A nucleotide sequence for regulating rice grain shape and weight and its application

Abstract

The present invention discloses a nucleotide sequence for regulating rice grain shape and weight and its application. The nucleotide sequence is shown in SEQ ID NO: 1. The nucleotide sequence of the gene is used to construct a "CaMV 35S-AtABI4" fusion gene, which is transformed into rice. The fusion gene increases the grain length, width, thickness and weight of the transgenic rice lines expressing heterologous AtABI4, and has potential application value in improving rice seed size and grain weight and increasing crop yield.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to a nucleotide sequence for regulating rice grain shape and weight and an application thereof. Background Art

[0002] Rice yield is a complex agronomic trait influenced by panicle number, number of grains per panicle, seed set rate, and grain weight. Grain shape is a key determinant of grain weight. Grain shape, which includes grain length, width, thickness, and aspect ratio, is a quantitative trait controlled by multiple genes. Rice grain shape is not only crucial to yield but also to rice quality. Rice seed development begins with double fertilization, where the sperm and egg cells fuse to form a diploid embryo, and the sperm and two polar nuclei combine to form a triploid endosperm. The embryo and endosperm are daughter tissues, and during endosperm development, maternal cytoplasmic genes also indirectly regulate grain traits. Therefore, rice grain size is regulated by different genetic systems, including maternal growth and endosperm development.

[0003] Grain shape and grain weight of rice are closely related, and grain weight is largely determined by grain shape. Currently, several key genes regulating grain shape have been cloned. These genes are mainly involved in multiple regulatory pathways such as plant hormone synthesis and signaling, ubiquitin-proteasome, mitogen-activated protein kinase (MAPK) signaling, G protein signaling, and epigenetic modification (Liu Xi et al., 2018). In plant hormone synthesis and signaling pathways, mutations in genes encoding key components of brassinosteroid (BR) biosynthesis and signaling, such as GW5, D2, D11, SLG, XIAO, and GS2, lead to shortened rice grain length, dwarfed plants, and reduced leaf inclination. The protein encoded by the GW5 gene interacts with GSK2, inhibiting kinase activity and leading to the accumulation of unphosphorylated OsBZR1 and DLT proteins, which affect rice grain shape by participating in BR synthesis (Liu et al., 2017). D2 and D11 encode cytochrome P450 oxygenases, which positively regulate cell elongation and grain length (Tanabe et al., 2005; Wu et al., 2016). SLG encodes a BAHD-like acyltransferase that inhibits cell elongation, and its mutation causes longer grains and increased leaf inclination (Feng et al., 2017). et al., 2016); GS2 encodes the growth regulator OsGRF4, which alters rice grain length and width by promoting cell division and expansion (Hu et al., 2015). GAD1 regulates rice grain length, grain number per panicle, and awn development by reducing endogenous cytokinin (CTK) levels by activating the expression of OsCKX2 and DST (Jin et al., 2016). In the ubiquitin-proteasome pathway, GW2 encodes a RING-type E3 ubiquitin ligase that negatively regulates cell division by anchoring substrates to the proteasome for degradation. GW2 mutations inhibit substrate degradation, activating spikelet hull cell division, and increasing spikelet hull width. Furthermore, grain filling rate is increased, resulting in endosperm enlargement, ultimately leading to increased grain width, weight, and yield (Bednarek et al., 2012; Song et al., 2007). Zhou et al. discovered that the MATE transporter SMG4 regulates rice grain size by forming a complex with COPII and CYP78As (CYP78As-SMG4-COPII) (Chunlei et al., 2023). Abscisic acid (ABA), a key plant hormone, is involved in processes such as seed germination, root differentiation, stomatal closure, photosynthetic inhibition, leaf senescence, and fruit ripening. In the ABA signaling pathway, ABI4 is a key transcriptional regulator, regulating the expression of downstream target genes by binding to their promoters (Kakan et al., 2021; Wind et al., 2013).Although many studies have shown that the model plant Arabidopsis thaliana AtABI4 is a transcription factor with multiple regulatory functions, playing an important role in regulating processes such as seed germination, lateral root development, ABA and sugar signaling responses, lipid synthesis and decomposition, and adverse stress responses, there are few reports on whether this gene is involved in regulating seed size and grain weight. The present invention cloned the Arabidopsis thaliana AtABI4 gene and found that heterologous expression of this gene can increase grain length, grain width, grain thickness, and grain weight in transgenic rice lines. This important discovery has potential application value in increasing rice seed size and grain weight and increasing crop yield through molecular genetic modification methods. Summary of the Invention

[0004] The present invention aims to isolate and clone the AtABI4 gene of the model plant Arabidopsis thaliana, use the nucleotide sequence of the gene to construct a "CaMV 35S-AtABI4" fusion gene, and transform it into rice, so that the grain length, grain width, grain thickness and grain weight of the transgenic rice strains expressing heterologous AtABI4 are increased, and provide a nucleotide sequence for regulating rice grain shape and grain weight and its application.

