OsPIGL gene and method for regulating and controlling grain length, thousand grain weight and drought tolerance of rice by using OsPIGL gene

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to an OsPIGL gene and a regulation and control method thereof for improving the grain length, thousand grain weight and drought tolerance of rice. The nucleotide sequence of the OsPIGL gene is as shown in SEQ ID NO: 1, and the CDS sequence is as shown in SEQ ID NO: 2; the specific application is that overexpression of the OsPIGL gene effectively improves the grain length, the thousand seed weight and the drought tolerance of the rice, and new gene resources and technical means are provided for rice breeding. The gene and the application of the gene provided by the invention are helpful for cultivating a new rice variety with excellent agronomic traits and stress resistance and improving the yield and quality of rice, and have important significance for guaranteeing grain safety.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and relates to an OsPIGL gene and the application of the gene in regulating rice grain length, 1000-grain weight and drought resistance. Background Art

[0002] Rice is one of the most important food crops in the world, and half of the world's population relies on rice for food. Rice grain shape characteristics are measured by grain length, grain width, grain thickness and length-to-width ratio, while grain size is mostly measured by thousand-grain weight (Li et al., Current Opinion in Plant Biology, 2016, 33: 23-32; Li et al., Annual Review of Plant Biology, 2019, 70: 435-463). Currently, multiple QTLs and genes related to rice grain shape such as grain length, grain width, and grain thickness have been identified, such as GS3, GW2, and qSW5 / GW5, which negatively regulate rice grain development, while GS5, TGW6, RGA1, and OsGIF1 positively regulate grain length (Li et al., Annual Review of Plant Biology, 2019, 70: 435-463). According to the cell signaling pathways involved, it can be divided into G protein signaling (RGS), ubiquitin-26S proteasome system (UPS), mitogen-activated protein kinase (MAPK) signaling, hormone signaling and transcription factor regulation (Li et al., Current Opinion in Plant Biology, 2016, 33: 23-32; Li et al., Annual Review of Plant Biology, 2019, 70: 435-463; Song et al., Nature Genetics, 2007, 39: 623-630; Zuo et al., Annual Review of Genetics, 2014, 48: 99-118). Recent studies have shown that overexpression of the DEP1 gene leads to large grains, while knocking out DEP1 leads to small grains and upright panicles (Sun et al., Nature Communications, 2018, 9: 851). In addition, GS3 and DEP1 directly interact with OsMADS1, a transcription factor that regulates rice husk development, enhancing its transcriptional activity and promoting the transcription of common target genes, thereby regulating grain size and shape (Liu et al., Nature Communications, 2018, 9: 1-12).MAPK signaling pathways have been shown to play an important role in plant grain development. For example, SMG1 encodes mitogen-activated protein kinase (OsMKK4), and the mutant smg1 has smaller grains. Similarly, the loss of DSG1, a gene encoding mitogen-activated protein kinase (OsMAPK6), affects cell proliferation, resulting in smaller grains, while the activation of OsMAPK6 increases the proliferation of husk cells, resulting in larger grains. SMG2 encodes OsMKKK10, and mutations in SMG2 result in the inhibition of spikelet husk cell proliferation, resulting in small and light grains. OsMKKK10 can sequentially phosphorylate and activate OsMKK4 and OsMAPK6, so OsMKKK10, OsMKK4 and OsMAPK6 work together in a cascade signal to regulate grain length (Xu et al., Molecular Plant, 2018, 11: 860-873).

[0003] In recent years, the drought and semi-arid land caused by global climate change have seriously affected the growth, development and yield of rice, posing a huge challenge to food security. More than one-third of the world's arable land is affected by drought stress, which ultimately leads to a reduction in rice production (Aslam et al., Rice Science, 2022, 29: 105-117). Therefore, improving rice drought tolerance is an important condition for stable rice production. A series of drought-tolerant genes have also been identified, most of which are also regulated by ABA. Their promoter regions have conserved ABA response elements and dehydration response elements DRE / CRT, and the expression of drought stress response genes is mainly driven by ABA. ABA induces the expression of many genes, and the enzymes encoded by these genes catalyze the biosynthesis of osmoprotectants (such as proline), stomatal proteins, ABA synthesis-related proteins and LEA proteins (Tang et al., Plant Physiol. 2012, 158: 1755-1768; Xiao et al., Theor Appl Genet. 2007, 115: 35-46; Mundy et al., The EMBO Journal. 1988, 7: 2279-2286; Gangul et al., Plant Signaling & Behavior, 2012, 7: 502-509). Therefore, increasing rice yield, especially rice yield under drought conditions, has become the key to solving food security. Improving rice grain length and panicle length, thereby increasing rice yield and enhancing its drought tolerance are all current rice breeding goals. Summary of the invention

