AMT4 gene and its application in regulating rice grain shape and weight

Abstract

The present invention provides an AMT4 gene and its application in regulating rice grain shape and grain weight. The nucleotide sequence of the AMT4 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the AMT4 gene is shown in SEQ ID NO.2. The AMT4 gene regulates rice grain shape and grain weight, and inhibiting the expression of the gene can increase the length of rice grains, reduce the width of grains, increase the length-to-width ratio of grains, and increase the thousand-grain weight of grains. The present invention provides an important theoretical basis for high-yield breeding of rice and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of agriculture and biotechnology, and more specifically to an AMT4 gene and application thereof in regulating rice grain shape and grain weight. Background Art

[0002] Rice ( Oryzasati goes to L. ) is one of the main food crops in the world. Nearly half of the world's population relies on rice as their staple food. In my country, rice is the largest staple food crop. In recent years, with the gradual increase in population, the rice planting area has continued to decline. In order to ensure rice food security, it is particularly important to increase rice yields. In addition, with the improvement of people's living standards and the opening of the rice market, the requirements for the appearance and cooking taste of rice are also increasing, especially in the international market. Therefore, improving rice quality has also become one of the important goals of breeding.

[0003] Rice yield is mainly composed of three factors: the number of effective tillers per unit area, the number of grains per panicle, and the thousand-grain weight. Grain size and shape are not only a stable agronomic trait, but also a key factor in determining rice yield and an important quality trait. Grain weight is an important yield component factor. Grain weight and grain shape are highly positively correlated, and they are both quantitative traits. Grain shape includes the length, width, thickness, and aspect ratio of the grain, which is also an important appearance quality trait. It also affects the commercial quality and processing quality of rice (brown rice rate, polished rice rate, whole polished rice rate, etc.). my country's national high-quality rice standards also stipulate that the aspect ratio of high-quality glazed rice varieties cannot be lower than 2.8. Grain shape has become an important indicator for breeders to select high-quality rice varieties. In recent years, the use of molecular biological methods to clone rice grain type genes has become a research hotspot. The cloned rice grain type genes have important theoretical and practical guiding significance for the improvement of rice grain type.

[0004] Rice grain shape is a typical quantitative trait, which is regulated by multiple genes and has a complex genetic basis. In recent years, a number of key genes controlling rice grain shape have been analyzed through a combination of GWAS, QTL decomposition and map-based cloning techniques. For example, the genes GS3 and GL3.1 that control rice grain length and the genes GW2, GW5, GS5, GW7 and GW8 that control rice grain width. The cloning and functional analysis of these grain shape genes provide theoretical support for molecular design breeding based on grain shape improvement to improve rice yield and quality. Summary of the invention

[0005] The purpose of the present invention is to provide an AMT4 gene and its application in regulating rice grain shape and grain weight.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A use of an AMT4 gene in regulating rice grain shape and grain weight, wherein the nucleotide sequence of the AMT4 gene is as shown in SEQ ID NO.1, or is a sequence that is completely complementary to SEQ ID NO.1, or is a nucleotide sequence that is a nucleotide sequence as shown in SEQ ID NO.1 that is substituted, deleted and / or added with one or more nucleotides and can encode a protein with the same function, or is a nucleotide sequence that encodes an amino acid sequence as shown in SEQ ID NO.2.

[0008] The invention discloses an application of an expression protein of an AMT4 gene in regulating rice grain shape and grain weight. The amino acid sequence of the expression protein of the AMT4 gene is as shown in SEQ ID NO.2, or the amino acid sequence as shown in SEQ ID NO.2 is replaced, deleted and / or one or more amino acids are added but the protein activity is the same.

[0009] A product for inhibiting the expression of the AMT4 gene and its use in regulating the grain shape and weight of rice grains. The nucleotide sequence of the AMT4 gene is as shown in SEQ ID NO.1, or is a sequence that is completely complementary to SEQ ID NO.1, or is a nucleotide sequence that is a nucleotide sequence as shown in SEQ ID NO.1 that is substituted, deleted and / or added with one or more nucleotides and can encode a protein with the same function, or is a nucleotide sequence that encodes an amino acid sequence as shown in SEQ ID NO.2.

