Application and method of rice gene OsLRR6 in improving yield and modifying plant type and grain type of rice

Abstract

The application discloses application and a method of a rice gene OsLRR6 in improving rice yield and improving plant type and grain type. The gene is a DNA sequence in SEQ ID No. 1. The application firstly finds that the gene is related to growth and yield of rice, and the mitogen-activated protein kinase OsMPK6 activity (phosphorylation level) in a rice leaf sheath can be improved by knocking out or silencing the gene, thereby effectively increasing the rice plant height, the aboveground part and the underground part (root) weight, the effective tiller number, the grain length and width and the thousand-grain weight, and finally significantly improving the yield of a single rice plant. The application can be applied to crop genetic breeding, and is widely applied in breeding of improving the rice yield and improving the rice plant type and the grain type.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant engineering, and particularly relates to application and method of rice gene OsLRR6 in improving yield and plant type and grain type of rice. BACKGROUND

[0002] Rice is an important food crop in the world and one of the most important food crops in Asia. With the continuous growth of population, how to improve rice yield and ensure food production and safety is an important scientific problem. Rice yield is mainly determined by the number of effective panicles per plant, the number of grains per panicle and grain weight; the grain type of rice is determined by the length, width and thickness of the grain, which not only affects the appearance of rice, but also affects the number of seeds per unit mass and the yield of rice. With the continuous deepening of rice functional genomics research, researchers have found some genes that regulate yield-related traits such as plant type and grain type of rice, and revealed that the molecular mechanism of yield formation involves complex biological processes such as mitogen-activated protein kinase (MPK) signaling pathway, G protein-coupled receptor and its signaling transduction pathway, plant hormone-mediated signaling pathway, and epigenetic modification. Even so, there are still few rice yield, plant type and grain type related genes that can be directly applied to breeding. Therefore, accelerating the discovery and utilization of rice yield-related genes and revealing their regulatory mechanisms can provide important gene resources, theoretical basis and technical support for molecular design breeding and genetic engineering breeding of high-quality and high-yield rice.

[0003] Plant MPK cascade pathway plays an important role in regulating plant growth and development. For example, in rice, smg1 plants with impaired OsMKK4 function exhibit a short grain phenotype similar to brassinosteroid-deficient mutant plants, and the relative expression of brassinosteroid synthesis genes is also significantly reduced compared with conventional rice, indicating that OsMKK4 may regulate plant height and spikelet development through brassinosteroid-mediated signaling pathway (Duan et al, 2014). OsMPK6 can directly interact with OsMKK4 and is located downstream of OsMKK4, and regulates cell proliferation and brassinosteroid signaling and homeostasis to positively regulate rice grain size (Liu et al, 2015). Rice receptor-like protein kinase OsERECTA (OsER1) acts upstream of the OsMKKK10-OsMKK4-OsMPK6 cascade signal, and regulates the phosphorylation level of OsMPK6 in combination with OsMKKK10 and OsMKK4, maintains the cytokinin homeostasis in young rice panicles by regulating local cell division, and participates in the morphological formation of rice panicle (Guo et al. 2018; 2020). The above research results show that the MPK pathway plays an important role in regulating plant type, grain size and panicle morphological formation of rice.

[0004] The rice plant with the expression of the gene OsLRR6 inhibited has stronger direct resistance to rice planthoppers than a conventional plant, indicating that OsLRR6 can be used as a negative regulation factor for rice to resist damage by rice planthoppers. However, it is still unclear how OsLRR6 regulates the growth and development of rice, whether it can improve the plant type and grain type of rice, and thus serve as a potential genetic resource for breeding new rice varieties with high quality and high yield. SUMMARY

[0005] The present application aims at the deficiencies of the prior art and provides an application and method of a rice gene OsLRR6 in improving the yield and plant type and grain type of rice.

[0006] The present application aims at the deficiencies of the prior art and provides an application and method of a rice gene OsLRR6 in improving the yield and plant type and grain type of rice.

