Novel gene LR for regulating and controlling amylose content of rice, protein coded by novel gene LR and application of novel gene LR

Abstract

The invention belongs to the technical field of plant breeding and biology, and particularly relates to how to reduce the amylose content of rice through a gene LR and application of the gene LR to creation of a novel rice germplasm with medium and low amylose content. The invention discloses a nucleotide sequence of a gene LR coding region and a coded protein sequence as shown in an ST.26 standard sequence nucleotide or amino acid sequence table. After the gene LR is knocked out by using a CRISPR-Cas9 technology, the amylose content of rice of the LR-knocked-out transgenic plant is obviously lower than that of a wild type variety; after genetic function complementation is carried out on the LR gene knockout transgenic plant by utilizing a transgenic function complementation technology, the rice amylose content of the functional complementation transgenic plant is not obviously different from that of a wild type variety. According to the invention, the LR gene can be utilized to create a novel rice germplasm with low rice amylose content.

Description

Technical Field

[0001] The present invention belongs to the field of plant breeding and biotechnology, and specifically relates to a new gene for regulating the amylose content of rice. LR and its encoded proteins and applications. Background Art

[0002] Rice ( Oryza sativa L.) is one of the most important food crops, and more than half of the world's population relies on rice as their staple food. In recent years, people's demand for high-quality rice has become increasingly urgent. Rice quality mainly includes cooking and eating quality, appearance quality, milling and processing quality, and nutritional quality. Studies have shown that the content of amylose is one of the most important factors affecting the cooking and eating quality, appearance quality, and milling and processing quality of rice. Therefore, the analysis of 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 the biological breeding of high-quality, high-yield, and stable yields of crops such as rice. The content of amylose in rice is a key factor in determining the cooking and eating quality of rice, and its genetic improvement has become an important part of current rice breeding research.

[0003] The genetic regulatory network of rice amylose content is very complex. At present, more than 100 QTLs related to rice amylose content have been located, including the wax gene encoding the granule-bound starch synthase GBSSI. Wx It is the major gene regulating the amylose content of rice, and there are many types of allele variation, including Wx a , Wx in , Wx b , Wx op , wx , Wx mq , Wx lv , Wx mp , Wx la and Wx mw In addition, 10 genes related to the amylose content of rice have been cloned, including three genes related to soluble starch synthase SSSI , ALK and SSIIIa , and 6 genes related to ADP glucose pyrophosphorylase: AGPL1 , AGPL2 , AGPL3 , AGPL4 , AGPS1 and AGPS2, and genes related to starch branching enzymes SBEIIb . These previous research results are still insufficient to analyze the genetic basis of rice amylose content, and many micro-effect genes are also involved in regulating the synthesis of rice amylose. Further analysis found that most of the research related to rice amylose content focused on enzymes related to starch synthesis in rice endosperm, while ignoring the impact of the external environment, such as biotic and abiotic stresses. Studies have shown that abiotic stresses such as pests and diseases, salt, drought and high temperature can affect rice filling and thus affect rice amylose content. Leucine-rich repeat proteins (LRR) have been shown to be closely related to plant immunity and abiotic stress. So far, there have been no reports on LRR proteins regulating rice amylose content in domestic and foreign studies. Summary of the invention

[0004] In view of this, the present invention aims at the above-mentioned problems existing in the current research on the content of amylose in rice and discloses a new gene for regulating the content of amylose in rice. LR , which encodes an LRR protein, is disclosed LR The coding region sequence of the gene and the encoded protein sequence are shown in SEQ ID NO.1 and SEQ ID NO.2 in the ST.26 standard sequence nucleotide or amino acid sequence table.

[0005] The present invention also discloses a method of utilizing LR The application method of the gene and its encoded protein for regulating the content of amylose in rice comprises the following steps: 1) Using CRISPR-Cas9 gene editing technology LR When constructing a CRISPR-Cas9 knockout vector, select LR The nucleotide forward sequence of one target site on the gene coding region is 5′→3′: as shown in SEQ ID No. 3 in the ST.26 standard sequence nucleotide or amino acid sequence table, and the nucleotide forward sequence of another target site is 5′→3′: as shown in SEQ ID No. 4 in the ST.26 standard sequence nucleotide or amino acid sequence table; 2) Obtain knockout LR After the knockout vector was constructed, a conventional japonica rice variety Nipponbare was selected as the transformation background for genetic transformation, and two homozygous knockout mutant transgenic lines were obtained, one of which was a knockout mutant. LRThe nucleotide forward sequence of the gene coding region is 5′→3′: as shown in SEQ ID No.5 in the ST.26 standard sequence nucleotide or amino acid sequence table, and the amino acid forward sequence is 5′→3′: as shown in SEQ ID No.7 in the ST.26 standard sequence nucleotide or amino acid sequence table; another knockout mutant LR The nucleotide forward sequence of the gene coding region is 5′→3′: as shown in SEQ ID No.6 in the ST.26 standard sequence nucleotide or amino acid sequence table. The amino acid forward sequence is 5′→3′: as shown in SEQ ID No.8 in the ST.26 standard sequence nucleotide or amino acid sequence table; 3) Obtain knockout LR Transgenic functional complementation homozygous strains of gene mutants. Transgenic functional complementation experimental technology is used to verify the functional complementation of a knockout mutant in step 2), and two homozygous LR Homozygous strains for gene transgenic functional complementation; 4) Determination of wild type and knockout LR The amylose content and main agronomic traits of transgenic mutants and functionally complementary transgenic homozygous rice lines of the gene were determined by using the national standard (GB / T 15683). LR The amylose content of rice in the mutant of the gene was significantly lower than that in the wild type, while the plant height and grain shape were not significantly different from those in the wild type. LR There was no significant difference in the amylose content of rice in the homozygous transgenic line with gene function complementation and that in the wild type.

