (Wx) gene is responsible for synthesizing amylose and regulating its content. Different Wx alleles have significantly different effects on the regulation of amylose content in rice and its quality. At present, a large number of Wx alleles have been discovered. Most of them slightly adjust the amylose content or produce glutinous rice. There are few very low amylose content rice resources, which limits the application of very low amylose content rice in agricultural and industrial production. Therefore, exploring more Wx alleles of rice for regulating very low amylose content is of great significance for analyzing the regulation of starch synthesis and rice quality breeding. Summary of the Invention

[0003] The present invention aims to provide a Wx allele of rice for regulating very low amylose content and its application in breeding, especially in the breeding of very low amylose content rice. It significantly reduces the amylose content, moderately reduces the grain transparency, changes the viscosity and digestion characteristics of starch, but does not affect the agronomic traits and yield of rice, showing strong application prospects in the breeding of very low amylose content rice. C2701T

[0004] The technical solution provided by the present invention is as follows:

[0005] A Wx allele of rice for regulating very low amylose content, on the basis of the Wx gene of the wild-type rice variety Wuyunjing 8, the 20th C on the 12th exon is replaced by T; the nucleotide sequence of the Wx allele of rice is as shown in SEQ ID No.1. C2701T C2701T

[0006] Furthermore, its coding region sequence is as shown in SEQ ID No.2.

[0007] The present invention also provides a method for detecting the Wx allele of rice for regulating very low amylose content C2701T ​​​​Primers for alleles, wherein the sequence of the forward primer of the primers is as shown in SEQ ID NO: 4, and the sequence of the reverse primer is as shown in SEQ ID NO: 5.

[0008] The present invention also provides the above-mentioned rice Wx C2701T protein encoded by the allele, on the basis of the wild-type Wuyunjing 8 GBSSI protein, the 506th amino acid threonine (Thr) is replaced by isoleucine (Ile); the above-mentioned rice Wx C2701T The amino acid sequence of GBSSI encoded by the allele is as shown in SEQ ID No. 3.

[0009] The present invention also provides the above-mentioned rice Wx C2701T Application of the allele in breeding of very low amylose content rice.

[0010] Furthermore, the above-mentioned rice Wx C2701T allele reduces the amylose content in the endosperm.

[0011] Furthermore, the rice Wx C2701T allele reduces the grain transparency.

[0012] Furthermore, the rice Wx C2701T allele changes the viscosity and digestion properties of starch.

[0013] Furthermore, the target base of the gene described in claim 1 or 2 is changed by gene editing technology to change the target base of the target rice, and a rice material with target base substitution is obtained; the amylose content of the rice with the target base substitution is lower than that of the target rice.

[0014] Furthermore, the T0 generation plants are amplified with primers having sequences SEQ ID NO: 4 and SEQ ID NO: 5.

[0015] The present invention provides a Wx allele that regulates very low amylose content, which has the 20th base C replaced by T in the 12th exon of the coding region of the Wx gene (Genebank: NC_029261.1, https: / / www.ncbi.nlm.nih.gov / nuccore / NC_029261.1 ) in the background of Wuyunjing 8, and its gene nucleotide sequence is as shown in SEQ ID No. 1, and the coding region sequence is as shown in SEQ ID No. 2. The Wx C2701T allele of the present invention can provide excellent germplasm resources for breeding very low amylose content rice.

[0016] Beneficial effects

[0017] Currently, there is no such Wx C2701TReport on gene allelic variation, which is a new mutation of the Wx allele. The present invention screened a Wx from the endosperm mutant library of japonica rice Wuyunjing 8 mutagenized by EMS. C2701T A new allele, and the Wx provided by the present invention C2701T The new allele encodes granule-bound starch synthase I (GBSSI), which is mainly responsible for the synthesis of amylose and plays a key role in regulating the structure and functional properties of starch. This Wx C2701T The new allele can significantly reduce the amylose content to an extremely low level and moderately reduce the transparency of rice grains; it can significantly change the viscosity and digestion properties of starch, and has no obvious effect on the agronomic traits and yield of rice, and has strong application in the breeding of rice with extremely low amylose content. Description of the Drawings

[0018] Figure 1 This is the endosperm phenotype of the wild-type rice variety Wuyunjing 8 and the homozygous mutant M137 carrying the Wx C2701T new allele in the present invention. A is the appearance phenotype of brown rice and milled rice, B is the iodine absorption spectrum of starch, and C is the amylose content.

