Application of OsPIU1 gene and its encoded protein in regulating rice grain size, leaf angle and salt tolerance

Abstract

The present invention belongs to the field of genetic engineering and specifically relates to OsPIU1 Application of the gene and its encoded protein in regulating rice grain size, leaf angle and salt tolerance. OsPIU1 It can regulate rice grain size, thousand-grain weight, leaf angle and salt tolerance, and can be used to improve rice grain size, thousand-grain weight, leaf angle and salt tolerance, or improve grain size, thousand-grain weight, leaf angle and salt tolerance at the same time. OsPIU1 The gene increases rice grain length, grain width and 1000-grain weight, while reducing leaf angle and increasing salt tolerance. OsPIU1 The gene makes rice grains smaller and reduces 1,000-grain weight, while increasing leaf angles and reducing salt tolerance. Therefore, the gene can be used in plant genetic improvement to cultivate superior strains and varieties.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to the application of OsPIU1 gene and its encoded protein in regulating rice grain size, leaf angle and salt tolerance. Background Art

[0002] Rice is an important food crop in my country, and its output accounts for about 42% of the total grain output. The stable and increased yield of rice is of great strategic significance to ensuring my country's food security. According to my country's actual situation, the country is promoting the adjustment of the planting industry structure, and increasing the yield and quality of rice while stabilizing the area. This requires the selection and breeding of better and more advanced rice varieties. Rice grain size, thousand-grain weight, leaf angle and stress resistance are all important factors affecting yield. Therefore, improving grain size, thousand-grain weight, leaf angle and improving salt tolerance are all rice breeding goals.

[0003] Grain size has always been a yield trait that breeders and plant geneticists have focused on, and a lot of research has been done on it. Grain size is mostly quantitatively inherited and controlled by multiple genes. Scientists have cloned or analyzed multiple genes that regulate grain size through forward and reverse genetics. GS3 is the first QTL controlling rice grain length obtained through map-based cloning, revealing the reason for the difference in grain length between indica and japonica rice (Fan C, Xing Y, Mao H, et al. GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice,encodes a putative transmembrane protein[J]. Theoretical and applied genetics, 2006, 112(6): 1164-1171.). Two mitogen-activated protein kinase genes, OsMKK4 and OsMAPK6, positively regulate rice grain size (Duan P, Rao Y, Zeng D, et al. SMALL GRAIN 1, whichencodes a mitogen-activated protein kinase kinase 4, influences grain size inrice[J]. The Plant Journal, 2014, 77(4): 547-557.; Liu S, Hua L, Dong S, et al. Os MAPK 6, a mitogen-activated protein kinase, influences rice grain sizeand biomass production[J]. The Plant Journal, 2015, 84(4): 672-681.).GW8 and GLW7 are two other QTLs controlling rice grain length, encoding plant-specific transcription factors OsSPL16 and OsSPL13, respectively (Wang S, Wu K, Yuan Q, et al. Control of grain size, shape and quality by OsSPL16 in rice [J]. Nature genetics, 2012, 44 (8): 950-954.; Si L, Chen J, Huang X, et al. OsSPL13 controls grain size in cultivated rice [J]. Nature genetics, 2016, 48 (4): 447-456.). GW2 is a major QTL controlling rice grain width, encoding a RING-type E3 ligase (Song XJ, Huang W, Shi M, et al. A QTL for rice grain width and weightencodes a previously unknown RING-type E3 ubiquitin ligase[J]. Naturegenetics, 2007, 39(5): 623-630.), and qTGW2 is a semi-dominant QTL locus controlling rice grain width and grain weight (Ruan B, Shang L, Zhang B, et al. Natural variation in the promoter of TGW2determines grain width and weight in rice[J]. New Phytologist, 2020, 227(2):629-640.). Rice plant type is also an important agronomic trait affecting rice yield. Leaf angle is a key trait affecting plant type, which can affect planting density and ventilation and light transmission of the population.Scientists have also analyzed many genes that regulate leaf angle, such as OsBUL1, RAV6 and POW1. Among them, POW1 can regulate both leaf angle and rice grain size (Jang S, An G, Li H Y. Rice leaf angle and grain size are affected by theOsBUL1 transcriptional activator complex[J]. Plant Physiology, 2017, 173(1):688-702.;Zhang X, Sun J, Cao X, et al. Epigenetic mutation of RAV6 affectsleaf angle and seed size in rice[J]. Plant Physiology, 2015, 169(3): 2118-2128.;Zhang L, Wang R, Xing Y, et al. Separable regulation of POW1 in grainsize and leaf angle development in rice[J]. Plant biotechnology journal,2021, 19(12): 2517-2531.). There are a lot of saline-alkali lands in my country. Improving the salt tolerance of rice can improve the availability of land, which is also an important condition for the stable yield of rice. At present, many salt-tolerant genes have been identified, such as OsCOIN , OsDREB2A , OsMYB2 , OsbZIP71 , OsbZIP23 It can cause the accumulation of osmoprotectants and antioxidants, increase the activity of sodium and potassium ion transporters, and thus regulate the salt tolerance of rice (Gumi AM, Guha PK, Mazumder A, et al. Characterization of OglDREB2A gene from African rice (Oryza glaberrima), comparative analysis and its transcriptional regulation under salinity stress[J]. 3 Biotech, 2018, 8(2): 1-16.). Summary of the invention