[0004] In view of this, the present invention provides a gene OsPIGL that can regulate rice grain length and 1000-grain weight and rice drought resistance. After overexpression in rice plants, this gene can increase grain length, 1000-grain weight, panicle length and single plant yield, while improving rice drought resistance, with one gene having multiple functions. The second purpose is to establish a method for regulating rice grain length, 1000-grain weight, panicle length and single plant yield, while improving rice drought resistance through genetic engineering. The third purpose is to use the method to cultivate high-yield and drought-resistant rice varieties in breeding.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a rice gene OsPIGL, whose nucleic acid sequence is shown in SEQ ID NO: 1, and CDS sequence is shown in SEQ ID NO: 2;

[0007] The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 3;

[0008] Also provided is a biological material of the rice gene OsPIGL, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium;

[0009] Furthermore, the present invention provides a method for regulating rice grain length, thousand-grain weight and drought tolerance, that is, using a genetic engineering method to overexpress the rice gene OsPIGL;

[0010] The method comprises the following steps:

[0011] S1: PCR amplification of rice OsPIGL gene

[0012] Using OsPIGL-F and OsPIGL-R as specific primers, the nucleic acid sequence of OsPIGL-F is shown in SEQ ID NO:8, and the nucleic acid sequence of OsPIGL-R is shown in SEQ ID NO:9, the rice OsPIGL gene is amplified by PCR method;

[0013] S2: Construction of pTCK303-OsPIGL overexpression vector

[0014] The PCR product in step S1 is connected to the pMD19-T vector under the action of T4 DNA ligase, and the connection product is transformed into Escherichia coli DH5α competent cells by heat shock method, and positive clones are screened on LB plates containing ampicillin, and plasmids are extracted. After PCR identification, sequencing is performed, and the correctly sequenced pMD19-T-OsPIGL vector is double-digested with SacI and KpnI, and the target fragment is recovered, and it is connected with the pTCK303 vector double-digested with SacI and KpnI under the action of recombinant T4 DNA ligase, and the connection product is transformed into DH5α competent cells, and positive clones are screened on LB plates containing kanamycin. The plasmid is extracted, and after PCR and restriction digestion identification, the correct pTCK303-OsPIGL overexpression vector is obtained;

[0015] S3: Transformation of vector into Agrobacterium

[0016] The vector obtained in step S2 is transformed into the Agrobacterium GV3101 strain, and colony PCR is performed using the primers described in step S1 to identify positive colonies;

[0017] S4: Acquisition and identification of transgenic seedlings

[0018] The wild-type rice callus tissue infected by the positive Agrobacterium in step S3 was screened and differentiated on a medium containing hygromycin to obtain tissue culture seedlings. The transgenic seedlings were identified simultaneously using the hygromycin primer Hyg-F nucleic acid sequence as shown in SEQ ID NO: 12, the Hyg-R nucleic acid sequence as shown in SEQ ID NO: 13, and the overexpression vector construction primers OsPIGL-F nucleic acid sequence as shown in SEQ ID NO: 8, and the OsPIGL-R nucleic acid sequence as shown in SEQ ID NO: 9.

[0019] The invention provides the application of the transgenic rice obtained according to the method in plant breeding.

[0020] The breeding methods include, but are not limited to, hybridization, backcrossing, selfing or asexual reproduction.

[0021] The beneficial effects of the present invention are:

[0022] Provided are a rice gene OsPIGL and related biological materials, enriching rice gene resources and providing diversified biological materials; also provided is a new way to effectively regulate rice grain length, 1000-grain weight and drought resistance, providing new technical means and operation methods for high-yield, high-quality and stress-resistant rice breeding, which is of great significance for promoting the development of rice genetic improvement and molecular breeding; through the method, a rice variety with increased rice grain length, improved 1000-grain weight, increased tiller number, longer panicle length, increased single-plant yield and enhanced drought resistance is bred, demonstrating the wide application potential and great value of the gene in rice breeding.

[0023] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0025] Figure 1 This is a homology comparison result of rice OsPIGL amino acid sequence (EEE61910) with switchgrass PvPIGL amino acid sequence (XP_039820641.1), sorghum SbPIGL amino acid sequence (XP_002448044.1) and corn ZmPIGL amino acid sequence (NP_001348355.1).