[0010] A method for regulating rice grain shape and grain weight, which comprises using genetic engineering technology to inhibit the expression of AMT4 gene in rice, thereby reducing the width of rice grains and increasing the length, length-width ratio and 1000-grain weight of rice grains; the nucleotide sequence of the AMT4 gene is as shown in SEQ ID NO.1, or is a sequence that is completely complementary to SEQ ID NO.1, or is a nucleotide sequence that is substituted, deleted and / or added with one or more nucleotides as the nucleotide sequence shown in SEQ ID NO.1 and can encode a protein with the same function, or is a nucleotide sequence that encodes an amino acid sequence as shown in SEQ ID NO.2.

[0011] Furthermore, the above-mentioned genetic engineering technology uses the AMT4 gene as the target gene, constructs a gene knockout vector, transforms it into rice, and cultivates the rice; the nucleotide sequence of the AMT4 gene is as shown in SEQ ID NO.1, or is a sequence that is completely complementary to SEQID NO.1, or is a nucleotide sequence as shown in SEQ ID NO.1 that is substituted, deleted and / or added with one or more nucleotides and can encode a protein with the same function, or is a nucleotide sequence encoding an amino acid sequence as shown in SEQID NO.2.

[0012] Furthermore, the above-mentioned gene knockout vector is constructed using Crispr / Cas9 editing technology.

[0013] Furthermore, the gene knockout vector is transformed into rice through Agrobacterium-mediated transformation.

[0014] Furthermore, the nucleotide sequence of the target site of the above-mentioned Crispr / Cas9 editing technology is shown in SEQ ID NO.3.

[0015] Furthermore, the nucleotide sequences of the adapter primers of the above target sites are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0016] The significant advantages of the present invention are:

[0017] The present invention discloses for the first time the biological function of the rice AMT4 gene, which regulates the grain shape and weight of rice grains. Inhibiting the expression of the gene increases the thousand-grain weight of grains, increases the grain length, reduces the grain width, and increases the grain length-width ratio. The present invention provides valuable gene resources for high-yield rice breeding. DETAILED DESCRIPTION

[0018] Nitrogen is one of the three main nutrients that are essential and in the greatest demand for rice growth and development. It is a component of proteins, amino acids, nucleic acids, and certain alkaline substances, accounting for about 1%-5% of the total dry matter of rice. It is a very important mineral nutrient element and is called a life element. Plants absorb and utilize nitrogen in the soil mainly in inorganic form, which is divided into two forms: ammonium nitrogen (NH4 + ) and nitrate (nitrate, NO3 - ). Since ammonium nitrogen requires less energy to absorb, studies have shown that plants prefer to use ammonium nitrogen first. Rice absorbs nitrogen from the soil and applies it to various physiological metabolisms in the body, including the synthesis of proteins, nucleic acids, and signal and storage molecules. Rice is an ammonium-loving crop and also prefers to use ammonium nitrogen first.

[0019] Rice absorbs ammonium nitrogen through ammonium transporter (AMT). There are 12 AMT genes predicted in rice (TIGR database, http: / / rice.plantbiology.msu.edu / ), and the proteins of all 12 AMT genes contain the ammonium transporter domain (Accession No. PF00909) (Pfam analysis, http: / / pfam.sanger.ac.uk / search / sequence), which is consistent with the function of ammonium ion transport. Most AMT proteins contain 10-11 transmembrane domains, with the N-terminus outside the membrane and the C-terminus inside the membrane, which is consistent with the crystal structure of other known AMT proteins. The rice AMT4 gene (SEQ ID NO.1) is located on chromosome 3 of rice. The protein is 300 amino acids long (SEQ ID NO.2) and contains 4 transmembrane domains, with the N-terminus outside the membrane and the C-terminus inside the membrane. Reports indicate that the rice AMT4 gene is expressed in all parts of the plant, but its specific biological function and expression regulation mechanism are unclear. Since the structure contains an ammonium transporter domain, it is predicted that the function of the rice AMT4 gene may be related to the absorption and / or transport of ammonium. Given the importance of nitrogen metabolism to plants and the wide range of its involvement, the function of the AMT4 gene may be related to many important traits of plants, including grain shape, grain weight, etc.

