CN111909939B - Application of rice gene LJS4-2 in controlling rice leaf pillow development and leaf angle - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and specifically relates to the application of rice gene LJS4-2 in controlling the development of rice leaf pillow and the size of leaf angle. The present invention obtains the gene LJS4‑2 (GENE ID: Os07g0674800 ). Knockout LJS4‑2 by gene knockout technology, the mutant showed significantly smaller angle than the wild type, and the plant type was erect. By freehand sections of the flag leaf pillow at the heading stage and staining with phloroglucinol staining, it was found that the phloroglucinol staining of the sclerenchyma cells in the cross-section of the leaf pillow of the mutant was redder than that of the wild type, indicating that the lignin accumulation was higher than that of the wild type. (Nipponbare) increases, and the mechanical strength of the leaf pillow increases. Therefore, knocking out the LJS4-2 gene through genetic engineering technology can change the mechanical strength of the plant leaf pillow and the formation of the leaf angle, thereby improving the plant shape and planting density, and increasing the yield.

Description

Application of rice gene LJS4-2 in controlling the development of rice pillow and the size of leaf angle

technical field

The invention belongs to the field of plant genetic engineering, and specifically relates to the application of rice gene LJS4-2 in controlling the development of rice leaf pillow and the size of leaf angle.

Background technique

Rice leaves are divided into three categories, including coleoptile, incomplete leaf and complete leaf. The coleoptile is the first white coleoptile to appear during germination and is a deformation of the leaf without chlorophyll. The incomplete leaf is the first green leaf to grow from the coleoptile, only the sheath, without the leaf and leaf pillow structure. A complete leaf is from the second green leaf, with blades, leaf sheaths, and leaf pillows. The main function of the leaves is as the organ of photosynthesis. Leaf sheaths wrap around the main stem and enhance the support of the stem. Leaf pillow is a kind of mechanical tissue unique to monocotyledonous grasses. It is a mechanical tissue connecting the leaf and the leaf sheath, including the leaf pillow band, leaf auricle, and ligule. The occipital band acts as a mechanical tissue that allows the blade to deviate from the main stem to form an angle when the blade and sheath have grown (Hoshikawa and Ichii, 1989). Therefore, the presence and development of the leaf pillow directly determine the size of the leaf angle, thereby affecting the rice plant type and yield.

With the growing population, the demand for food is also increasing. Improving rice yield has always been the focus and focus of rice breeding research and gene function research. In 1968, Donald proposed the concept of crop ideal plant type breeding, that is, the selection of traits related to plant photosynthesis, growth and grain yield (Donald, 1968). The traits affecting rice plant type mainly include plant height, number of tillers, panicle shape, grain shape, leaf shape, leaf angle and tiller angle, etc. Among them, the size of leaf angle is an important agronomic trait that determines the uprightness of leaves (Feng Rongkun, 2006) . Erect leaves can enhance the light-harvesting capacity of photosynthesis, serve as a nitrogen source for grain filling, and can also increase planting density, thereby increasing leaf area index and rice yield (Sakamoto, et al., 2006).

Contents of the invention

The purpose of the present invention is to obtain the gene LJS4-2 that regulates the development of the paddy leaf pillow and the size of the leaf angle.

Another object of the present invention is to provide the application of LJS4-2 in controlling rice leaf pillow development and leaf angle.

In order to achieve the above object, the present invention clones the gene LJS4-2 for regulating the development of the leaf pillow from the rice leaf pillow tissue through the method of reverse genetics.

Specifically, the primer sequences for cloning the LJS4-2 gene are as follows:

LJS4-2-F CGGGATCCATGTGTGGCGGCGCGATCATTT

LJS4-2-R GCGTCGACCATCGGCACGGCCGTGTGGAT

Specifically, the PCR conditions for cloning the LJS4-2 gene were: denaturation at 94°C for 3 min, 30 s at 94°C, 1 min at 55°C, 35 cycles at 68°C for 2 min, and extension at 68°C for 10 min.

Specifically, the total volume of the PCR reaction system used to clone the LJS4-2 gene is 50 μl, and the template is 1ul of Nipponbare cDNA (about 50ng), 5 μl of 10×KOD enzyme reaction buffer, 2 μl of 25mM MgCl ₂ , 5 μl of 5mM dNTP, and 5 uM primers 5μl (2.5μl for each primer), 1μl KOD enzyme, add ddH2O (sterile deionized water) to 50μl.

