CN-121992011-A - Rice grain-specific promoter in early stage of grouting and application thereof

CN121992011ACN 121992011 ACN121992011 ACN 121992011ACN-121992011-A

Abstract

The invention discloses a rice pre-grouting grain specific promoter and application thereof, wherein the promoter is named proASP < 1>, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1. The promoter has high strict space-time expression specificity, and activation expression is limited in seeds in the early stage of rice grouting (4-12 days after flowers), and particularly, gene expression is efficiently driven in endosperm and aleurone layers, and leakage expression is avoided in other tissues such as roots, stems, leaves, leaf sheaths, glumes and the like and seeds in the middle and later stages of grouting. The invention provides basic and precious molecular tools for gene function research, quality improvement (such as protein and starch synthesis regulation) and bioreactor development in the key window period of rice grain development, and has important scientific research and application values.

Inventors

CHEN LIN
WANG ZITENG
ZHANG WEIYAN
ZHU YAN
HU HAOHAO
WANG YU
Sha Huilin
Zou Heshu
DING YANFENG

Assignees

南京农业大学

Dates

Publication Date: 20260508
Application Date: 20251219

Claims (8)

1. The rice pre-grouting grain-specific promoter with the nucleotide sequence shown in SEQ ID NO.1 is applied to at least one of the following (a 1) - (a 4): (a1) Driving the target gene to specifically express in the rice grain in the early stage of grouting; (a2) The rice yield is improved; (a3) Improving the nutrition quality of rice grains; (a4) And the environmental adaptability of the rice is enhanced.
2. The use of a biological material related to a rice pre-grouting grain-specific promoter having a nucleotide sequence shown in SEQ ID NO. 1 in at least one of the following (a 1) to (a 4): (a1) Driving the target gene to specifically express in the rice grain in the early stage of grouting; (a2) The rice yield is improved; (a3) Improving the nutrition quality of rice grains; (a4) The rice environmental adaptability is enhanced; the biological material related to the rice pre-grouting grain-specific promoter is at least one of the following (b 1) - (b 9): (b1) An expression cassette containing the rice pre-grouting grain-specific promoter; (b2) A recombinant vector comprising the rice pre-grouted grain-specific promoter, or a recombinant vector comprising the expression cassette of (b 1); (b3) A recombinant microorganism comprising the rice pre-grouted grain-specific promoter, or a recombinant microorganism comprising the expression cassette of (b 1), or a recombinant microorganism comprising the recombinant vector of (b 2); (b4) A transgenic rice cell line comprising said rice pre-grain-filling specific promoter, or a transgenic rice cell line comprising said expression cassette of (b 1), or a transgenic rice cell line comprising said recombinant vector of (b 2); (b5) Transgenic rice tissue containing the rice pre-grouting grain-specific promoter, or transgenic rice tissue containing the expression cassette of (b 1), or transgenic rice tissue containing the recombinant vector of (b 2); (b6) Transgenic rice organs containing the rice pre-grouting grain-specific promoter, or transgenic rice organs containing the expression cassette of (b 1), or transgenic rice organs containing the recombinant vector of (b 2); (b7) Transgenic rice plants containing the rice pre-grouting grain-specific promoter, or transgenic rice plants containing the expression cassette of (b 1), or transgenic rice plants containing the recombinant vector of (b 2); (b8) A regenerable cell, tissue culture or protoplast produced therefrom of the transgenic rice plant of (b 7); (b9) Propagation material of a transgenic rice plant according to (b 7).
3. The use according to claim 2, wherein the expression cassette is a pre-grouting grain-specific gene expression cassette comprising a rice pre-grouting grain-specific promoter having the nucleotide sequence shown in SEQ ID NO. 1, a target gene coding sequence operably linked downstream of the promoter, and a transcription terminator.
4. The use according to claim 3, wherein the gene of interest is a reporter gene, a gene related to starch synthesis, a gene related to mineral element enrichment, a gene related to stress resistance or a gene related to hormonal regulation.
5. The use according to claim 2, wherein the backbone vector of the recombinant vector is the binary vector pBWA (V) HG or a derivative thereof.
6. The use according to claim 1 or 2, characterized in that a plant recombinant expression vector comprising the rice pre-grain specific promoter and a gene of interest is introduced into rice to obtain transgenic rice, said specific promoter being capable of driving the specific expression of the gene of interest operably linked thereto in the pre-grain of said transgenic rice.
7. A method, characterized in that the method is at least one of the following (c 1) - (c 4): (c1) Driving the target gene to specifically express in the rice grain in the early stage of grouting; (c2) The rice yield is improved; (c3) Improving the nutrition quality of rice grains; (c4) The rice environmental adaptability is enhanced; And (3) introducing a plant recombinant expression vector containing a rice pre-grouting grain specific promoter with a nucleotide sequence shown as SEQ ID NO. 1 and a target gene into rice to obtain transgenic rice, wherein the specific promoter can drive the target gene operably connected with the specific promoter to specifically express in the pre-grouting grain of the transgenic rice.
8. The method according to claim 7, characterized in that it comprises in particular the following steps: (1) Cloning a rice pre-grouting seed specific promoter shown in SEQ ID NO. 1; (2) Inserting the specific promoter into a plant expression vector to construct a recombinant expression vector containing proASP 1:1 target genes; (3) Transforming agrobacterium, and introducing the recombinant expression vector into rice by a rice callus transformation method; (4) Screening and identifying to obtain transgenic rice positive plants; (5) And (3) verifying the specific expression of the target gene in the grain in the early stage of grouting by using GUS staining or qRT-PCR.