[0020] Based on this, the applicant knocked out the AMT4 gene in rice and statistically analyzed the grain shape and weight of the rice grains of the knocked-out plants and found that knocking out the AMT4 gene in rice can significantly increase the length of rice grains, reduce the width of grains, increase the length-to-width ratio of grains, and increase the thousand-grain weight of grains.

[0021] Example 1: Editing the rice AMT4 gene using CRISPR / Cas9 technology

[0022] Open the CRISPR-P v2.0 webpage (http: / / rice.hzau.edu.cn / cgi-bin / rice2 / CRISPR_rice), enter the CRISPR design subprogram, select the corresponding PAM type (NGG), the proposed promoter (U6), and the target reference genome (Oryza sativa (MSU)). Enter the gene name in Locus Tag, or enter the position information of the target gene in Position, or paste the target gene sequence shown in SEQ ID NO.1 in Sequence, and click Submit to run the program. In the target design results, you can view the sequence, position, positive and negative strands, GC content, potential off-target sites, and valuation information of all candidate targets. Select targets with the highest valuation possible and the target located near the 5' end of the target gene exon or in the important structural domain region of the target gene protein as candidate target genes. At the same time, it is required that the candidate target has no potential off-target sites as much as possible or the potential off-target sites have a low score and are located in the non-gene region of the genome. The target site sequence finally selected was 5'-ggacaacgcgtggccgctag-3', and the linker primers of the target site were 5'-GCCggacaacgcgtggccgctag-3' and 5'-AAACctagcggccacgcgttgtc-3'. The AMT4 gene knockout vector was constructed according to the reference (Zeng Dongchang, Ma Xingliang, Xie Xianrong, et al. Operation methods for construction and mutation analysis of plant CRISPR / Cas9 multi-gene editing vector [J]. Chinese Science: Life Sciences, 2018, 48(07): 783-794). After the linker primers were annealed to form a double-stranded linker, the intermediate vector containing the promoter OsU6a was first cut into linearized fragments, and then the sgRNA expression cassette fragment was amplified by overlapping PCR. Finally, it was spliced ​​with the plant expression vector pYLCRISPR / Cas9Pubi-H by cutting and ligating to construct the final AMT4 gene knockout vector.

[0023] The AMT4 gene knockout vector was transferred into the callus tissue of rice Minghui 86 through Agrobacterium-mediated transfer. 12 to 15 days after the rice panicle bloomed, the young seeds were selected, the husks were removed, and then they were washed in 75% alcohol solution for 0.5 to 1 minute for disinfection and sterilization, and then further soaked in 10 to 30% sodium hypochlorite solution for 15 to 30 minutes for sterilization again; then, they were rinsed with sterile water 3 to 4 times in the clean bench, poured out, and air-dried on sterile paper; finally, the young embryo part was picked out from the sterilized young seeds with tweezers and dissecting needles, inoculated on the induction medium, and placed at 27°C. The cells were cultured in a light-proof incubator for 10 to 15 days. The embryonic sheath and other tissues were then removed, leaving only the terminal cell bulge, and the cells were transferred to a new induction medium. The cells were then cultured at 27°C in a light-proof environment. Subculture was performed every 20 days, and embryonic callus tissues that had undergone three to six subcultures were selected for the next step of genetic transformation. At this time, the callus tissue had grown rapidly. The embryonic callus tissues that were in the vigorous growth period were selected and placed in a sterilized culture dish. OD 600 The callus tissue was then transferred to a sterile culture dish in a clean bench, rinsed three times with sterile water, and then washed once with sterile water containing 250 mg / L carbenicillin to kill excess Agrobacterium cells. Finally, the callus tissue was dried with sterile filter paper and set aside. The embryonic callus tissue infected with Agrobacterium was transferred to a culture dish containing 250 mg / L carbenicillin and 50 mg / L The resistant callus was selected on the screening medium of hygromycin, and cultured at 28℃ in the dark. After the infected callus was cultured on the screening medium for 20-30 days, the resistant callus growing on the edge was transferred to the new screening medium for further screening 1-2 times. The resistant callus obtained by screening on the screening medium was then transferred to the differentiation medium, cultured at 28℃ in the dark for 7-10 days, and then moved to the light (14 hours / day) for culture, and then gradually grew green spots and finally differentiated into seedlings. After the seedlings were fully differentiated, they were transferred to the rooting medium and cultured at 28℃ in the light (14 hours / day) for 14-21 days for rooting and screening. After the rooting, the resistant seedlings were washed off the root culture medium, and moved to 1 / 10 MS nutrient solution for hydroponics for 3-5 days to grow new roots, and then transplanted to the greenhouse or net room paddy field. After screening and differentiation, 25 green seedlings were obtained. After further molecular identification, 10 mutant plants with successful knockout of the AMT4 gene were obtained.