The present invention obtains the LJS4-2 gene sequence comprising the nucleotide described in SEQ ID NO.1 through the above method.

The present invention also obtains the amino acid sequence encoded by the LJS4-2 gene, as shown in SEQ ID NO.2.

The genes identified by the method of bioinformatics in the present invention are genes that regulate the development of the leaf pillow, and the expression specificity of the gene LJS4-2 in the development of the leaf pillow is verified by using the RNA-seq data of the leaf pillow in different periods.

Application of LJS4-2 gene in grass crop improvement.

Application of LJS4-2 gene in improving rice plant structure and increasing rice yield.

The application of the LJS4-2 gene in regulating the development of the rice pillow and the size of the leaf angle. During the application, a CRISPR/CAS9 vector containing the LJS4-2 gene was constructed and transferred into Nipponbare, and the LJS4-2 gene was knocked out. The obtained transgene The line shows that the leaf angle is smaller than that of the wild type (Nipponbare). Therefore, this method can increase the lignin accumulation in the sclerenchyma tissue of the leaf pillow, improve the mechanical strength of the leaf pillow, reduce the leaf angle, improve the plant shape and planting density, and increase Yield, realized the regulation of rice leaf pillow development and leaf angle.

Compared with prior art, the beneficial effect of the present invention is:

1. The LJS4-2 gene is a gene specifically expressed in the later stage of rice leaf pillow development, which makes it possible to use this gene to only change the angle of the crop without affecting other traits.

2. The LJS4-2 gene is an effective gene for changing the leaf angle. It can change the lignin accumulation and mechanical strength of the sclerenchyma of the leaf pillow, and then change the leaf angle of the crop.

3. At present, there are not many genes that regulate the size of the leaf angle by changing the mechanical strength of the leaf pillow. This gene reveals that it participates in the regulation of the leaf angle by controlling the lignin accumulation and mechanical strength of the leaf pillow sclerenchyma .

4. At present, there are relatively few studies on the mechanism of plant upright plant type increasing yield. After LJS4-2 was successfully knocked out by CRISPR/Cas9, rice yield increased by increasing the number of fertile panicles per unit area under high density (as shown in Figure 6 Show).

Description of drawings

Figure 1 shows the results of RNA-seq sequencing, in which Figure 1a shows the specific expression of LJS1-1 in the early stage of rice leaf pillow development, Figure 1b shows the specific expression of LJS4-1 in the late development of rice leaf pillow, and Figure 1c shows LJS3-1 and its homologs The gene LJS3-1L is specifically expressed in the S3 and S4 stages of rice leaf pillow development, respectively. Figure 1d shows that LJS5-1 is specifically expressed in the late stage of rice leaf pillow development. Figure 1e shows that LJS1S2-1 is specifically expressed in the early stage of rice leaf pillow development. Figure 1f It shows that LJS4-2 is specifically expressed in the late stage of rice leaf pillow development. Figure 1g shows that LJS5-2 and its homologous gene LJS5-2L are specifically expressed in the S4 and S5 stages of rice leaf pillow development;

Figure 2 shows the leaf angle phenotype and plant phenotype at heading stage of the CRISPR/CAS9 knockout target gene of transgenic rice seedlings and the leaf angle statistics of the knockout transgenic rice seedlings and wild type (Nipponbare), in which the target gene in Figure 2a Because LJS1-1 and homologous gene LJS1-1L thereof; Fig. 2b target gene is LJS4-1 ; Fig. 2c target gene is LJS3-1 and homologous gene LJS3-1L thereof; Fig. 2d target gene is LJS5-1 ; Fig. 2e Target gene is LJS1S2-1 and its homologous gene LJS1S2-1L ; Figure 2f target gene is LJS4-2 ; Figure 2g target gene is LJS5-2 and its homologous gene LJS5-2L ;

Figure 3 is a diagram of the cytological structure changes of the flag leaf pillow of seedlings of transgenic rice and wild type (Nipponbare) seedlings at the heading stage of CRISPR/CAS9 knockout of the target gene, in which the target gene in Figure 3a is LJS1-1 and its homologous gene LJS1- 1L ; Figure 3b target gene is LJS4-1 ; Figure 3c target gene is LJS3-1 and its homologous gene LJS3-1L ; Figure 3d target gene is LJS5-1 ; Figure 3e target gene is LJS1S2-1 and its homologous gene LJS1S2-1L ; Figure 3f target gene is LJS4-2 ; Figure 3g target gene is LJS5-2 and its homologous gene LJS5-2L ;