[0026] Figure 2 Schematic diagram of the T-DNA insertion loss-of-function mutant (ospigl) of the OsPIGL gene (A), PCR identification diagram of the T-DNA insertion mutant ospigl (B), and T-DNA plasmid structure diagram (C).

[0027] Figure 3 Schematic diagram of the construction of the overexpression vector of the OsPIGL gene driven by Ubi.

[0028] Figure 4 The expression level of OsPIGL gene in OsPIGL overexpressing rice lines and loss-of-function mutant ospigl (A), grain length phenotype (B), grain length (C), grain width (D) and 1000-grain weight (E) of transgenic rice lines overexpressing OsPIGL and knocking out OsPIGL. Among them, ospigl is a T-DNA insertion loss-of-function mutant line, WT is a wild-type line, and OE5, OE8, OE11 and OE12 are genetically stable OsPIGL overexpressing lines.

[0029] Figure 5 The panicle length phenotype (A, B), number of effective panicles per plant (C), number of primary stalks (D), number of secondary stalks (E), and yield per plant (F, G) of the OsPIGL overexpressing rice lines and the loss-of-function mutant ospigl.

[0030] Figure 6 The OsPIGL gene was induced to express in response to abiotic stress, and the OsACTIN1 gene was used as an internal reference.

[0031] Figure 7 Shown are the drought tolerance phenotypes of OsPIGL-overexpressing rice lines and the loss-of-function mutant ospigl (A), the survival rate statistics after rehydration after drought stress (B), and the water loss rate statistics of detached leaves (C).

[0032] Figure 8 This is the subcellular localization map of OsPIGL protein. OsPIGL protein is localized in the endoplasmic reticulum. DETAILED DESCRIPTION

[0033] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0034] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0035] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0036] Example 1 Homology comparison between OsPIGL and PIGL proteins from different plant sources

[0037] The OsPIGL gene number is LOC_Os04g58210, located on chromosome 4, the DNA nucleotide sequence of the gene is 3772bp in length, including 9 exons and 8 introns, the nucleic acid sequence is shown in SEQ ID NO:1; the coding sequence CDS is 789bp in length, the nucleic acid sequence is shown in SEQ ID NO:2; the gene encodes 262 amino acids, the amino acid sequence is shown in SEQ ID NO:3. According to its amino acid sequence, it is a N-Acetylglucosaminyl-phosphatidylinositoldeacetylase (also called P Hosphatidyl i nositol G lycan Anchor Biosynthesis,Class L , PIGL), so the gene was named OsPIGL. Rice OsPIGL has a high homology with PIGL from corn, sorghum, and switchgrass ( Figure 1 ).

[0038] Example 2 Mutant strains of ospigl

[0039] Preparation of loss-of-function mutants of OsPIGL. Purchase commercial T-DNA plasmids with the following structure: Figure 2 C. The T-DNA plasmid vector was transformed into the Agrobacterium GV3101 strain by heat shock method, and the callus of wild rice variety (Oryza sativa ssp. Japonica cv Dongjin) was infected with Agrobacterium containing T-DNA plasmid. Rice genetic transformation was carried out according to the method reported by Nishimura et al. (Nature Protocols, 2006, 1: 2796-2802). After screening and differentiation on LB medium, tissue culture seedlings were obtained, genomic DNA was extracted, and PCR amplification was performed using primers P1 (nucleic acid sequence as shown in SEQ ID NO: 6) and P2 (nucleic acid sequence as shown in SEQ ID NO: 7) or P1 (nucleic acid sequence as shown in SEQ ID NO: 6) and RB (nucleic acid sequence as shown in SEQ ID NO: 5) to identify the ospigl mutant. PCR amplification identified the mutant ospigl as a homozygous mutant ( Figure 2 A, B).

[0040] The mutant strain was planted in the experimental field for two generations, and the phenotype was observed to obtain a genetically stable mutant expression strain of ospigl.

[0041] Example 3 OsPIGL overexpression strain

[0042] PCR amplification of OsPIGL gene CDS. Based on the sequence of SEQ ID NO: 2, specific primers OsPIGL-F (nucleic acid sequence as shown in SEQ ID NO: 8) and OsPIGL-R (nucleic acid sequence as shown in SEQ ID NO: 9) containing the multiple cloning sites Sac I and Kpn I of the basic vector pTCK303 were designed using Primer 5.0 software, and then the reverse transcribed cDNA of mixed mRNA of leaves of wild-type rice (Oryza sativa ssp. Japonica cv Dongjin) grown for 4 weeks and 5 cm long panicles of rice at the heading stage were used as templates, and PCR amplification of the target fragment was performed using a high-fidelity DNA polymerase. The PCR reaction program was: pre-denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, for a total of 36 cycles; and finally extension at 72°C for 10 minutes.

