A rice amylose content regulating gene lr and the protein encoded by the gene and applications thereof

Abstract

The application belongs to the field of plant breeding and biotechnology, and particularly relates to how to reduce the amylose content of rice by using gene LR and the application of the gene in creating new germplasm of rice with low amylose content. The application discloses the nucleic acid sequence of the coding region of gene LR and the protein sequence coded by the gene, as shown in the ST.26 standard sequence nucleotide or amino acid sequence table. After the gene LR is knocked out by using the CRISPR-Cas9 technology, it is found that the amylose content of the rice of the transgenic plant with the knocked-out LR is significantly lower than that of the wild type variety. After the transgenic plant with the knocked-out LR gene is subjected to genetic function complementation by using the transgenic function complementation technology, the amylose content of the transgenic plant with the function complementation is not significantly different from that of the wild type variety. The application can create new germplasm of rice with low amylose content by using the LR gene.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding and biotechnology, specifically relating to a gene LR that regulates the amylose content of rice, its encoded protein, and its applications. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops, with more than half of the world's population relying on it as a staple food. In recent years, the demand for high-quality rice has become increasingly urgent. Rice quality mainly includes its cooking and eating quality, appearance quality, milling and processing quality, and nutritional quality. Existing research has shown that amylose content is one of the most important factors affecting the cooking and eating quality, appearance quality, and milling and processing quality of rice. Therefore, elucidating the genetic mechanism of rice amylose content is one of the keys to achieving high-quality rice breeding, and will provide a theoretical basis for high-quality, high-yield, and stable-yield bio-breeding of rice and other crops. The amylose content of rice is a key factor determining the cooking and eating quality of rice, and its genetic improvement has become an important aspect of current rice breeding research.

[0003] The genetic regulatory network of rice amylose content is highly complex. Currently, over 100 QTLs associated with rice amylose content have been identified. Among them, the waxy gene Wx, encoding the grain-binding starch synthase GBSSI, is the major gene regulating rice amylose content. It exhibits numerous allelic variants, including Wx... a Wx in Wx b Wx op wx, Wx mq Wx lv Wx mp Wx la and Wx mwIn addition, 10 genes related to rice amylose content have been cloned. These include three genes related to soluble starch synthases (SSSI, ALK, and SSIIIa), six genes related to ADP-glucose pyrophosphorylase (AGPL1, AGPL2, AGPL3, GPL4, AGPS1, and AGPS2), and one gene related to starch branching enzyme (SBEIIb). These previous findings are still insufficient to fully elucidate the genetic basis of rice amylose content, as many minor genes also participate in regulating rice amylose synthesis. Further analysis reveals that most studies on rice amylose content focus on enzymes related to starch synthesis in the rice endosperm, neglecting the influence of external environmental factors, such as biotic and abiotic stresses. Existing research has demonstrated that abiotic stresses such as pests, diseases, salt, drought, and high temperatures affect rice grain filling and thus rice amylose content. Leucine-rich repeats (LRRs) have been shown to be closely related to plant immunity and abiotic stress. To date, there are no reports in domestic or international research on how LRR protein regulates the amylose content in rice. Summary of the Invention

[0004] In view of the above-mentioned problems existing in current research on rice amylose content, this invention discloses a gene LR that regulates rice amylose content. This gene encodes an LRR protein, and the coding region sequence of the LR gene and the encoded protein sequence are disclosed, as shown in SEQ ID NO.1 and SEQ ID NO.2. This invention also discloses an application method for regulating rice amylose content using the LR gene and its encoded protein, comprising the following steps:

[0005] 1) Knockout of the LR gene was performed using CRISPR-Cas9 gene editing technology. When constructing the CRISPR-Cas9 knockout vector, the forward nucleotide sequence of one target site in the coding region of the LR gene was selected as 5′→3′: as shown in SEQ ID No. 3, and the forward nucleotide sequence of the other target site was also 5′→3′: as shown in SEQ ID No. 4;

[0006] 2) Obtaining homozygous transgenic mutant lines with the LR gene knocked out. After constructing the knockout vector, a conventional japonica rice variety, Nipponbare, was selected as the transformation background for genetic transformation, resulting in two homozygous knockout mutant transgenic lines. One knockout mutant had a 5′→3′ nucleotide sequence in the LR gene coding region, as shown in SEQ ID No. 5, and a 5′→3′ amino acid sequence, as shown in SEQ ID No. 7. The other knockout mutant had a 5′→3′ nucleotide sequence in the LR gene coding region, as shown in SEQ ID No. 6, and a 5′→3′ amino acid sequence, as shown in SEQ ID No. 8.

[0007] 3) Obtain homozygous transgenic functional complementation lines of the LR gene knockout mutant. Functional complementation of one of the knockout mutants from step 2) was verified using transgenic functional complementation techniques, resulting in two homozygous LR gene transgenic functional complementation lines.

[0008] 4) The amylose content and major agronomic traits in rice from wild-type, LR gene knockout transgenic mutants, and functionally complementary homozygous transgenic lines were determined. Using the national standard (GB / T 15683) to determine amylose content, it was found that the amylose content in rice from LR gene knockout mutants was significantly lower than that in wild-type, while plant height and grain shape showed no significant difference from wild-type. However, the amylose content in rice from LR gene functionally complementary homozygous transgenic lines showed no significant difference from wild-type.

[0009] This invention provides a gene LR that regulates amylose content in rice, its encoded protein, and its applications. The LR gene positively regulates amylose content in rice, and knocking out this gene can significantly reduce the amylose content in rice. This invention discloses the creation of a gene with lower amylose content by knocking out the LR gene and thereby altering the LR protein, which can be applied to rice quality improvement and has significant application value. Attached Figure Description

[0010] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0011] Figure 1 illustrates the technical method of knocking out the LR gene using CRISPR-Cas9 gene editing technology according to this invention. Figure A shows the map of the CRISPR knockout vector used; Figure B shows the information of the two target sites selected in the coding region of the LR gene and the nucleotide mutation types of the two homozygous knockout mutants obtained; Figure C shows the protein changes of the two homozygous knockout LR gene mutants. NIP represents the wild type (Nipponbare), and Cr-lr-1 and Cr-lr-2 are two homozygous knockout mutants. Figure 2 shows the amylose content of rice in the knockout LR gene mutants and functionally complementary transgenic homozygous lines obtained by the technical method of this invention. WT represents the wild type (Nipponbare), Cr-lr-1 and Cr-lr-2 are two homozygous knockout mutants, and CP-1 and CP-2 are two functionally complementary transgenic homozygous lines. ** indicates extremely significant (P < 0.01), ns indicates no significant difference; Figure 3 shows the plant height and grain shape of the knockout LR gene mutants obtained by the technical method of this invention. NIP is wild-type (Nipponbare), while Cr-lr-1 and Cr-lr-2 are two homozygous knockout mutants.

Claims

1. Genes LR Its application in regulating the amylose content of rice and the protein it encodes, characterized by, Knockout using CRISPR-Cas9 gene editing technology LR The gene can significantly reduce the amylose content in rice; LR The accession number of the gene in the rice genome is LOC_Os03g40250 The nucleic acid sequence of its coding region and the protein sequence it encodes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. The application according to claim 1, characterized in that, Two gene editing techniques were obtained using CRISPR-Cas9 gene editing technology. LR Gene knockout mutants, one type of knockout mutant LR The forward nucleotide sequence of the gene coding region is 5′→3′: as shown in SEQ ID No. 5, another knockout mutant LR The forward nucleotide sequence of the gene coding region is 5′→3′, as shown in SEQ ID No. 6.

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