Figure 4a is Real-time PCR verification of target gene regulation to promote and inhibit the expression of lignin synthesis target genes in transgenic rice after CRISPR/CAS9 knockout target genes, where the target gene in Figure 4a is LJS4-1 , and Figure 4b The target gene is LJS3-1 and its homologous gene LJS3-1L , the target gene in Figure 4c is LJS1S2-1 and its homologous gene LJS1S2-1L , and the target gene in Figure 4d is LJS4-2 ;

Fig. 5 is the seedling phenotype and expression level analysis of pLJS1S2-1::LJS1S2-1 transgenic rice transgenic lines;

Figure 6 shows the effect of CRISPR/CAS9 knockout target gene transgenic rice on yield, wherein the target gene in Figure 6a is LJS1-1 and its homologous gene LJS1-1L, the target gene in Figure 6b is LJS4-1 , and the target gene in Figure 6c is Because LJS3-1 and its homologous gene LJS3-1L , the target gene in Figure 6d is LJS5-1 , the target gene in Figure 6e is LJS1S2-1 and its homologous gene LJS1S2-1L , the target gene in Figure 6f is LJS4-2 , and Figure 6g The target gene is LJS5-2 and its homologous gene LJS5-2L .

Detailed ways

The present invention clones the gene LJS1-1 (GENE ID: Os01g0922800 ) and its homologous gene LJS1-1L (GENE ID: Os08g0531900 , homology is 45.83%), Gene LJS4-1 (GENE ID: Os03g0182800 ), gene LJS3-1 (GENE ID: Os04g0549700 ) and its homologous gene LJS3-1L (GENE ID: Os02g0656600, homology is 57.58%), gene LJS5-1 (GENE ID : Os06g0166400 ), gene LJS1S2-1 (GENE ID: Os06g0181700 ) and its homologous gene LJS1S2-1L (GENEID: Os02g0797100, homology 71.59%), gene LJS4-2 (GENE ID: Os07g0674800 ), gene LJS5-2 (GENE ID: Os10g0536100 ) and its homologous gene LJS5-2L (GENE ID: Os03g0122600 , homology is 63.88%), the genes identified by bioinformatics methods are all genes that regulate leaf pillow development, and use different The RNA-seq data of the stage occipital validated the expression specificity of the obtained genes during the development of the occipital. The present invention transfers the above gene into Nipponbare through a transgenic method, and finds that the target gene can regulate the size of the rice leaf angle, improve the plant type and planting density, increase the yield, and realize the regulation of the rice leaf pillow development and the size of the leaf angle.

The specific implementation method is as follows, it should be noted that, in the following gene synthesis process or rice application test, if not specified, all are routine test methods and technical means in this field; the reagents or biological materials involved, if not specified Instructions, all have been published or are commercially available products that can be purchased directly.

Example

(1) The application test of LJS1-1 gene and its homologous gene LJS1-1L in controlling the development of rice leaf pillow and the size of leaf angle, the specific steps are as follows:

1. Acquisition of gene LJS1-1 and its homologous gene LJS1-1L regulating rice leaf pillow development and leaf angle

1.1 The total volume of the reaction system is 50 μl, the template is Nipponbare cDNA 1ul (about 50ng), 10×KOD enzyme reaction buffer 5μl, 25mM MgCl ₂ 2μl, 5mM dNTP 5μl, 5 μM primer 5μl (using step-by-step PCR method, using primer LJS1-1-F and LJS1-1-R and LJS1-1L-F and LJS1-1L-R (2.5 μl each primer), 1 μl KOD enzyme, add _ddHO (sterile deionized water) to 50 μl .

1.2 The reaction program is: denaturation at 94°C for 5min, 30s at 94°C, 1min at 55°C, 35cycles at 68°C for 2min, and extension at 68°C for 10min.

1.3 The primers used are as follows:

LJS1-1-F CGGGATCCATGGCGCGGAGGGGGAGA

LJS1-1-R GCGTCGACTGCACTTCTCTTCCTCCTGCC

LJS1-1L-F CGGGATCCATGGAGGGAGGAGGGAGGAGG

LJS1-1L-R GCGTCGACAGAGCTCACTCCTGATCTTGGCT

2. Using RNA-seq data to verify the specific expression of gene LJS1-1 and its homologous gene LJS1-1L in rice leaf pillow

Rice Nipponbare seedlings growing 4, 5, 6, 7, and 9 days from soaking under normal conditions are defined as the first (S1), second (S2), third (S3), and third stages of leaf pillow development. Fourth period (S4), fifth period (S5). The leaves and occipital of the first complete leaf were taken respectively, and the total RNA was extracted using Tiangen RNAprep pure Plant Kit (Tiangen), followed by RNA-seq sequencing.