[0043] Construction of OsPIGL overexpression vector. The above RT-PCR product was subjected to agarose gel electrophoresis, and then the gel was cut and recovered and purified according to the instructions of the DNA gel recovery kit; the purified DNA fragment was connected to the pMD19-T vector under the action of T4 DNA ligase at 16°C for 5 hours, and the ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method, and positive clones were screened on LB plates containing ampicillin, and plasmids were extracted. After PCR identification, sequencing was performed. The correctly sequenced pMD19-T-OsPIGL vector was double-digested with SacI and KpnI, and the target fragment was recovered. It was connected with the pTCK303 vector double-digested with SacI and KpnI at 16°C for 5 hours under the action of recombinant T4 DNA ligase, and the ligation product was transformed into DH5α competent cells. Positive clones were screened on LB plates containing kanamycin, and plasmids were extracted. After PCR and enzyme digestion identification, the correct pTCK303-OsPIGL overexpression vector was obtained. The specific vector structure is as follows Figure 3 shown.

[0044] The overexpression vector pTCK303-OsPIGL was transformed into Agrobacterium. The overexpression vector pTCK303-OsPIGL obtained above was transformed into Agrobacterium GV3101 strain by heat shock method, and the primers OsPIGL-F (sequence shown in SEQ ID NO: 8) and OsPIGL-R (sequence shown in SEQ ID NO: 9) were used to perform colony PCR to identify positive colonies.

[0045] Obtaining an OsPIGL overexpression strain. Callus tissue of a wild-type rice variety (Oryza sativa ssp. Japonica cv Dongjin) was infected with Agrobacterium containing the pTCK303-OsPIGL plasmid, and rice genetic transformation was performed according to the method reported by Nishimura et al. (Nature Protocols, 2006, 1: 2796-2802). After screening and differentiation on a medium containing hygromycin, tissue culture seedlings were obtained. Hygromycin primers Hyg-F (nucleic acid sequence shown in SEQ ID NO: 12) and Hyg-R (nucleic acid sequence shown in SEQ ID NO: 13) and overexpression vector construction primers OsPIGL-F (nucleic acid sequence shown in SEQ ID NO: 8) and OsPIGL-R (nucleic acid sequence shown in SEQ ID NO: 9) were used to simultaneously identify transgenic seedlings to obtain positive OsPIGL overexpression strains.

[0046] The obtained overexpression strains were planted in the experimental field for two generations, and the phenotypes were observed to obtain genetically stable OsPIGL overexpression strains.

[0047] Example 4 Analysis of Rice Grain Length and Thousand-Grain Weight

[0048] The expression level of OsPIGL gene in overexpressed rice lines and loss-of-function mutant ospigl was detected. After the overexpressed transgenic plants were planted for 2 generations, 4 genetically stable transgenic lines (OE5, OE8, OE11, OE12) were selected. The expression level of the gene in the OsPIGL overexpression lines and mutant ospigl was detected by reverse transcription quantitative PCR (RT-qPCR), with quantitative primers F (nucleic acid sequence as shown in SEQ ID NO: 10) and R (nucleic acid sequence as shown in SEQ ID NO: 11), and OsACTIN1 gene was used as an internal reference. The results are shown in Figure 4 A, quantitative primer position as Figure 2 As indicated by the red arrow in A. The differences among the strains were analyzed by Tukey's HSD test (P<0.05, one-way ANOVA), and different lowercase letters indicate significant differences.

[0049] Depend on Figure 4 A shows that the expression of OsPIGL gene was significantly upregulated in the over-fertilized rice lines, while the expression level of OsPIGL gene in the mutant ospigl was significantly reduced and almost undetectable.

[0050] Functional identification of OsPIGL gene in improving agronomic traits related to rice yield. First, the grain phenotype and thousand-grain weight of OsPIGL overexpression rice lines and loss-of-function mutants were analyzed. The four stable overexpression lines (OE5, OE8, OE11, OE12) were used as materials, and 5 strains were selected from each overexpression line, and 5 strains were selected from the wild type WT and mutant ospigl. 100-300 grains were randomly selected from each plant, and the grain length and width were measured. The results were repeated 5 times and the average value was taken as the final result. 1000 seeds were randomly selected to measure the thousand-grain weight. The results were repeated 5 times and the average value was taken as the final result. Figure 4 The differences among the strains were analyzed by Tukey's HSD test (P<0.05, one-way ANOVA), and different lowercase letters indicate significant differences.