[0004] The present invention proposes a gene that can regulate both rice grain size and rice leaf angle and salt tolerance OsPIU1 After being overexpressed in rice plants, it increases grain length, grain width and 1,000-grain weight while reducing leaf angle and improving salt tolerance. It has multiple functions of one gene. This gene can be used to cultivate rice varieties with large grains, excellent plant shape and salt tolerance, which can play an important role in ensuring national food security.

[0005] A gene for regulating rice grain size, leaf angle or salt tolerance, or simultaneously regulating grain size, leaf angle and salt tolerance of the present invention OsPIU1 , whose nucleotide sequence is shown in SEQ ID NO:1, gene OsPIU1 The CDS sequence is shown in SEQ ID NO:2.

[0006] The present invention also provides the above-mentioned rice grain size regulation or leaf angle regulation or salt tolerance regulation, or simultaneous regulation of grain size, leaf angle and salt tolerance genes. OsPIU1 The amino acid sequence of the encoded protein is shown in SEQ ID NO:3.

[0007] In addition, the present invention also provides a OsPIU1 or a recombinant expression vector of its CDS, and a gene OsPIU1 Specifically, the recombinant expression vector contains the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4, and the recombinant expression vector is an overexpression vector. The nucleotide sequence shown in SEQ ID NO:4 is to adapt to the recombinant expression vector, while the nucleotide sequence shown in SEQ ID NO:2 OsPIU1 The gene CDS sequence is the sequence after codon optimization.

[0008] The present invention also provides the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4. OsPIU1 Gene sequence, or OsPIU1 protein in improving rice breeding. Specifically, the above recombinant vector is transformed into rice, which can increase the length, width and thousand-grain weight of rice grains more efficiently, reduce the leaf angle, and improve the survival rate of plants under salt stress. In the above recombinant vector, Gt13a or Ubi promoter gene sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4 can be used. OsPIU1 Gene knockout or reduction OsPIU1 Gene expression can reduce rice grain length, grain width and 1000-grain weight, increase leaf angle, and reduce plant survival rate under salt stress.

[0009] The beneficial effects of the present invention are:

[0010] Genes of the present invention OsPIU1 It can regulate rice grain size, thousand-grain weight, leaf angle and salt tolerance, and can be used to improve rice grain size, thousand-grain weight, leaf angle and salt tolerance, or improve grain size, thousand-grain weight, leaf angle and salt tolerance at the same time. OsPIU1 The gene increases rice grain length, grain width and 1000-grain weight, while reducing leaf angle and increasing salt tolerance. OsPIU1 The gene makes rice grains smaller and reduces 1,000-grain weight, while increasing leaf angles and reducing salt tolerance. Therefore, the gene can be used in plant genetic improvement to cultivate superior strains and varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Started for Gt13a OsPIU1 Gene overexpression vector.

[0012] Figure 2 Launched for Ubi OsPIU1 Gene overexpression vector.

[0013] Figure 3 for OsPIU1 Gene knockout vectors.

[0014] Figure 4 For overexpression OsPIU1 Figure 1 shows the grain size phenotype of knockout rice. KO-1, KO-2 and KO-3 are knockout lines, Gt-23, Gt-28 and Gt-30 are overexpression lines driven by Gt13a, and OX-13, OX-15 and OX-17 are overexpression lines driven by Ubi.