Through bioinformatics analysis, the differential gene sets specifically expressed in the five stages and the corresponding GO terms of functional enrichment analysis were obtained, and the differential gene sets were selected, including the single-stage specific expression gene sets M01-M05 and leaf pillow development-related GO terms gene collection, extract the promoter sequence, use MEME software to analyze the enriched motifs (motifs) on the promoter, and select the top 10 motifs that meet E-value ≤ 10 ^-6 as possible transcription factor binding sites (pTFBSs); then use TOMTOM software and JASPAR CORE database to compare pTFBSs with known TFBSs, and select known TFBSs that meet the conditions of q-value ≤ 0.05 and p-value ≤ 10 ^-4 , so as to obtain the known TFBSs corresponding to pTFBSs TFs; then use BLASTP (e-value ≤ 10 ^-10 ) to find the homologous genes of these known TFs in rice; the definition of candidate TFs meets two conditions, the first is that plantTFDB is identified as belonging to the corresponding transcription factor family, the second The second is that TFs and their corresponding target genes specifically expressed in the same period have a high correlation of expression trends (PCC ≥ 0.9). The present invention obtains the gene LJS1-1 and its homologous gene LJS1-1L by analyzing the differential gene set M01 specifically expressed in the S1 phase as above . Finally, the expression levels of the gene LJS1-1 and its homologous gene LJS1-1L in the leaves and occipital of the five stages were obtained. The gene LJS1-1 was found to be specifically expressed in the developing S1 of the occipital (Fig. 1a).

3. Regulation of rice leaf pillow by LJS1-1 gene and its homologous gene LJS1-1L

3.1 Construction of CRISPR/Cas9 vector

Using a fragment containing the CRISPR target site sequence of the gene LJS1-1 and its homologous gene LJS1-1L , the fragment was cloned into the pCXUN-CAS9 vector using the KpnI restriction site. For details, see the literature He, Y., Zhang, T., Yang, N., Xu, M., Yan, L., Wang, L., Wang, R., and Zhao, Y. (2017). Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of Promoters for CRISPR/Cas9 mediated genome editing. Journal of genetics and genomics = Yi chuan xue bao 44,469-472.

Wherein, the total volume of the reaction system for obtaining the CRISPR target site fragment of the gene LJS1-1 and its homologous gene LJS1-1L is 50 μl, and the template used by the LJS1-1 gene is 1 μl (about 50ng) of the U6 carrier; the LJS1-1L gene The templates used were 1 μl of U3 vector (about 50ng), 5 μl of 1×KOD enzyme reaction buffer, 2 μl of 25mM MgCL2, 5 μl of 5 mM dNTP, 5 μl of 5 uM primers (2.5 μl for forward and reverse primers), 1 μl of KOD enzyme, Add ddH2O (sterile deionized water) to 50 μl.

The reaction program was: denaturation at 94°C for 5min, 30s at 94°C, 1min at 55°C, 35cycles at 68°C for 2min, and extension at 68°C for 10min.

The primers used are:

LJS1-1-U6F GGTGCGGTTCTCCAAGAGGAgttttagagctagaaatagcaagtta

LJS1-1-U6R TCCTCTTGGAGAACCGCACCAACCTGAGCCTCAGCGCAGC

LJS1-1L-U3F ATCGCACCTGCCGGCTCGTCgttttagagctagaaatagcaagtta

LJS1-1L-U3F GACGAGCCGGCAGGTGCGATgccacggatcatctgcacaactc

The sequence fragment sequence of the LJS1-1 target site and the sequence fragment sequence of the LJS1-1L target site were obtained, and the fragments were cloned into the pCXUN-CAS9 vector using the KpnI restriction site.

Take 2 μl of the pCXUN-CAS9 vector containing LJS1-1 and LJS1-1L targets, add it to 50 μl EHA105 competent, and mix well. Add it to the pre-cooled electric shock cup for electric shock transformation. Parameter setting of electric excitation instrument: voltage 2.45 kV, resistance 200Ω, capacitance 200 μF.