[0007] Further, the rice gene OsLRR6 has the DNA sequence of SEQ ID No. 1.

[0008] Further, the yield of rice is the thousand-grain weight and the yield per plant; the plant type is the plant height, the mass of the aboveground and underground parts, and the effective tiller number; and the grain type is the length and width of the rice grain.

[0009] The present application also provides a method for improving the yield and plant type and grain type of rice by reducing the expression level of the rice gene OsLRR6.

[0010] Further, the reduction of the expression level of the rice gene OsLRR6 specifically refers to the mutation or reduction of the transcription level of the rice gene OsLRR6 to obtain a rice strain with the gene OsLRR6 knocked out, mutated, knocked down, or silenced.

[0011] Further, the CRISPR or RNAi technology is used to knock out, mutate, or reduce the transcription level of the rice gene OsLRR6 to obtain a rice strain with the gene OsLRR6 knocked out, mutated, knocked down, or silenced.

[0012] Further, the rice gene OsLRR6 has the DNA sequence of SEQ ID No. 1.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] The application plants the gene OsLRR6 knockout mutant, the gene OsLRR6 silencing mutant and normal rice (wild type, WT) in an artificial climate chamber and a natural environment in a field and carries out a comprehensive phenotype observation test, finds that knocking out and silencing the gene OsLRR6 can significantly improve the rice plant height, the aboveground part and the underground part quality, the effective tiller number, the grain length and width and the thousand-grain weight, finally significantly increases the rice single plant yield, verifies the application of knocking out, mutating or silencing the gene OsLRR6 in improving the rice yield. The kinase activity detection finds that knocking out the gene OsLRR6 significantly improves the activity (phosphorylation level) of OsMPK6 in the outer leaf sheath of rice, indicates that OsLRR6 acts on the upstream of the MPK cascade pathway, regulates the activity of OsMPK6 and then negatively regulates the yield of rice and improves the rice plant type. The application provides important gene resources, theoretical basis and technical support for the molecular design breeding and genetic engineering breeding of high-quality and high-yield rice. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0016] Figure 1 is a schematic diagram of a vector for constructing an OsLRR6 gene mutation and gene silencing rice plant; wherein, Figure 1 A in the above is a schematic diagram of a CRISPR / Cas9 vector for mutating the OsLRR6 gene, Figure 1 B in the above is a schematic diagram of an RNAi vector for silencing the OsLRR6 gene.

[0017] Figure 2 is a silencing effect diagram of OsLRR6 in normal rice (WT), gene mutation lines and gene silencing lines; wherein, Figure 2 A in the above is the relative expression amount of OsLRR6 in the rice line with the mutated OsLRR6 gene, Figure 2 B in the above is the relative expression amount of OsLRR6 in the rice line with the silenced OsLRR6 gene.

[0018] Figure 3 is the growth condition of normal rice (WT) and gene OsLRR6 mutation lines (CR1, CR2, CR3) planted in a room for 30 days; wherein, Figure 3 A in the above is a plant growth phenotype photo, Figure 3 B in the above is a plant height statistical diagram, Figure 3 C in the above is a root length statistical diagram, Figure 3D is a statistical chart of the above-ground part mass of rice in the figure, Figure 3 E is a statistical chart of the underground part mass of rice in the figure.

[0019] Figure 4 is the growth condition of normal rice (WT) and gene OsLRR6 mutant lines (CR1, CR2, CR3) planted indoors for 40 days; wherein, Figure 4 A is a plant growth phenotype photo in the figure, Figure 4 B is a plant height statistical chart in the figure, Figure 4 C is a root length statistical chart in the figure, Figure 4 D is an above-ground part mass statistical chart of rice in the figure, Figure 4 E is an underground part mass statistical chart of rice in the figure.

[0020] Figure 5 is the growth condition of normal rice (WT) and gene OsLRR6 silencing lines (Ri-12, Ri-23) planted indoors for 30 days; wherein, Figure 5 A is a plant growth phenotype photo in the figure, Figure 5 B is a plant height statistical chart in the figure, Figure 5 C is a root length statistical chart in the figure, Figure 5 D is an above-ground part mass statistical chart of rice in the figure, Figure 5 E is an underground part mass statistical chart of rice in the figure.