[0015] Figure 5 for OsPIU1 Graph showing the regulation of leaf angle. KO-1, KO-2 and KO-3 are knockout strains, and OX-13, OX-15 and OX-17 are overexpression strains driven by Ubi.

[0016] Figure 6 for OsPIU1 Figure 2. Regulating salt tolerance. KO-1, KO-2 and KO-3 are knockout strains, and OX-13, OX-15 and OX-17 are Ubi-activated overexpression strains. DETAILED DESCRIPTION

[0017] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0018] Example 1 OsPIU1 Gene sequence optimization

[0019] Obtained from the RAP-DB website OsPIU1 Gene CDS sequence, see SEQ ID NO: 2, because the sequence contains the restriction site of pTCK303 vector Sac

[0020] Example 2 Gt13a startup OsPIU1 Construction of gene overexpression vector

[0021] Use plus Kpn I restriction site primer OsPIU1-OX-F, Sac I restriction site primer OsPIU1-OX-R, using SEQ ID NO:4 as a template, amplified the optimized OsPIU1 Gene CDS sequence, agarose gel electrophoresis, gel excision and recovery.

[0022] Linearize the expression vector p1301 containing the endosperm-specific promoter Gt13a and ligate the optimized OsPIU1 The gene CDS sequence was connected to the expression vector p1301 to construct a Gt13a-promoted OsPIU1 Gene overexpression vector, the structural schematic diagram of the overexpression vector is as follows Figure 1 shown.

[0023] OsPIU1-OX-F ( Kpn I): 5'-GGGGTACCATGCAAATCTCTCGTTAAGACTCTTACTGGT-3';

[0024] OsPIU1-OX-R ( Sac I): 5'-CGAGCTCTTATTGTCCACCTCTAAGTCTAA-3'.

[0025] Example 3 Ubi-enabled OsPIU1 Construction of gene overexpression vector

[0026] Use plus Kpn I restriction site primer OsPIU1-OX-F, Sac I restriction site primer OsPIU1-OX-R, using SEQ ID NO:4 as a template, amplified the optimized OsPIU1 Gene CDS sequence, agarose gel electrophoresis, gel excision and recovery.

[0027] The expression vector pTCK303 containing the promoter Ubi was linearized, and the optimized OsPIU1 gene CDS sequence was ligated to the expression vector pTCK303 by ligase to construct the Ubi-promoted OsPIU1 Gene overexpression vector, the structural schematic diagram of the overexpression vector is as follows Figure 2 shown.

[0028] OsPIU1-OX-F ( KpnI): 5'-GGGGTACCATGCAAATCTTCGTTAAGACTCTTACTGGT-3';

[0029] OsPIU1-OX-R ( Sac I): 5'-CGAGCTCTTATTGTCCACCTCTAAGTCTAA-3'.

[0030] Example 4 OsPIU1 Construction of gene knockout vector

[0031] exist OsPIU1 The PAM sequence (GGG) is determined at 102 bp from ATG on the gene exon, based on the intermediate vector sk-gRNA Aar I restriction site, designed primers and added a ggca linker to the 5' end of the forward primer, and added an aaac linker to the 5' end of the reverse primer to form primers OsPIU1-Cas9-F and OsPIU1-Cas9-R, which were synthesized by Shanghai Bioengineering.

[0032] Dilute OsPIU1-Cas9-F and OsPIU1-Cas9-R to 100 μM with ultrapure water, take 20 μL of each and mix, put into PCR instrument, place at 100℃ for 5 min, take out and place at room temperature for 10 minutes to form guide RNA. Aar I enzyme to linearize sk-gRNA and ligate it to the guide RNA using T4-DNA ligase. Kpn I and Bgl Ⅱ enzymes were used to digest the sk-gRNA intermediate vector and Pc1300-cas9 vector with guide RNA, gel cutting, recovery, and purification. Finally, T4-DNA ligase was used to connect the guide RNA into the Pc1300-cas9 vector to construct OsPIU1 Gene knockout vector, the structure diagram of the knockout vector is as follows Figure 3 shown.

[0033] OsPIU1-Cas9-F: 5'-GGCAGCTAAGATCCAAGATAAGGA-3';

[0034] OsPIU1-Cas9-R: 5'-AAACTCCTTATCTTGGATCTTAGC-3'.