3.2 Rice genetic transformation

The rice transformation in the following method adopts the genetic transformation method mediated by Agrobacterium EHA105, and the specific steps are as follows:

3.2.1 Callus induction

Hull the rice seeds, soak the plump and clear grains in 70% ethanol for 1 min, rinse with sterile water 1-2 times; then use NaClO solution containing 2% active chlorine (40 ml NaClO Add 60 ml of water to the solution), add 1-3 drops of Tween 20, and soak for more than 30 minutes (generally 40 minutes, up to 1 hour). Shake occasionally, then rinse 4-5 times with sterile water. Pour it on a sterilized flat plate and filter paper to blot it dry for about 1 hour; put it on N6D solid medium (10 grains/25 ml/bottle), with the embryo facing up or in contact with the medium, at 28°C, dark culture for 25 ~30d. N6D medium: N6 salt and vitamins, 0.5g/l casein hydrolyzate, 30g/l sucrose, 2mg/l 2,4-D, 2.5g/l Phytagel (Sigma), pH 5.8.

3.2.2 Culture of Agrobacterium and its co-culture with rice callus

Take a sterilized small spoon to scrape the Agrobacterium, use the back of the spoon to stick the bacteria body on the tube wall and pat lightly to disperse, OD ₆₀₀ =0.8～1.0; let the previously cultured callus dry on sterile filter paper, and then concentrate it in a Transfer the plate into the bacterial solution at one time, gently rotate the centrifuge tube to distribute the bacterial solution evenly, and let it stand for about 15-20 minutes; pour out the bacterial solution, and put the callus on sterile filter paper for about 1.5 hours to ensure that the bacterial solution Blot dry, transfer to 1/2 N6D AS medium, culture in dark at 20°C for 2-3 days, if there is a bacterial film on the contact part of the callus and the medium, then it can be sterilized; 1/2 N6D AS medium: N6D2 , 10g/l glucose, 100~400μmol/l acetosyringone (add when used), pH5.2.

3.2.3 Removal of Agrobacterium

Put the co-cultured callus into a 50 ml centrifuge tube, wash it with sterile water for more than 3 times until the liquid is relatively clear, pour out the sterile water, N6D+Cn 500 mg/L (or AP500ml/L), 100 rpm ,, 15-20 min, 2-3 times; pour the callus on sterile filter paper and blot it dry for about 2 hours, depending on the situation; transfer the dried callus into N6D-AS, add cephalosporin Cn 250 mg /L, 28°C, dark culture for 7-10 days.

3.2.4 Screening of callus

Pick out the calli not contaminated by Agrobacterium, add Cn250 mg/L and Hn (50 mg/L) for the first time, 15-20 days; All the healed wounds were transferred again, 15-20 days. For the third selection of new callus, use Hn to screen for 15-20 days; the frequency of use must be in accordance with the above arrangement, but it should be ensured that the time for calli to be screened on Hn is at least 45 days, and the new callus selected for the third time It is best to screen the callus for 20 days; N6D screening medium: N6D+Cn250 mg/L+Hn50 mg/L, pH=5.8-5.9.

3.2.5 Differentiation and rooting

All the calli selected for the fourth time were transferred into MS, Hn 50 mg/L, cultured in dark, and pre-differentiated (pH 5.9) for 12-15 days. Select the fresh callus with good growth, move it into MS (PH 6.0), and culture it in light for 15-20 days, you can see green shoots grow out, usually change the medium every 15 days; select the green shoots that grow more than 1cm , peel off the excess callus around, cut off the root (about 0.5cm long) and move it into a test tube, 1/2 MS rooting culture. MS differentiation medium: MS salt and vitamins, 2g/l casein hydrolyzate, 30g/l sucrose, 25g/L sorbitol, 2mg/l 6-BA, 0.5mg/l NAA, 0.2mg/l zeatin (Zeatin ), 0.5mg/l KT, 3.0g/l Phytagel, pH 5.8, 50mg/l hygromycin B, 200mg/l cephalosporin. 1/2 MS rooting medium: 1/2 MS salt, MS vitamins, 30g/l sucrose, 1mg/l paclobutrazol, 0.5mg/l NAA, 50mg/l hygromycin, 2.5g/l Phytagel, pH5.8.