[0019] Figure 2 This is the viscosity and digestion properties of rice flour and starch of the wild-type Wuyunjing 8 and the homozygous mutant M137 carrying the Wx C2701T new allele in the present invention. A is the viscosity property of rice flour, B is the viscosity property of starch, C is the digestion property of native starch, and D is the digestion property of gelatinized starch.

[0020] Figure 3 This is the Wx of the wild-type Wuyunjing 8 and the homozygous mutant M137 carrying the Wx C2701T new allele in the present invention. A is the sequence analysis of the Wx C2701T gene, B is the amino acid sequence analysis of GBSSI, and C is the conservation analysis of the target amino acid with mutations in GBSSI of different species. C2701T gene sequence analysis, B is the amino acid sequence analysis of GBSSI, and C is the conservation analysis of the target amino acid with mutations in GBSSI of different species.

[0021] Figure 4 This is the functional verification of the Wx C2701T new allele in the present invention. A is the sequence analysis of the target site of the T0 generation mutant strain, B is the appearance phenotype of the iodine staining and non-staining of the grains and their cross-sections of the T1 generation homozygous mutant strain, and C is the iodine absorption spectrum of the grain starch of the T1 generation homozygous mutant strain.

[0022] Figure 5 This is the agronomic traits of the wild-type Wuyunjing 8 and the homozygous mutant M137 carrying the Wx C2701T new allele in the present invention. A is the effective panicle number per plant of rice, B is the number of filled grains per panicle, C is the 1000-grain weight of brown rice, D is the grain length of brown rice, E is the grain width of brown rice, and F is the grain thickness of brown rice. Detailed implementation manners

[0023] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following detailed description of the specific implementation manners of the present invention will be given in conjunction with specific embodiments.

[0024] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0025] 1. Experimental materials

[0026] Wild-type rice Wuyunjing 8 and its derived mutant M137, and wild-type rice Zhonghua 11 and its derived mutant ZH11 (Wx C2701T ) were used as experimental materials. M137 was screened from an EMS-induced endosperm mutant library of Wuyunjing 8, and ZH11 (Wx C2701T ) is a homozygous mutant created using the CRISPR / Cas9 single-base editing system (CBE) in the background of japonica rice Zhonghua 11 (ZH11) with the same Wx mutant gene carried by M137.

[0027] 2. Analysis of grain phenotypes and amylose content

[0028] A mutant M137 with a dark endosperm phenotype was screened from an EMS-induced endosperm mutant library of Wuyunjing 8. The phenotypes of brown rice and polished rice were observed under reflected light and transmitted light respectively. It was found that the brown rice and polished rice grains of Wuyunjing 8 were plump and transparent, with a small amount of chalkiness; while the transparency of the grains of M137 decreased, the brown rice grains showed a dark endosperm state, and the polished rice grains were semi-transparent ( Figure 1 A). The rice quality determination method of the People's Republic of China's agricultural industry standard (NY / T 83-2017) quantifies the transparency of rice into 5 grades: Grade 1 (>70%), Grade 2 (61-70%), Grade 3 (46-60%), Grade 4 (31-45%), and Grade 5 (<31%). The transparency of polished rice was analyzed using a rice appearance quality analyzer, and it was found that the transparency of the polished rice of Wuyunjing 8 was Grade 1, while that of M137 was Grade 3. During 2019-2021, significant differences were found in the starch iodine absorption spectra of Wuyunjing 8 and M137, and the absorbance of M137 decreased significantly ( Figure 1B). The amylose content of Wuyunjing 8 and M137 was analyzed by using concanavalin A (Con A) precipitation method (determined by the amylose / amylopectin assay kit from Megazyme, Ireland). The amylose content of M137 was 7.5%, which was significantly decreased by 50.0% compared with that of Wuyunjing 8 (15.1%) Figure 1 C). According to the previous classification criteria, the amylose content of M137 was between 5 - 12%, belonging to the very low amylose rice type. The above results indicate that M137 is a very low amylose rice mutant with translucent endosperm.