[0035] Example 5 OsPIU1 Related expression vectors for rice transformation

[0036] 1. OsPIU1 Related expression vectors for transformation of Agrobacterium:

[0037] (1) Take the Gt13a startup OsPIU1 Gene overexpression vector, Ubi-promoted OsPIU1 Gene overexpression vectors and OsPIU1 1 μg of gene knockout vector was added to 200 μL of Agrobacterium competent cells, mixed gently, placed in ice bath for 30 min, placed in liquid nitrogen for 5 min, taken out and placed in 37°C water bath for 5 min, and then quickly placed in ice bath for 2 min;

[0038] (2) Add 800 μL of LB liquid medium (without antibiotics) and shake gently at 28°C for 5 h;

[0039] (3) Centrifuge the cultured bacterial liquid, remove the supernatant, resuspend the cells, apply them on AB medium (Kan 50 mg / L, Rif 50 mg / L), invert, and culture at 28°C in the dark for 3 days.

[0040] 2. Rice callus induction:

[0041] (1) Hull the rice seeds, soak them in 70% ethanol for 1 min, and rinse them with sterile water five times;

[0042] (2) Soak the above seeds in 30% NaClO (the stock solution has an effective chlorine content of 10%, and add one drop of Tween-20 for every 50 mL) for 15 minutes to sterilize them, and then wash them with sterile water for 5 times;

[0043] (3) Repeat step (2) but do not add Tween-20 to the NaClO;

[0044] (4) Place the above seeds on sterilized filter paper and absorb the moisture on the surface of the seeds;

[0045] (5) Sow the above seeds on N6D medium and culture at 32°C with continuous light for 5 days.

[0046] 3. Agrobacterium infection and transformation seedling screening:

[0047] (1) Scrape the Agrobacterium cultured on AB medium into AAM culture medium, suspend it, and adjust the concentration to OD600≈0.1;

[0048] (2) Soak the seeds pre-cultured in step 2 in the Agrobacterium solution and shake gently for about 1.5 minutes;

[0049] (3) The above seeds were inoculated on N6D-As co-cultivation medium and co-cultivated in a dark box at 25°C for 3 days;

[0050] (4) Rinse the co-cultured seeds with sterile water (containing 400 mg / L carboxybenzyl) 10 times, and then soak them in sterile water for 30 min to completely remove Agrobacterium;

[0051] (5) Place the above seeds on sterilized filter paper to dry, then inoculate on N6D-S screening medium (containing 50 mg / L hygromycin / herbicide and 400 mg / L carboxybenzyl) and keep them in continuous light at 32°C for 14 days;

[0052] (6) Transfer the vigorously growing callus on the above medium to RE-III medium (containing 50 mg / L hygromycin / herbicide and 250 mg / L carboxybenzyl) to induce differentiation at 28°C with continuous light for 14 days;

[0053] (7) The seedlings differentiated on the above culture medium were transferred to a test tube containing HF medium (containing 50 mg / L hygromycin / herbicide and 200 mg / L carbenicillin) to induce rooting and cultured at 28°C with 16 h light per day for 14 days;

[0054] (8) After the seedlings in the test tube have grown stably, add 1 cm deep sterile water at room temperature into the test tube. Incubate the tube at 28°C and humidity >50% for 4 days. Remove the seedlings, rinse the root culture medium with water, and then transplant them into the soil for growth.

[0055] 4. Identification of transgenic seedlings:

[0056] (1) OsPIU1 Identification of overexpressing material

[0057] Extract the DNA from the leaves of the transformed seedlings, design hygromycin resistance gene-specific primers based on the vector information, perform PCR amplification on the transformed strains, and the transgenic positive plants are those that amplify the hygromycin resistance gene fragment. Then extract the RNA of the positive plants and use OsPIU1 Gene-specific primers were used for quantitative PCR analysis to obtain OsPIU1 Overexpressing strains.

[0058] Hygromycin resistance gene amplification primers:

[0059] Hyg-F: 5'-ACGGTGTCGTCCATCACAGTTTGCC-3';

[0060] Hyg-R: 5'-TTCCGGAAGTGCTTGACATTGGGGA-3'.

[0061] OsPIU1 Expression analysis primers:

[0062] OsPIU1-OX-F: 5'-GCCGGTAAACAACTCGAA-3';

[0063] OsPIU1-OX-R: 5'-CCAGCGAAAATAAGCCTCTG-3'.