4. Transplanting, expression level identification and phenotypic analysis

The rooted transgenic plants were each genetically constructed 20 lines, transplanted in the greenhouse, and the leaves of the CRISPR transgenic plants of the gene LJS1-1 and its homologous gene LJS1-1L were extracted to extract DNA, and the edited fragments were amplified to identify mutant individual plants.

The primers used are as follows:

LJS1-1-genomeF CATCCGCCTCGTCAAATGC

LJS1-1-genomeR CGGGATAGCAGAACGAAATGG

LJS1-1L-genomeF GGATTCCCTCACCACCACATTA

LJS1-1L-genomeR CGCAGTGGAGTGGAGTACAT

5. Yield Assay

In the field, LJS1-1-cri/LJS1-1L-cri and wild-type Ni were tested for yield under different planting densities. The normal density (Normal, N) is planted with a row spacing of 30cm and a plant spacing of 15cm, with an average of 22.2 rice plants per square meter. Gaomi (Dense, D) was planted with a row spacing of 15cm and a plant spacing of 15cm, with an average of 44.4 rice plants per square meter. Each treatment was repeated three times in randomized blocks. Each cell is 2m long and 2m wide. For the investigation of traits, the intermediate plants except the surrounding plants were selected for statistics. Data processing was carried out using SPSS17.0. Multiple comparisons were performed using Tukey's Honest Significant Difference test (P<0.05).

(2) The application test of LJS4-1 gene in controlling the development of rice leaf pillow and the size of leaf angle, the test method is the same as above.

The primers used in step 1.3 are:

LJS4-1-F CGGGATCCATGTGCGGCGGTGCAATCCTC

LJS4-1-R GCGTCGACGTCGAGCAGAAGAGAGGCCTG

In step 2., LJS4-1 was obtained by performing the above analysis on the set of cell wall-related GO term genes with differentially expressed genes specific to the S4 phase. Finally, the expression levels of LJS4-1 in the occipital of the five periods were obtained. The gene LJS4-1 was found to be specifically expressed in the developing S4 of the occipital (Fig. 1b).

In step 3.1, the template of gene LJS4-1 is U6 vector, and the primers used are:

LJS4-1-U6F GGGGGACGACACACATGACAgttttagagctagaaatagcaagtta

LJS4-1-U6R TGTCATGTGTGTCGTCCCCCAACCTGAGCCTCAGCGCAGC

The primers used in step 4. are:

LJS4-1-genomeF CAATCCTCGCCGATTTCACC

LJS4-1-genomeR GCTCTTCTTGCTCGCCTTC

(3) The application test of LJS3-1 gene and its homologous gene LJS3-1L in controlling the development of rice leaf pillow and the size of leaf angle, the test method is the same as above.

The primers used in step 1.3 are:

LJS3-1-F CGGGATCCATGGAAGCAGACGCGAGCCATA

LJS3-1-R GCGTCGACCTCGGCCCACAAGAGTGGCTCA

LJS3-1L-F CGGGATCCATGGAAGCTGCCGCGATCC

LJS3-1L-R GCGTCGACGTCAGGCTGCACGGGCGC

In step 2., LJS3-1 and LJS3-1L were respectively obtained by performing the above analysis on the differentially expressed gene sets M03 and M04 specifically expressed in the S3 and S4 phases. Finally, the expression levels of LJS3-1 and its homologous gene LJS3-1L in the leaf occipital of the five stages were obtained. It was found that the gene LJS3-1 and its homologous gene LJS3-1L were specifically expressed in S3 and S4 of the developing leaf occipital, respectively (shown in Figure 1c).

The template used by the LJS3-1 gene in step 3.1 is 1ul of U6 vector (about 50ng); the template used by the LJS3-1L gene is 1ul of U3 vector (about 50ng), and the primers used are:

LJS3-1-U6F GGCCGCTCTCTTGCGCTTCTgttttagagctagaaatagcaagtta

LJS3-1-U6R AGAAGCGCAAGAGAGCGGCCAACCTGAGCCTCAGCGCAGC

LJS3-1L-U3F ACAAGCAGCTCAAGCGGAAGgttttagagctagaaatagcaagtta

LJS3-1L-U3R CTTCCGCTTGAGCTGCTTGTgccacggatcatctgcacaactc

The primers used in step 4. are:

LJS3-1-genomeF CAGACCGCACTTCCATCGA

LJS3-1-genomeR GATCTCCGACACCCACTTCC

LJS3-1L-genomeF CATCTCCTTCCTGCGGTATTCT

LJS3-1L-genomeR AGCCAGATGCGCGACTTCT

(4) The application test of LJS5-1 gene in controlling the development of rice leaf pillow and the size of leaf angle, the test method is the same as above.