[0005] The present invention provides an Arabidopsis thaliana AtABI4 open reading frame (ORF) sequence capable of increasing grain length, width, thickness and weight of transgenic rice, the nucleic acid sequence of which is the sequence shown in SEQ ID NO: 1;

[0006] As an example of specific application, the present invention provides a method for cloning the Arabidopsis thaliana AtABI4 gene, and the specific steps are as follows:

[0007] (1) Extract total RNA from Arabidopsis plants and reverse transcribe it into cDNA;

[0008] (2) Using cDNA as a template, the open reading frame (ORF) sequence of the AtABI4 gene was amplified by PCR;

[0009] (3) Recover the PCR amplification product.

[0010] The present invention also provides a method for heterologously expressing the AtABI4 gene to increase grain length, width, thickness, and weight in transgenic rice lines. Specifically, the open reading frame (ORF) sequence of the cloned AtABI4 gene is used to construct a "CaMV 35S-AtABI4" fusion gene for rice transformation, thereby obtaining a transgenic rice line in which the AtABI4 gene is heterologously constitutively expressed. The target gene in the fusion gene can be any target gene required for basic research or for improving rice traits such as grain length, width, thickness, and weight.

[0011] As an example of specific application, the present invention provides a construction of a "CaMV 35S-AtABI4" fusion gene and its application in increasing grain length, width, thickness, and weight in transgenic rice. The specific operation process is as follows:

[0012] (1) The open reading frame (ORF) sequence fragment of the AtABI4 gene amplified by PCR was digested with Nco I / BstE II;

[0013] (2) The pCAMBIA1301 expression vector plasmid was digested with Nco I / BstE II enzymes to recover the large vector fragment;

[0014] (3) The open reading frame (ORF) sequence fragment of the AtABI4 gene obtained in the first step and the large fragment of the pCAMBIA1301 vector obtained in the second step were mixed and ligated under the catalysis of ligase to complete the construction of the "CaMV 35S-AtABI4" fusion gene on the pCAMBIA1301 vector.

[0015] The designed PCR amplification primer sequences for the open reading frame (ORF) sequence of the AtABI4 gene are as follows, wherein the upstream primer introduces an Nco I restriction enzyme site, and the downstream primer introduces a BstE II restriction enzyme site:

[0016] Upstream primer: 5'-CATG CCATGG ACCCTTTAGCTTCCCAACA-3' (introducing Nco I restriction enzyme site)

[0017] Downstream primer: 5'-CATG GGTAACC TTAATAGAATTCCCCCAAG-3' (introducing a BstE II restriction enzyme site)

[0018] The "CaMV 35S-AtABI4" fusion gene in the application is constructed and constitutively expressed in transgenic rice. The operation process is as follows: rice callus tissue is transformed by Agrobacterium infection, the resistant seedlings obtained are harvested and seeds are propagated, DNA is extracted from leaves of hygromycin-resistant rice lines and tested by PCR, and RNA is extracted from the propagated offspring and tested by real-time fluorescence quantitative PCR. Homozygous transgenic rice lines heterologously expressing AtABI4 are screened, and the grain length, width, thickness and weight of the grains are quantitatively measured.

[0019] Beneficial effects of the present invention:

[0020] Experimental results show that transforming the cloned AtABI4 into rice significantly increases grain length, width, thickness, and weight in transgenic rice lines heterologously expressing AtABI4. The fusion gene construct "CaMV 35S-AtABI4" of the present invention can be used as a genetic resource to genetically improve transgenic rice with increased grain length, width, thickness, and weight, and has potential applications in increasing rice seed size and grain weight, as well as crop yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide 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 improper limitations on the present invention.

[0022] Figure 1 These are PCR molecular identification images of exogenous gene integration in resistant rice lines (variety: Nipponbare) transformed with the "CaMV 35S-AtABI4" fusion gene. M: DL10000. Figure 1 is an electrophoresis image of PCR products using wild-type Nipponbare rice DNA as a template. Figures 2-10 are electrophoresis images of PCR products using DNA from resistant rice lines transformed with the "CaMV 35S-AtABI4" fusion gene as a template.