Description

Rice grain-specific promoter in early stage of grouting and application thereof Technical Field The invention belongs to the field of agricultural genetic engineering and biotechnology, and particularly relates to a rice-derived pre-grouting grain-specific promoter and application thereof in construction of transgenic plants and research of gene functions. Background Rice (Oryza sativa l.) is one of the most important food crops worldwide, and its grain filling process directly determines yield and quality. The pre-grouting phase (usually 0-12 days after flowering) is the stage of dividing the seed cells and the preliminary formation of starch bodies and has a decisive effect on the final grain weight and nutrient accumulation. Therefore, the accurate regulation of the expression of the key genes in the stage is of great significance for improving the yield and quality of rice. Promoters are key cis-acting elements that regulate gene expression in space-time, and are typically located upstream of a gene, and are recognized and bound by RNA polymerase and transcription factors to regulate gene expression. Promoters are generally classified into constitutive promoters, inducible promoters and tissue-specific promoters. Traditional constitutive promoters such as 35S, ubi and ACTIN are not limited by plant development and induced by exogenous substances when used for driving gene expression, and the nonspecific high-expression exogenous genes can cause interference to each growth stage and tissue part of the plant, so that fine time-space regulation of different tissue development cannot be achieved. At present, a plurality of rice tissue-specific promoters have been reported, such as endosperm-specific promoters OsGt, osGluB-1, chloroplast-specific promoters OsRbcS, etc. However, these promoters are mostly expressed continuously throughout the grouted period or non-grain site, and lack precise regulatory capability for the early grouted period. There is a lack of a promoter element in the prior art that efficiently and specifically initiates transcription in rice pre-grain. Therefore, the development of a promoter which is activated only in the grain in the early stage of grouting and is silenced in other tissues or development period has important application value for realizing accurate space-time expression of genes, avoiding non-target effect and improving transgene efficiency. Disclosure of Invention The invention aims to provide a rice-derived pre-grouting grain specific promoter and application thereof. The promoter is named proASP1, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1 (full length 3100 bp). The promoter is derived from a 5' upstream regulatory region of Os11g0184800 in a rice variety (such as Japanese sunny) genome, can efficiently start transcription in seeds at early stage (0-12 days) of rice grouting, and has high tissue and development stage specificity. The aim of the invention can be achieved by the following technical scheme: in a first aspect, the invention claims the use of a rice pre-grouting grain-specific promoter having the nucleotide sequence shown in SEQ ID NO. 1 in at least one of the following (a 1) to (a 4): (a1) Driving the target gene to specifically express in the rice grain in the early stage of grouting; (a2) The rice yield is improved; (a3) Improving the nutrition quality of rice grains; (a4) And the environmental adaptability of the rice is enhanced. In a second aspect, the present invention claims the use of a biological material related to a rice pre-grouting grain-specific promoter having a nucleotide sequence as shown in SEQ ID NO:1 in at least one of the following (a 1) to (a 4): (a1) Driving the target gene to specifically express in the rice grain in the early stage of grouting; (a2) The rice yield is improved; (a3) Improving the nutrition quality of rice grains; (a4) The rice environmental adaptability is enhanced; the biological material related to the rice pre-grouting grain-specific promoter is at least one of the following (b 1) - (b 9): (b1) An expression cassette containing the rice pre-grouting grain-specific promoter; (b2) A recombinant vector comprising the rice pre-grouted grain-specific promoter, or a recombinant vector comprising the expression cassette of (b 1); (b3) A recombinant microorganism comprising the rice pre-grouted grain-specific promoter, or a recombinant microorganism comprising the expression cassette of (b 1), or a recombinant microorganism comprising the recombinant vector of (b 2); (b4) A transgenic rice cell line comprising said rice pre-grain-filling specific promoter, or a transgenic rice cell line comprising said expression cassette of (b 1), or a transgenic rice cell line comprising said recombinant vector of (b 2); (b5) Transgenic rice tissue containing the rice pre-grouting grain-specific promoter, or transgenic rice tissue containing the expression cassette of (b 1), or transgenic rice tissue contain