[0021] Figure 6 is the growth condition of normal rice (WT) and gene OsLRR6 mutant lines (CR1, CR2) at the mature stage in the field; wherein, Figure 6 A is a plant growth condition photo at the mature stage in the figure, Figure 6 B is a grain length photo in the figure, Figure 6 C is a grain width photo in the figure; Figure 6 D is a grain length statistical chart in the figure, Figure 6 E is a grain width statistical chart in the figure, Figure 6 F is a thousand-grain weight statistical chart in the figure, Figure 6 G is a seed setting rate statistical chart in the figure, Figure 6 H is an effective tiller number statistical chart in the figure, Figure 6 I is a single plant rice yield statistical chart in the figure.

[0022] Figure 7 is the growth condition of normal rice (WT) and gene OsLRR6 silencing lines (Ri-12, Ri-23) at the mature stage in the field; wherein, Figure 7 A is a plant growth condition photo at the mature stage in the figure, Figure 7 B is a grain length photo in the figure, Figure 7 C is a grain width photo in the figure; Figure 7 D is a grain length statistical chart in the figure, Figure 7E grain width statistical chart in D, Figure 7 F is a thousand-grain weight statistical chart in D, Figure 7 G is a seed setting rate statistical chart in D, Figure 7 H is an effective tiller number statistical chart in D, Figure 7 I is a single rice yield statistical chart in D.

[0023] Figure 8 is the activity (phosphorylation level) of OsMPK6 in the outer leaf sheath of normal rice (WT) and gene OsLRR6 mutant lines (CR1, CR2) planted in a room for 30 days.

[0024] The asterisk in the figure indicates that the gene OsLRR6 mutant line or the OsLRR6 silenced line has a significant difference compared with normal rice (WT) (*, P < 0.05; **, P < 0.01; Student's t-test). DETAILED DESCRIPTION

[0025] The present application studies the target gene and the role in improving the yield of rice by using tissue culture, transgenic and other molecular biology techniques, combined with plant-related growth phenotype determination methods. The results prove that knocking out or silencing the gene OsLRR6 in rice can improve the activity (phosphorylation level) of OsMPK6 in the leaf sheath of rice, and finally significantly improve the plant height, effective tiller number, grain length and width, thousand-grain weight and single rice yield of rice.

[0026] The present application will be described in detail below in combination with the accompanying drawings. The features in the following examples and embodiments can be combined with each other without conflict.

[0027] Example 1: Obtaining of rice materials

[0028] The rice varieties used in the present application are Xiushui 11 (XS11, WT) and OsLRR6 gene mutant transgenic lines (CR1, CR2, CR3) or silenced transgenic lines (Ri-12, Ri-23) constructed by taking XS11 as a receptor.

[0029] The method for obtaining the OsLRR6 gene mutant lines (CR1, CR2, CR3) mainly includes the following steps: (1) predicting the target sequence of OsLRR6 by using the CRISPR-GE website, annealing and synthesizing double-stranded sequences, then fusing the target sequence with U3b promoter to form an SgRNA expression box, and then connecting into a pHUN4c12s vector to form a pHUN4c12s-koOsLRR6 knockout vector, which is identified by plasmid enzyme digestion and sequencing, then electroporated into Agrobacterium LBA4404 and stored at -80 DEG C for standby; (2) using the plant transformation method mediated by Agrobacterium, target gene sequence amplification, PCR sequencing, and real-time fluorescent quantitative PCR method to screen the three homozygous OsLRR6 gene mutant homozygous lines (T2) obtained without T-DNA insertion traces, which can refer to the patent literature with the authorized publication number CN112126710B; specifically, CR1 and CR2 are mutated to 5'-CCTTGTAATCCCTGCACCTGGT-3' and 5'-CCTTGTATCCCTGCACCTGGT-3' at the target point 5'-CCTTGTAAATCCCTGCACCTGGT-3', and CR3 is mutated to 5'-AGGTCCTCTGATTCCAGCTGG-3' at the target point 5'-AGGTCCTCTGATTCCACAGCTGG-3'.