[0024] Example 2: AMT4 gene increases grain length, decreases grain width, increases length-to-width ratio and increases thousand-grain weight

[0025] The AMT4 gene knockout mutant plants obtained above were planted in Shoushan Base, Fuzhou City, Fujian Province, and the harvested seeds were analyzed by YTS-5D digital seed copying instrument of Wuhan Gufeng Optoelectronics Technology Co., Ltd. The results are shown in Table 1. After further calculation, compared with the controlled Minghui 86, the grain length of the AMT4 gene knockout mutant plants increased by 5% on average, the grain width decreased by 8% on average, the grain length-to-width ratio increased by 8% on average, and the thousand-grain weight increased by 5% on average. There were no significant differences in other field phenotypes.

[0026] Table 1 Results of seed analysis

[0027]

[0028] The above detailed description of the present invention does not limit the present invention. Those skilled in the art may make various changes and modifications based on the present invention, and these changes and modifications should all fall within the scope of the appended claims of the present invention.

Claims

1. The application of AMT4 gene in regulating rice grain shape and grain weight is characterized by: The nucleotide sequence of the AMT4 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2; the regulation refers to knocking out the AMT4 gene in rice, reducing the width of rice grains, and increasing the length of rice grains, the length-to-width ratio of grains, and the thousand-grain weight of grains.

2. Application of a product for inhibiting the expression of the AMT4 gene in regulating the grain shape and weight of rice grains, characterized in that: The nucleotide sequence of the AMT4 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the AMT4 gene is shown in SEQ ID NO.2; the regulation refers to reducing the width of rice grains and increasing the length of rice grains, the length-to-width ratio of grains, and the thousand-grain weight of grains; The product is a gene knockout vector constructed using Crispr / Cas9 editing technology with AMT4 gene as the target gene; The gene knockout vector is transformed into rice through Agrobacterium-mediated transformation; The Crispr / Cas9 editing technology acts on the target site of the nucleotide sequence as shown in SEQ ID NO.3; The sequences of the adapter primers of the target site are shown in SEQ ID NO.4 and SEQ ID NO.

5.

3. A method for regulating rice grain shape and grain weight, characterized in that: AMT4 gene with a nucleotide sequence as shown in SEQ ID NO.1 is used as a target gene, a gene knockout vector is constructed, and the gene knockout vector is transformed into rice, and the rice is cultivated, thereby reducing the width of rice grains and increasing the length of rice grains, the length-to-width ratio of grains, and the thousand-grain weight of grains; The gene knockout vector is constructed using Crispr / Cas9 editing technology; The gene knockout vector is transformed into rice through Agrobacterium-mediated transformation; The Crispr / Cas9 editing technology acts on the target site of the nucleotide sequence as shown in SEQ ID NO.3; The sequences of the adapter primers of the target site are shown in SEQ ID NO.4 and SEQ ID NO.5.

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