[0051] Depend on Figure 4 BE showed that compared with the wild type, the grains of OsPIGL overexpression lines (OE5, OE8, OE11, OE12) became longer and the 1000-grain weight increased, while the grains of its loss-of-function mutant ospigl became shorter and the 1000-grain weight decreased, that is, the overexpression of the OsPIGL gene can significantly increase the length and 1000-grain weight of rice grains.

[0052] Example 5 Analysis of rice panicle length, number of stalks and yield per plant

[0053] Analysis of panicle length, number of branches and yield per plant of OsPIGL overexpression rice lines and loss-of-function mutant ospigl. Twenty plants were selected from each of the OsPIGL mutant, wild type WT and two OsPIGL overexpression lines (OE5 and OE8). The main panicle of each plant was selected for panicle length, number of primary branches and number of secondary branches, and the average value was calculated. At the same time, 20 plants were selected from each line for analysis of yield per plant, and the average value was calculated. The results are shown in Figure 5 The differences among the strains were analyzed by Tukey's HSD test (P<0.05, one-way ANOVA), and different lowercase letters indicate significant differences.

[0054] Depend on Figure 5 It can be seen that compared with the wild type, the panicle length of the OsPIGL overexpression line increased, the number of primary and secondary branches increased, and the yield per plant increased significantly; while the panicle length of the OsPIGL functional loss mutant ospigl decreased, the number of primary and secondary branches decreased, and the yield per plant decreased. In other words, the overexpression of the OsPIGL gene can increase the panicle length, number of branches and yield per plant of rice.

[0055] Example 6 Analysis of the expression level of OsPIGL gene in response to various abiotic stresses

[0056] Two-week-old rice seedlings (Oryza sativa ssp. Japonica cv Dongjin) were treated with drought, high temperature, low temperature, high salt, flooding, and nitrate for different time periods. Total leaf RNA was extracted and reverse transcribed into cDNA. Reverse transcription quantitative PCR (RT-qPCR) was used to detect the expression level of OsPIGL gene after various stress treatments. Three biological replicates were selected at each time point for each treatment, and the average value was calculated. The results are shown in Figure 6 OsACTIN1 gene was used as internal reference. * indicates significant difference in Student's t-test (*P<0.05), with 0h as control.

[0057] Depend on Figure 6 It can be seen that the expression level of OsPIGL gene increased significantly after drought and high temperature stress, that is, the expression of OsPIGL gene was induced by drought and high temperature stress.

[0058] Example 7 Analysis of rice drought tolerance

[0059] Functional study on OsPIGL gene in improving drought tolerance of rice. The transgenic lines of OsPIGL were tested for drought tolerance. To ensure the rigor of the experimental results, the wild type, OsPIGL overexpression rice lines (OE5, OE8) and the loss-of-function mutant ospigl were planted in the same container filled with soil. After 3 weeks of cultivation, drought stress treatment (no watering) was carried out. After all lines were drought-wilted, watering was resumed. After 2 weeks of recovery, the survival rates of different transgenic lines were counted. Five independent repeated experiments were performed, with no less than 24 lines in each experiment. The survival rate was the average of 5 independent repeated experiments. The results are shown in Figure 7 AB. The differences among the strains were analyzed by Tukey's HSD test (P<0.05, one-way ANOVA), and different lowercase letters indicate significant differences.

[0060] Depend on Figure 7 AB shows that after drought stress treatment and rehydration, the survival rate of the OsPIGL overexpression strain was significantly higher than that of the wild type, while the survival rate of the ospigl loss-of-function mutant was significantly reduced, that is, the overexpression of the OsPIGL gene can improve the drought tolerance of rice.