[0030] In addition, the method for obtaining the OsLRR6 gene silencing transgenic lines (Ri-12, Ri-23) mainly includes the following steps: (1) analyzing the specific sequence in the OsLRR6 cDNA sequence by using the NCBI website, and designing specific primers to amplify the fragment by PCR, then connecting the amplified OsLRR6 specific fragment to the left and right sides of the intron in the pCAMBIA1301-Ri vector respectively, obtaining the pCAMBIA1301-OsLRR6-Ri gene silencing vector, which is identified by plasmid enzyme digestion and sequencing, then electroporated into Agrobacterium EHA105 and stored at -80 DEG C for standby; (2) obtaining T0 transgenic plants by using the plant tissue culture technology mediated by Agrobacterium. The T0 rice plants are grown in the field, the T1 rice seeds harvested after selfing are soaked in water for 10 days, the tender root tip parts of each seedling are cut separately, and then immersed in GUS dye (Solarbio, item number G3061) for staining, and the plants with blue root tips are positive plants. The lines with a GUS color separation ratio (blue: no color) close to 3:1 are selected, 10 blue plants are randomly selected from the lines and transferred to the field for further breeding to obtain T2 seeds. After the T2 generation seeds are soaked in water for 10 days, 50 seedlings are randomly selected from each line for GUS staining, and the final confirmation that all the root tips of the seedlings in the corresponding line are blue is a homozygous line, which can be further planted and expanded for use in subsequent tests.

[0031] AsFigure 1 As shown, the constructed OsLRR6 gene knockout vector and gene silencing vector are pHUN4c12s-koOsLRR6 and pCAMBIA1301-OsLRR6-Ri, respectively.

[0032] Example 2: Detection of silencing effect of OsLRR6 knockout and silencing lines

[0033] The outer sheaths (five biological replicates) of 30-day-old WT plants, OsLRR6 gene knockout lines (CR1, CR2, CR3), and OsLRR6 gene silencing lines (Ri-12, Ri-23) were collected, respectively, and stored at -80°C for later use. The collected rice tissues were ground with liquid nitrogen, and about 0.1 g of ground rice tissue powder was weighed.

[0034] Total RNA was extracted using a TaKaRa plant RNA extraction kit (TaKaRa, Catalog No. 9769), and the concentration, purity, and quality of the obtained RNA were evaluated using a ultramicro protein nucleic acid spectrophotometer (BioDrop). 0.5 μg of rice total RNA was reverse transcribed into cDNA using a PrimeScript TM RT Master Mix (TaKaRa, Catalog No. RR036) reverse transcription kit, and the specific operation was carried out according to the product instruction.

[0035] Fluorescence quantitative PCR (qRT-PCR) detection uses TB Premix Ex Taq TM II (TaKaRa, Catalog No. RR820) enzyme premix to prepare the reaction system, and CFX96 TM Real-Time system (Bio-RAD) quantitative PCR instrument to detect the fluorescence signal. Rice gene OsACTIN (TIGR ID: LOC_Os03g50885) was used as an internal reference to detect the relative expression of OsLRR6 gene in each rice line. The specific reaction system and procedure are shown in the product instruction, and the quantitative PCR primer sequences are as follows:

[0036] OsACTIN-F: 5'-GGACAGGTTATCACCATTGGT-3'

[0037] OsACTIN-R: 5'-CCGCAGCTTCCATTCCTATG-3'

[0038] OsLRR6-F: 5'-GTTCCATGTCACCTGCAACAAT-3'

[0039] OsLRR6-R: 5'-CTGATAGACCTGCTAACCCCAAA-3'

[0040] As shown in Figure 2 , the results show that the relative expression of OsLRR6 gene in 30-day-old OsLRR6 gene mutant lines CR1, CR2 and CR3 is reduced by 91.31%, 93.44% and 95.37% respectively compared with WT plants; and the relative expression of OsLRR6 gene in 30-day-old OsLRR6 gene silencing lines Ri-12 and Ri-23 is reduced by 85.96% and 81.54% respectively.