[0006] The present invention provides a new gene for regulating the content of amylose in rice LR and its encoded protein and application, LR The gene positively regulates the amylose content of rice, and knocking out the gene can significantly reduce the amylose content of rice. LR The gene can then change the LR protein to create a gene with lower amylose content, which can be used to improve rice quality and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 The present invention utilizes CRISPR-Cas9 gene editing technology to knock out LR A is a map of the CRISPR knockout vector used; B is a map of the LRInformation on the two target sites selected in the gene coding region and the types of nucleotide mutations of the two homozygous knockout mutants obtained; Figure C shows the protein changes of the two homozygous knockout LR gene mutants. NIP is the wild type (Nipponbare), Cr-lr-1 and Cr-lr-2 There are two homozygous knockout mutants.

[0008] Figure 2 The knockout obtained by the technical method of the present invention LR Amylose content of rice in gene mutants and functionally complemented transgenic homozygous lines. WT is wild type (Nipponbare), Cr-lr-1 and Cr-lr-2 There are two homozygous knockout mutants. CP-1 and CP-2 Two homozygous transgenic lines with complementary functions. ** indicates extremely significant ( P <0.01), ns means the difference is not significant.

[0009] Figure 3 The knockout obtained by the technical method of the present invention LR Plant height and grain shape of gene mutants. NIP is the wild type (Nipponbare), Cr-lr-1 and Cr-lr-2 There are two homozygous knockout mutants.

Claims

1. A new gene regulating the amylose content in rice LR and the protein encoded by it, characterized in that Said LR The accession number of the gene in the rice genome is LOC_Os03g40250 The nucleic acid sequence of its coding region and the encoded protein sequence are shown as SEQ ID NO.1 and SEQ ID NO.2 in the ST.26 standard sequence nucleotide or amino acid sequence table, respectively.

2. A method of using the method according to claim 1 LR The application of the gene and its encoded protein in regulating the content of amylose in rice is characterized in that: Knockout using CRISPR-Cas9 gene editing technology LR The gene can significantly reduce the amylose content of rice.

3. The use according to claim 2, characterized in that: Knockout LR After the gene was knocked out, the main agronomic traits of the mutant did not change significantly.

4. The use according to claim 2, characterized in that: When constructing a CRISPR-Cas9 knockout vector, select LR The nucleotide forward sequence of one target site on the gene coding region is 5′→3′: as shown in SEQ ID No.5 in the ST.26 standard sequence nucleotide or amino acid sequence table, and the nucleotide forward sequence of another target site is 5′→3′: as shown in SEQ ID No.6 in the ST.26 standard sequence nucleotide or amino acid sequence table.

5. The use according to claim 2, characterized in that: Using CRISPR-Cas9 gene editing technology to obtain two LR A knockout mutant, one of which is LR The forward sequence of the nucleotides in the gene coding region is 5′→3′: as shown in SEQ ID No.7 in the ST.26 standard sequence nucleotide or amino acid sequence table. Another knockout mutant LR The forward sequence of the nucleotides in the gene coding region is 5′→3′: as shown in SEQ ID No. 8 in the ST.26 standard sequence nucleotide or amino acid sequence table.

6. The use according to claim 2, characterized in that: Using CRISPR-Cas9 gene editing technology to obtain two LR Gene knockout mutants, wherein the amino acid forward sequence of one knockout mutant is 5′→3′: as shown in SEQ ID No.9 in the ST.26 standard sequence nucleotide or amino acid sequence table, and the amino acid forward sequence of another knockout mutant is 5′→3′: as shown in SEQ ID No.10 in the ST.26 standard sequence nucleotide or amino acid sequence table.

Images