[0023] Figure 2 This is an analysis of the relative expression levels of the target gene AtABI4 in transgenic rice lines transformed with the "CaMV 35S-AtABI4" fusion gene. NIP, OEAtABI4-1, OEAtABI4-2, and OEAtABI4-3 represent wild-type rice and three independent transgenic rice lines transformed with the AtABI4 gene, respectively. *P﹤0.05.

[0024] Figure 3 AB are the grain length and width phenotypes of seeds of wild-type rice (variety: Nipponbare, NIP) and transgenic rice lines transformed with the "CaMV 35S-AtABI4" fusion gene (OEAtABI4-1, OEAtABI4-2, and OEAtABI4-3), scale bar = 1 cm; CF is the quantitative statistical measurement of grain length, grain width, grain thickness, and grain weight of wild-type rice (NIP) and transgenic rice lines transformed with the "CaMV 35S-AtABI4" fusion gene, *P < 0.05. DETAILED DESCRIPTION

[0025] This section will describe in detail the specific embodiments of the present invention. The preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the accompanying drawings is to supplement the description of the text part of the specification with graphics, so that people can intuitively and vividly understand each technical feature and the overall technical solution of the present invention, but it should not be understood as a limitation on the scope of protection of the present invention.

[0026] Example 1

[0027] Cloning of the AtABI4 Gene from the Model Plant Arabidopsis thaliana

[0028] (1) Total RNA from Arabidopsis thaliana plants was extracted using the guanidine isosulfate-phenol method.

[0029] (2) Specific primers were designed based on the known open reading frame (ORF) sequence of the Arabidopsis thaliana AtABI4 gene, and an Nco I restriction enzyme site was introduced into the upstream primer and a BstE II restriction enzyme site was introduced into the downstream primer.

[0030] Upstream primer: 5'-CATG CCATGG ACCCTTTAGCTTCCCAACA-3' (introducing Nco I restriction enzyme site)

[0031] Downstream primer: 5'-CATG GGTAACC TTAATAGAATTCCCCCAAG-3' (introducing a BstE II restriction enzyme site)

[0032] (3) Take 1 μg RNA as the template for reverse transcription and use P 2853 ImProm-II RT TM Reverse transcription was performed, P 2853 The primer sequences, reverse transcription procedure, and reverse transcription system are as follows.

[0033] P 2853 Primer sequence: 5'-GCGAATTCTTTTTTTTTTTTTTTTT-3'

[0034] Reverse transcription procedure:

[0035] 72℃5min; 25℃5min; 42℃60min; 80℃20min; keep warm at 4℃.

[0036] Reverse transcription system:

[0037]

[0038] (4) Using the reverse transcribed cDNA as a template, PCR amplification was performed using the above primers to obtain the open reading frame (ORF) sequence fragment of the AtABI4 gene.

[0039] PCR reaction program: 94°C for 3 min; 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min; and insulation at 4°C.

[0040] PCR reaction system:

[0041]

[0042] (5) Recover the target DNA fragment by agarose gel electrophoresis.

[0043] (6) The recovered PCR product was subjected to DNA sequencing, and its nucleic acid sequence is shown in Sequence Table 1.

[0044] Example 2

[0045] Construction of the "CaMV 35S-AtABI4" fusion gene using the pCAMBIA1301 expression vector

[0046] (1) The pCAMBIA1301 plasmid was extracted from Escherichia coli and the large fragment of the vector was recovered after double digestion with Nco I / BstE II.

[0047] (2) The open reading frame (ORF) sequence fragment of the AtABI4 gene recovered in Example 1 was double-digested with Nco I / BstEII, and the digested fragments were recovered by agarose gel electrophoresis.

[0048] (3) The two fragments recovered above were ligated at 16°C for 24 h under the catalysis of ligase to complete the construction of the "CaMV 35S-AtABI4" fusion gene on the pCAMBIA 1301 expression vector.

[0049] Connection system:

[0050]

[0051] (4) Transform the ligation mixture into E. coli DH5α competent cells as follows:

[0052] E. coli DH5α competent cells were prepared according to conventional CaCl2 induction and transformation methods. 10 μl of the ligation product was used to transform the competent cells, which were then evenly spread on a plate containing Amp, X-gal, and IPTG and cultured in an inverted manner at 37°C for 12 h.

[0053] (5) PCR reaction was performed using the plasmid as a template to identify the "CaMV 35S-AtABI4" fusion gene in the plasmid. The size of the amplified fragment was 471 bp. The primers used were as follows:

[0054] Upstream primer: 5'-GCGATAAAGGAAAGGCCATCG-3'

[0055] Downstream primer: 5'-TTACGTGGCTCTCGGATCTCGG-3'

[0056] (6) Plasmids were extracted from positive clones and transformed into Agrobacterium EHA105 using conventional methods to obtain engineered Agrobacterium for rice transformation.