[0041] Example 3: Determination of indoor rice growth phenotype

[0042] An appropriate amount of conventional rice WT, OsLRR6 gene knockout lines (CR1, CR2, CR3) and OsLRR6 gene silencing lines (Ri-12, Ri-23) rice seeds were placed in tissue culture bottles (10 cm high, 8 cm wide), soaked with propanil for 24 hours, then washed clean, soaked in clean water for 24 hours, then drained the water, and placed the seeds in a petri dish to continue germination, and washed every day. After 10 days, the seedlings with uniform growth were selected, wrapped with sponge at the base, and planted on a plastic plate (50 cm long, 35 cm wide, 3 mm thick) with 72 round holes (2 cm in diameter) evenly distributed, then transferred to a plastic basket (45 cm long, 30 cm wide, 15 cm high) containing rice nutrient solution, so that the roots of the rice seedlings were in contact with the rice nutrient solution, and placed in a greenhouse with a temperature of 28±2℃, light for 14 hours, and humidity of 60-75% for further culture, and the rice nutrient solution was replaced every week.

[0043] When the plants grew to 30 and 40 days old, the plant height, root length, aboveground part and underground part (root system) mass of the rice plants were measured. Mass and length measurements were made using an electronic balance and a ruler, respectively. At the same time, photos of 30-day-old and 40-day-old rice plant growth were taken. The above experiments were all carried out in a greenhouse (26±2℃; light for 14 hours; humidity 60-75%).

[0044] As shown in Figure 3 , Figure 4 and Figure 5 , the results show that the plant height, aboveground part mass and root weight of 30-day-old OsLRR6 gene mutant lines are increased by 5.37%, 26.36% and 21.30% respectively compared with WT plants, and the plant height, aboveground part mass and root weight of 40-day-old OsLRR6 gene mutant lines are increased by 5.24%, 35.69% and 39.86% respectively; the plant height, aboveground part mass and root weight of 30-day-old OsLRR6 gene silencing lines are increased by 10.43%, 20.12% and 28.27% respectively; and the root length shows no significant difference among the lines.

[0045] Example 4: Field rice growth phenotype, yield determination

[0046] The field test was carried out in Zhejiang University Changxing Agricultural Science and Technology Park (Zhejiang University Transgenic Plant Test Base) in Sian Town, Changxing County, Huzhou City, Zhejiang Province. An appropriate amount of rice seeds was soaked with prochloraz for three days, then evenly spread on the field seedbed to germinate. After 30 days, the seedlings were transplanted to the field. The method of randomized block design was used, with a spacing of 20 cm between seedlings. Each strain was set up with 3 replicates, and each plot was 1 m 2 (1 m x 1 m). Necessary fertilization and pesticide application were carried out during the growth of rice. After the seeds matured, the growth phenotype of the rice plants was photographed in the field, and the effective tiller number of the rice was recorded. According to the Z-shaped sampling method, 10 clusters of rice were randomly selected in each plot and the panicles were cut off. The single cluster of rice panicles was placed in an independent net bag, and then taken back to the laboratory for drying to constant weight. The yield mainly investigated the grain length, grain width, thousand-grain weight, seed setting rate, and single cluster of rice yield. The specific measurement and calculation methods are as follows: grain length and grain width were measured with vernier calipers; the seed setting rate was represented by the ratio of mature seeds per cluster to the total number of grains (including the number of empty grains); the yield per cluster was equal to the total weight of mature seeds per plant; the thousand-grain weight = (total weight of mature seeds / total number of mature seeds) x 1000.