The primers used in step 1.3 are:

LJS5-1-F CGGGATCCATGGATAGGAGGGAGGCCACC

LJS5-1-R GCGTCGACCTCGTCGTCGGAGGTGTCCG

In step 2., LJS5-1 was obtained by performing the above analysis on the differentially expressed gene set M05 specific to S5 . Obtain the expression level of LJS5-1 in the occipital of the five stages. The gene LJS5-1 was found to be specifically expressed in the developing S5 of the occipital (Fig. 1d).

The primers used in step 3.1 are:

LJS5-1-U6F GCGAGCCGAACAAGCGGTCGgttttagagctagaaatagcaagtta

LJS5-1-U6R-CGACCGCTTGTTCGGCTCGCAACCTGAGCCTCAGCGCAGC

The primers used in step 4. are:

LJS5-1-genomeF GCGAGGATGGATAGGAGGGA

LJS5-1-genomeR TAGAACACGGCGGTGTCGTA

(5) Application test of LJS1S2-1 gene and its homologous gene LJS1S2L-1L in controlling rice leaf pillow development and leaf angle, the test method is the same as above.

The primers used in step 1.3 are:

LJS1S2-1-F CGGGATCCATGGCGCGGCCGCAGCA

LJS1S2-1-R GCGTCGACGCAGGAGATCTCCATGGAGAAGT

LJS1S2-1L-F CGGGATCCATGGCGAGGCCGCAGCAACGAT

LJS1S2-1L-R GCGTCGACGTAGCAGATCTCCATGGAGAAG

In step 2., LJS1S2-1 and LJS1S2L-1L were obtained by performing the above analysis on the differential gene set M06 with specific expression in both S1 and S2 phases. Finally, the expression levels of LJS1S2-1 and LJS1S2L-1L in leaves and occipital of five stages were obtained. The genes LJS1S2-1 and LJS1S2L-1L were found to be specifically expressed in the developing S1 and S2 of the occipital (Fig. 1e).

In step 3.1, the gene LJS1S2-1 uses the template as the U6 vector, and the gene LJS1S2L-1 uses the template as the U3 vector, and the primers used are:

LJS1S2-1-U6F GGCACGCGCGTACGACGAGGgttttagagctagaaatagcaagtta

LJS1S2-1-U6R CCTCGTCGTACGCGCGTGCCAACCTGAGCCTCAGCGCAGC

LJS1S2-1L-U3F AGGCCGCAGCAACGATACCGgttttagagctagaaatagcaagtta

LJS1S2-1L-U3R CGGTATCGTTGCTGCGGCCTgccacggatcatctgcacaactc

Step 3. also includes the construction of the plant expression vector pLJS1S2-1::LJS1S2-1, the specific steps are:

Using a step-by-step method, first connect the LJS1S2-1 promoter (the method for obtaining the LJS1S2-1 promoter sequence is the same as above) to the pCAMBIA1300 vector backbone (LJS1S2-1-proF and LJS1S2-1- proR) to obtain pCAMBIA1300-pLJS1S2-1 , and then clone the full-length cDNA of LJS1S2-1 into pCAMBIA1300-pLJS1S2-1 (LJS1S2-1-OE-F and LJS1S2-1-OE-R) using BamHI/SalI to obtain The plant expression vector pLJS1S2-1::LJS1S2-1 was transformed into Nipponbare. The primers used are as follows:

LJS1S2-1-OE-F CGGGATCCATGGCGCGGCCGCAGCA

LJS1S2-1-OE-R GCGTCGACGCAGGAGATCTCCATGGAGAAGT

LJS1S2-1-proF CTATGACATGATTACgaattcTGGTTGGCTTGGCTGTGAT

LJS1S2-1-proR CCGCTGCGTGGGGGTTggtaccTGCCGACGTCCTCGAGCTCG

Step 4. Construct 20 lines of each genetically rooted transgenic plant, and transplant them into the greenhouse. Extract DNA from the leaves of the CRISPR transgenic plants of LJS1S2-1, and amplify the edited fragments to identify homozygous individual plants (LJS1S2-1-genomeF and LJS1S2- 1-genomeR). For pLJS1S2-1::LJS1S2-1 transgenic plants, plant protein was extracted from the leaves, and the expression level was identified by western blot. The primers used are as follows:

LJS1S2-1-genomeF GCTCATGGGTCTCCGAGAT

LJS1S2-1-genomeR GAGCTCTGGTCCACGTACTGCTCCT

LJS1S2-1L-genomeF TCGGCGAAGTGCTCGATCA

LJS1S2-1L-genomeR CGAACGTGCCCAGCCATAT

(6) The application test of LJS4-2 gene in controlling the development of rice leaf pillow and the size of leaf angle, the test method is the same as above.