[0061] The water loss rate of leaves of OsPIGL-overexpressing rice lines and loss-of-function mutants was measured. Flag leaves of OsPIGL-overexpressing rice lines (OE5, OE8), wild type (WT) and loss-of-function mutant ospigl grown for 2 months were selected for in vitro leaf water loss determination, that is, the leaves were placed in a room with mild light, and the leaf weight was measured after being placed at room temperature for different periods of time. The water loss rate of leaves at each time point was calculated based on (initial leaf weight - leaf weight at different time points) / initial leaf weight x 100%. The experiment was repeated three times independently, each involving 20 plants, and the average value was calculated. The results are shown in Figure 7 C. * indicates that there are significant differences between the OsPIGL overexpression rice lines, loss-of-function mutants and wild type tested by Student's t-test (*P<0.05, **P<0.01, ***P<0.001)

[0062] Depend on Figure 7 C It can be seen that the water loss rate of leaves in the OsPIGL overexpression strain is significantly lower than that in the wild type, while the water loss rate of the OsPIGL functional loss mutant is significantly higher than that in the wild type, that is, the overexpression of the OsPIGL gene can increase the water loss rate of rice leaves. This is consistent with the enhanced drought tolerance of the OsPIGL overexpression strain and the reduced drought tolerance of the OsPIGL functional loss mutant.

[0063] Example 8 Detection of subcellular localization of OsPIGL protein

[0064] In order to determine the localization of OsPIGL protein in cells, the coding region of OsPIGL was fused with the pCAMBIA1301 vector containing GFP. The constructed p35S:OsPIGL-GFP vector was transformed into Agrobacterium GV3101 and co-injected with Agrobacterium containing p35S:AtCalnexin-RFP (endoplasmic reticulum localization marker) into Nicotiana benthamiana leaves grown for 4 weeks. The fluorescence signal of the fusion protein was observed under a laser confocal microscope for 2-3 days to determine the subcellular localization of the protein. The results are shown in Figure 8 .

[0065] Depend on Figure 8 It can be seen that the green fluorescence of OsPIGL-GFP overlaps with the red fluorescence of the endoplasmic reticulum marker protein AtCalnexin-RFP, indicating that the OsPIGL protein is localized in the endoplasmic reticulum of rice cells.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. The rice gene OsPIGL is characterized by: The nucleic acid sequence of the rice gene OsPIGL is shown in SEQ ID NO:1, and the CDS sequence is shown in SEQ ID NO:

2.

2. The rice gene OsPIGL according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

3.

3. A biological material containing the rice gene OsPIGL according to claim 1 or 2, wherein the biological material is any one of recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacteria.

4. A method for regulating rice grain length, 1000-grain weight and drought tolerance using the gene according to claim 1 or 2, characterized in that: The CDS sequence of the rice OsPIG gene according to claim 1 is overexpressed by genetic engineering methods.

5. The method according to claim 4, characterized in that: The method comprises the following steps: S1: PCR amplification of rice OsPIGL gene Using OsPIGL-F and OsPIGL-R as specific primers, the nucleic acid sequence of OsPIGL-F is shown in SEQ ID NO:8, and the nucleic acid sequence of OsPIGL-R is shown in SEQ ID NO:9, the rice OsPIGL gene is amplified by PCR method; S2: Construction of pTCK303-OsPIGL overexpression vector The PCR product in step S1 is connected to the pMD19-T vector under the action of T4 DNA ligase, and the connection product is transformed into Escherichia coli DH5α competent cells by heat shock method, and positive clones are screened on LB plates containing ampicillin, and plasmids are extracted. After PCR identification, sequencing is performed, and the correctly sequenced pMD19-T-OsPIGL vector is double-digested with SacI and KpnI, and the target fragment is recovered, and it is connected with the pTCK303 vector double-digested with SacI and KpnI under the action of recombinant T4 DNA ligase, and the connection product is transformed into DH5α competent cells, and positive clones are screened on LB plates containing kanamycin. The plasmid is extracted, and after PCR and restriction digestion identification, the correct pTCK303-OsPIGL overexpression vector is obtained; S3: Transformation of vector into Agrobacterium The vector obtained in step S2 is transformed into the Agrobacterium GV3101 strain, and colony PCR is performed using the primers described in step S1 to identify positive colonies; S4: Acquisition and identification of transgenic seedlings The wild-type rice callus tissue infected by the positive Agrobacterium in step S3 was screened and differentiated on a medium containing hygromycin to obtain tissue culture seedlings. The transgenic seedlings were identified simultaneously using the hygromycin primer Hyg-F nucleic acid sequence as shown in SEQ ID NO: 12, the Hyg-R nucleic acid sequence as shown in SEQ ID NO: 13, and the overexpression vector construction primers OsPIGL-F nucleic acid sequence as shown in SEQ ID NO: 8, and the OsPIGL-R nucleic acid sequence as shown in SEQ ID NO:

9.

6. Use of the transgenic rice obtained according to the method of claim 5 in rice breeding.

7. The use according to claim 6, characterized in that: Breeding methods include hybridization, backcrossing, selfing or asexual reproduction.

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