[0029] 3. Analysis of functional properties of rice flour and starch

[0030] The rapid viscosity analyzer can analyze the viscosity changes of starch during gelatinization. Parameters such as peak viscosity, hot paste viscosity, final viscosity, breakdown value, and setback value can be obtained from the RVA curve. The RVA results of rice flour and starch of Wuyunjing 8 and M137 both showed that the peak viscosity and breakdown value of M137 were significantly increased, while the hot paste viscosity, final viscosity, and setback value were significantly decreased, showing significantly different viscoelastic properties Figure 2 A,B). The digestion characteristics of starch in the human body can be simulated in vitro using α - amylase and amyloglucosidase. According to the digestion rate of starch, it can be divided into three categories: rapidly digestible starch (RDS) digested within 20 min, slowly digestible starch (SDS) digested within 20 - 120 min, and resistant starch (RS) not digested within 120 min. The digestion results of this study showed that compared with Wuyunjing 8, the RDS content of M137 native starch increased from 27.2% to 31.6%, an increase of 16.2%; the RDS content of gelatinized starch increased from 37.2% to 56.2%, an increase of 51.1% Figure 2 C,D). Therefore, the M137 starch is easily hydrolyzed.

[0031] 4. Identification of Wx new allele

[0032] Wx is mainly responsible for the synthesis of amylose. Usually, Wx gene mutation can cause a lower amylose content. Therefore, in this invention, the Wx gene of M137 was first sequenced. The genomic DNA of leaves of Wuyunjing 8 and M137 was extracted by CTAB method. Using the leaf DNA as a template, full - length segmented PCR amplification of the Wx gene was carried out. After the amplification products were identified by agarose gel electrophoresis, they were entrusted to Nanjing Qingke Biotechnology Company for sequencing. The sequencing results showed that the 20th base C (the 2701st base of the Wx gene) in the 12th exon of the Wx gene of M137 was replaced by TFigure 3 A), and it was named Wx C2701T , and its gene sequence is shown in SEQ ID No.1. This mutation causes the 506th amino acid of the GBSSI protein to be replaced by hydrophobic isoleucine (Ile) from hydrophilic threonine (Thr)( Figure 3 B), and its amino acid sequence is shown in SEQ ID No.3. The present invention further pasted the amino acid sequence of the wild type into the NCBI( http: / / www.ncbi.nlm.nih.gov / ) database for BlastP comparison, downloaded the homologous proteins of GBSSI in common species, and used the DNAMAN software to analyze the conservation of the mutant amino acids in different species. The results showed that the 506th threonine of GBSSI in rice was highly conserved in the GBSSI proteins of monocotyledonous and dicotyledonous species, including wild rice, barley, wheat, sorghum, millet, broomcorn millet, cassava, potato, grape, Chenopodium album, Eucalyptus grandis( Figure 3 C). Therefore, the variation at this site has a certain impact on the function of the GBSSI protein. After consulting the literature and relevant databases, the Wx mutation site of M137 has not been reported at present. Therefore, the mutant M137 is a new Wx allelic variation, and this site is highly conserved.

[0033] 5. Functional verification of the new Wx allele

[0034] To verify whether the semi-transparent phenotype of M137 grains and the extremely low amylose content are caused by the mutated Wx, the present invention commissioned Baige Gene Technology Co., Ltd. to replace the target base of Wx in ZH11 using the CRISPR / Cas9 single-base substitution editor under the background of japonica rice ZH11, and 70 T0 generation transgenic seedlings were obtained. The leaf DNA of the T0 generation transgenic positive plants was amplified using the Wx sequencing primers (forward primer: 5’-GGAGAAGTATCCGGGCAAGG-3’ (SEQ ID NO:4), reverse primer: 5’-CACCTTCTTCACGTCGCTTG-3’ (SEQ IDNO:5)). The results showed that the target base substitution occurred in 3 transgenic seedlings, that is, the 20th base C in the 12th exon of Wx was replaced by T( Figure 4 A). Therefore, 3 homozygous mutant strains with the replacement of the target amino acid of GBSSI were obtained under the background of ZH11. The mature grains of the above homozygous mutants in the T1 generation were analyzed, and it was found that the transparency of their grains decreased and showed a semi-transparent phenotype( Figure 4 B). Using the iodine colorimetric method, it was found that there were also obvious differences in the starch iodine absorption spectra between the mature grains of the above homozygous mutants in the T1 generation and ZH11, and the amylose content of the former decreased significantly( Figure 4C). These results indicate that the target base substitution of Wx under the ZH11 background produced a mutant phenotype consistent with M137, confirming that the new Wx allele is the regulatory gene for the semi-transparent grain phenotype and extremely low amylose content of M137 grains.