[0057] Example 3

[0058] Preparation of transgenic rice

[0059] (1) The "CaMV 35S-AtABI4" fusion gene constructed in Example 2 was transformed into rice (variety: Nipponbare). The specific transformation method was to infect rice callus with Agrobacterium tumefaciens. DNA was extracted from rice plant leaves with hygromycin resistance. PCR was used to identify the integration of the exogenous AtABI4 fragment into the wild-type rice genome. The size of the amplified fragment was 471 bp. The primers used were as follows:

[0060] Upstream primer: 5'-GCGATAAAGGAAAGGCCATCG-3'

[0061] Downstream primer: 5'-TTACGTGGCTCTCGGATCTCGG-3'

[0062] PCR reaction program: 94°C for 3 min; 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min; and insulation at 4°C.

[0063] The PCR reaction system is as follows:

[0064]

[0065] PCR test results such as Figure 1 As shown, the target bands could be amplified in the genomes of the nine hygromycin-resistant seedling lines tested (lanes 2-10), indicating that the exogenous AtABI4 fragment had been integrated into the genome of the wild-type rice.

[0066] (2) Real-time fluorescence quantitative RT-PCR detection of transgenic rice lines: Three transgenic rice lines identified by PCR (OEAtABI4-1, OEAtABI4-2, and OEAtABI4-3) were propagated. Total RNA of the transgenic rice lines was extracted and reverse transcribed into cDNA according to the method of Example 1. The relative expression level of AtABI4 was detected by real-time fluorescence quantitative RT-PCR using the following primers, PCR reaction procedure, and reaction system:

[0067] Detection primers for AtABI4 gene:

[0068] Upstream primer: 5'-CTCAACGCAAACGCAAAGGCAA-3'

[0069] Downstream primer: 5'-TTACGTGGCTCTCGGATCTCGG-3'

[0070] Detection primers for OsUBI internal standard gene:

[0071] Upstream primer: 5'-AACCAGCTGAGGCCCAAGA-3'

[0072] Downstream primer: 5'-ACGATTGATTTAACCAGTCCATGA-3'

[0073] PCR reaction procedure:

[0074] 95℃: 2 min; 95℃: 10 s, 60℃: 30 s, 40 cycles.

[0075] The PCR reaction system is as follows:

[0076]

[0077] Real-time fluorescence quantitative RT-PCR results Figure 2 As shown in the data, the expression levels of the AtABI4 gene in three transgenic rice lines (OEAtABI4-1, OEAtABI4-2, and OEAtABI4-3) were significantly increased compared with those in wild-type rice (NIP), indicating that OEAtABI4-1, OEAtABI4-2, and OEAtABI4-3 are three transgenic rice lines in which the AtABI4 gene is heterologously expressed in rice.

[0078] Example 4

[0079] Quantitative determination of grain length, width, thickness and weight in transgenic rice lines heterologously expressing the AtABI4 gene

[0080] Seeds of wild-type rice (NIP) and three homozygous transgenic lines expressing the AtABI4 gene (OEAtABI4-1, OEAtABI4-2, and OEAtABI4-3) were harvested after germination, seedling growth, transplanting, tillering, heading, and grain filling. The seeds were sun-dried under natural conditions. The seeds harvested from each line were phenotyped and quantitatively measured for grain length, width, thickness, and weight. The phenotype scanning results are shown in Figure 2. Figure 3 AB shows the quantitative determination results. Figure 3 As shown in CF, the experimental results showed that the heterologous expression model plant Arabidopsis thaliana AtABI4 significantly increased the grain length, grain width, grain thickness and grain weight of three transgenic rice lines. Among them, the increase in grain length of different lines ranged from 8.48% to 16.14%, the increase in grain width ranged from 5.52% to 8.60%, the increase in grain thickness ranged from 3.10% to 7.10%, and the increase in grain weight ranged from 5.10% to 14.90%.

[0081] Under the premise that no conflict occurs, those skilled in the art may freely combine and superimpose the above-mentioned additional technical features.

[0082] The above descriptions are only preferred embodiments of the present invention. Any technical solution that achieves the purpose of the present invention by substantially the same means shall fall within the scope of protection of the present invention.

Claims

1. A method for regulating rice grain shape and weight AtABI4 The application of genes is characterized by: Using this gene to construct CaMV 35S-AtABI4 " fusion gene, which was transformed into Nipponbare rice, and the grain length, width, thickness and weight of the transgenic Nipponbare rice strain were increased. AtABI4 The coding sequence of the gene is shown in SEQ ID NO: 1.

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