[0047] As shown in Figure 6 and Figure 7 , the results showed that the OsLRR6 mutant and silencing strains were stronger in the field compared to WT plants. Among them, the tiller number, grain length, grain width, thousand-grain weight, and single plant rice yield of the OsLRR6 mutant strain were increased by 15.46%, 8.05%, 1.91%, 4.32%, and 17.23%, respectively; the tiller number, grain length, grain width, thousand-grain weight, and single plant rice yield of the OsLRR6 gene silencing strain were increased by 20.29%, 12.79%, 1.67%, 7.99%, and 15.43%, respectively.

[0048] Example 5: Determination of OsMPK6 kinase activity in rice leaf sheath tissue

[0049] The outer leaf sheaths of 30-day-old WT plants and OsLRR6 knockout strains (CR1, CR2) were collected (five biological replicates), and stored at -80°C for later use. The leaf sheath tissue samples of the same strain were mixed together in equal amounts, ground with liquid nitrogen, and 0.12 g of rice sample was weighed for later use.

[0050] 200 μL protein extraction solution was added to the sample, and after standing on ice for 10 min, centrifuged at 14000 rpm for 20 min at 4°C, and the supernatant was the soluble total protein. The protein extraction solution used was: 100 mM HEPES (pH = 7.5), 5 mM EDTA, 5 mM EGTA, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerophosphate sodium, 1 mM PMSF, 10% glycerol, protease inhibitor (Roche, item number 04693132001), and phosphatase inhibitor (Roche, item number 04906845001).

[0051] The protein concentration was determined using Protein Assay Dye Reagent Concentrate (Bio-Rad; item number 5000006). 50 μg of protein was mixed with loading Buffer (Fdbio, item number FD006) and then incubated at 95°C for 5-10 min, separated by 10% SDS-PAGE, and then wet transferred to a nitrocellulose membrane (PALL, item number 66485). Anti-Plant beta Actin Mouse mAb antibody (Engibody, item number AT0004) and phospho-p44 / 42 MAPK Rabbit mAb (Cell Signaling Technologies, item number 4370) were used as the primary antibody, and horseradish peroxidase-labeled anti-mouse or anti-rabbit (MultiSciences, item number GAM007 or GAR007) was used as the secondary antibody. After color development using EZ-ECL chemiluminescence developing solution (Biological industries, item number 20-500), the activity of OsACTIN and OsMPK6 was detected.

[0052] As shown in Figure 8 the results showed that the activity of OsMPK6 in the outer sheath of OsLRR6 mutant plants was significantly higher than that of WT plants.

[0053] The above examples are only used to illustrate the design ideas and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and the protection scope of the present application is not limited to the above examples. Therefore, any equivalent changes or modifications made in accordance with the principles and design ideas disclosed by the present application are within the protection scope of the present application.

Claims

1. Use of increasing the expression level of a rice gene OsL RR6 in rice to increase yield and to increase plant height, shoot and root mass, effective tiller number, grain length and width in rice.

2. Use according to claim 1, wherein The DNA sequence of the rice gene OsLRR6 is shown as SEQ ID No.

1.

3. The use according to claim 1, wherein The rice yield is thousand-grain weight and yield per plant.

4. A method for increasing yield and increasing plant height, shoot and root mass, effective tiller number, grain length and width in rice, characterized by, Reducing the expression level of rice genes OsLRR6 to increase rice yield and to increase rice plant height, aboveground and belowground mass, effective tiller number, rice grain length and width.

5. The method of claim 4, wherein, The method for reducing the expression level of the rice gene OsLRR6 Specifically, the method comprises the following steps: mutating the rice gene OsLRR6 or reducing the transcription level of the rice gene, to obtain a rice strain with a knockout, mutation, knockdown or silencing of the rice gene OsLRR6 .

6. The method of claim 4, wherein, Rice lines with knock-out, mutation, knock-down or silencing of the genes OsLRR6 obtained by knocking out, mutating or reducing the transcription level of the genes OsLRR6 using CRISPR or RNAi technology.

7. The method of claim 4, wherein, The DNA sequence of the rice gene OsLRR6 is shown as SEQ ID No. 1.

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