[0035] 6. Agronomic trait analysis of rice

[0036] To compare whether the agronomic traits of mutant M137 changed compared with the wild type Wuyunjing 8, the present invention selected 45 individual plants to analyze the effective panicle number per plant of rice and 30 individual plants to analyze the filled grain number per panicle of rice, and found that there were no significant differences in these parameters between M137 and Wuyunjing 8 ( Figure 5 A,B). By weighing the 1000-grain weight of brown rice using an analytical balance, it was found that there was no significant difference in the 1000-grain weight of brown rice between M137 and Wuyunjing 8 ( Figure 5 C); randomly selecting 30 grains of brown rice and measuring their grain length, grain width and grain thickness using a vernier caliper, it was found that the grain length of brown rice of M137 was slightly reduced compared with the wild type, and there were no significant changes in grain width and grain thickness ( Figure 5 D,E,F). The yield was analyzed by planting in plots (67 m 2 ). For Wuyunjing 8, it was 59.6 kg, and for M137, it was 57.7 kg. The above results indicate that the Wx C2701T allelic mutation of M137 does not affect the agronomic traits of rice.

[0037] Sequence Listing

[0038] SEQ ID No.1

[0039] Wx C2701T New allele DNA of Wx:

[0040]

[0041] SEQ ID No.2

[0042] Wx C2701T Novel allelic cDNA:

[0043]

[0044] SEQ ID No.3

[0045] Wx C2701T Amino acid sequence encoded by the new allele:

[0046] MSALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQQRSVQRGSRRFPSVVVYATGAGMNVVFVGAEMAPWSKTGGLGDVLGGLPPAMAANGHRVMVISPRYDQYKDAWDTSVVAEIKVADRYERVRFFHCYKRGVDRVFIDHPSFLEKVWGKTGEKIYGPDTGVDYKDNQMRFSLLCQAALEAPRILNLNNNPYFKGTYGEDVVFVCNDWHTGPLASYLKNNYQPNGIYRNAKVAFCIHNISYQGRFAFEDYPELNLSERFRSSFDFIDGYDTPVEGRKINWMKAGILEADRVLTVSPYYAEELISGIARGCELDNIMRLTGITGIVNGMDVSEWDPSKDKYITAKYDATTAIEAKALNKEALQAEAGLPVDRKIPLIAFIGRLEEQKGPDVMAAAIPELMQEDVQIVLLGTGKKKFEKLLKSMEEKYPGKVRAVVKFNAPLAHLIMAGADVLAVPSRFEPCGLIQLQGMRYGTPCACASIGGLVDTVIEGKTGFHMGRLSVDCKVVEPSDVKKVAATLKRAIKVVGTPAYEEMVRNCMNQDLSWKGPAKNWENVLLGLGVAGSAPGIEGDEIAPLAKENVAAP

[0047] SEQ ID NO:4

[0048] Wx C2701T Primer before PCR amplification of the new allele sequence

[0049] ggagaagtatccgggcaagg

[0050] SEQ ID NO:5

[0051] Wx C2701T Primer after PCR amplification of the new allele sequence

[0052] caccttcttcacgtcgcttg。

Claims

1. A rice variety with extremely low amylose content for regulation Wx C2701T The allele is characterized in that Based on the wild-type rice variety Wuyunjing 8 Wx gene, the 20th C on the 12th exon is replaced by T; the nucleotide sequence of the rice Wx C2701T allele is shown in SEQ ID No.

1.

2. The rice for regulating very low amylose content according to claim 1 Wx C2701T allele, characterized in that Its coding region sequence is as shown in SEQ ID No.

2.

3. The rice according to claim 1 or 2 Wx C2701T The protein encoded by the allele, characterized in that Based on the wild-type Wuyunjing 8 GBSSI protein, the 506th amino acid threonine (Thr) is replaced by isoleucine (Ile); the rice Wx C2701T The amino acid sequence of the GBSSI protein encoded by the allele is shown in SEQ ID No.

3.

4. Use of the allele in breeding of rice with very low amylose content according to claim 1 Wx C2701T ​ 5. The application according to claim 4, characterized in that, The rice mentioned above Wx C2701T The allele reduces the amylose content in the endosperm.

6. The application according to claim 4, characterized in that, rice Wx C2701T Alleles reduce grain transparency.

7. The application according to claim 4, wherein rice Wx C2701T Alleles alter the viscous and digestion properties of starch.

8. The application according to claim 4, characterized in that, Changing the target base of the gene described in claim 1 in the target rice by gene editing technology to obtain a rice material with the target base substituted; the amylose content of the rice with the target base substituted is lower than that of the target rice.

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