[0064] (2) Knockout identification

[0065] DNA was extracted from the T0 generation lines obtained by transgenic plants, and knockout identification primers were designed around 300 bp before and after the PAM sequence for PCR amplification. The amplified products were sent to Shanghai Bioengineering for sequencing, and then the sequences were compared with the rice RAP-DB data to identify the knockout plants and mutation types.

[0066] The knockout identification primers are as follows:

[0067] PIU1-KO-JD-F: 5'-TTTGATCCGTTGTTGTGTCC-3';

[0068] PIU1-KO-JD-R: 5'-GGGATGCCCTCTTTGTCC-3'.

[0069] Example 6 Analysis of transgenic plant seed phenotypes

[0070] Select the overexpression gene initiated by Gt13a OsPIU1 and Ubi-initiated overexpression OsPIU1 as well as OsPIU1 After the knockout lines were harvested, 3 lines were selected, 5 lines were selected from each line, 100-300 grains were randomly selected from each line, and the grain length and width were measured using a rice appearance quality tester (JMWT12, Dongfu Jiuheng, Beijing). The results were repeated 5 times and the average 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 was taken as the final result. Figure 4 .

[0071] Depend on Figure 4 It can be seen that compared with the wild type, OsPIU1 Overexpression of seeds made them longer and wider, and their thousand-grain weight increased. OsPIU1 The grains of the knockout strains became shorter and narrower, and the thousand-grain weight was reduced.

[0072] Example 7 Analysis of leaf angle phenotype of transgenic plants

[0073] In the field, a protractor was used to measure the wild type, OsPIU1 The angles of the flag leaves of the overexpression and knockout strains were measured for 20 strains in each strain, and the final results were averaged. Figure 5 .

[0074] Depend on Figure 5 Compared with the wild type OsPIU1 The leaf angle of the overexpression strain became smaller. OsPIU1 The leaf angle of the knockout strain increased.

[0075] Example 8 Analysis of salt tolerance of transgenic plants

[0076] Soak the rice seeds in 10% sodium hypochlorite for 10 min for disinfection, and then wash them with distilled water. Place two layers of filter paper in the culture dish, sprinkle the seeds evenly, submerge the seeds in 20 ml of distilled water, and place the culture dish in a dark incubator at 28°C for about 2 days. After the seeds germinate, select seeds with consistent growth and move them into a 96-well hydroponic box filled with clean water. Culture them in an artificial climate chamber (28°C light 14 h / dark 10 h) with clean water for 5 days, then use 1 / 2 times the complete nutrient solution to culture until the one-leaf and one-heart stage, and then change the complete nutrient solution every 3 days. After the seedlings are cultured to the five-leaf stage, they are treated with 200mmol / L NaCl stress. The survival rate was counted 10 days after the treatment, and the results are shown in the table. Figure 6 .

[0077] Depend on Figure 6 It can be seen that after salt stress treatment, compared with the wild type, OsPIU1 The survival rate of overexpression lines was improved. OsPIU1 The survival rate of knockout strains was reduced.

[0078] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. OsPIU1 Application of genes in regulating rice grain size, leaf angle and / or salt tolerance, It is characterized in that OsPIU1 The nucleotide sequence of the gene is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

4.

2. Application of OsPIU1 protein in regulating rice grain size, leaf angle and / or salt tolerance, It is characterized in that OsPIU1 The amino acid sequence of the protein is shown in SEQ ID NO:

3.

3. The use according to claim 1 or 2, It is characterized in that The rice grain size includes the rice grain length, grain width and thousand-grain weight.

4. The use according to claim 1, It is characterized in that Increase OsPIU1 Gene expression increased rice grain length, grain width and 1000-grain weight, and reduced OsPIU1 Gene expression or knockout OsPIU1 Genes reduce grain length, grain width and thousand-grain weight.

5. The use according to claim 1, It is characterized in that Reduce or knock out OsPIU1 Gene expression increases rice leaf angle and upregulates OsPIU1 Gene expression reduces the angle of rice leaves.

6. The use according to claim 1, It is characterized in that Reduce or knock out OsPIU1 Gene expression reduces salt tolerance in rice, upregulates OsPIU1 Gene expression increases salt tolerance in rice.

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