The primers used in step 1.3 are:

LJS4-2-F CGGGATCCATGTGTGGCGGCGCGATCATTT

LJS4-2-R GCGTCGACCATCGGCACGGCCGTGTGGAT

The LJS4-2 gene sequence containing the nucleotides described in SEQ ID NO.1 is obtained, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

In step 2., LJS4-2 was obtained by performing the above analysis on the cell wall-related GO term gene set with differentially expressed genes specific to the S4 phase. Finally, the expression levels of LJS4-2 in the occipital of the five stages were obtained. The gene LJS4-2 was found to be specifically expressed in S4 of the occipital development (shown in Figure 1 f)

In step 3.1, the template of gene LJS4-2 is U3 vector, and the primers used are:

LJS4-2-U3F ACGGCCGCCGCCTGATGCCAgttttagagctagaaatagcaagtta

LJS4-2-U3R TGGCATCAGGCGGCGGCCGTgccacggatcatctgcacaactc

The sequence fragment tggca tcaggcggcg gccgt of the LJS4-2 target site was obtained.

The primers used in step 4. are:

LJS4-2-genomeF CAGAGGAGCCGACCAAGAAG

LJS4-2-genomeR GGCGTCGTAGTCCATGAACT

(7) The application test of LJS5-2 gene and its homologous gene LJS5-2L in controlling the development of rice leaf pillow and the size of leaf angle, the test method is the same as above.

The primers used in step 1.3 are:

LJS5-2-F CGGGATCCATGGTGCGGGGGAGGACGGA

LJS5-2-R GCGTCGACACCTGTCTCCGACCGGTTGGA

LJS5-2L-F CGGGATCCATGGTGCGGGGGAAGACGCAGA

LJS5-2L-R GCGTCGACAGAATGGGGCATCGCTTGGCTA

In step 2., LJS5-2 and LJS5-2L were obtained by analyzing the differentially expressed gene set M05 specific to S5 as above . Finally, the expression level of LJS5-2 in the occipital of the five stages. The gene LJS5-2 was found to be specifically expressed in the S5 stage of leaf occipital development (shown in Figure 1g).

The template used by the gene LJS5-2 gene in step 3.1 is U6 vector, the template used by LJS5-2L gene is U3 vector, and the primers used are:

LJS5-2-U6F GGATTGAGAACCCGACGAGCgttttagagctagaaatagcaagtta

LJS5-2-U6R GCTCGTCGGGTTTCCAATCCAACCTGAGCCTCAGCGCAGC

LJS5-2L-U3F ATTCGTAGAGCTTGCCGCGCgttttagagctagaaatagcaagtta

LJS5-2L-U3R GCGCGGCAAGCTCTACGAATgccacggatcatctgcacaactc

The primers used in step 4. are:

LJS5-2-genomeF CCTCGTCTCGTCTCGTCTCT

LJS5-2-genomeR TGCTTTATAGCGGTCGATGGT

LJS5-2L-genomeF-CGTGTGGTTGGTTGGTTCA

LJS5-2L-genomeR CTGATGCTAATGAGGCTTCTCT.

With reference to the test methods of the LJS1-1 gene in (1) and its homologous gene LJS1-1L above, relevant application tests were carried out on the genes in (2) to (7), and the test results are shown in Figures 1 to 6.

Test Results and Conclusion

1. From Figure 1a, it can be seen that LJS1-1 is specifically expressed in the early stage of leaf pillow development; from Figure 1b, it can be seen that LJS4-1 is specifically expressed in the S4 stage of leaf pillow development; from Figure 1c, it can be seen that LJS3-1 and Its homologous gene LJS3-1L is specifically expressed in the S3 and S4 stages of leaf pillow development; it can be seen from Figure 1d that LJS5-1 is specifically expressed in the late stage of leaf pillow development; it can be seen from Figure 1e that LJS1S2-1 is It is specifically expressed in the early stage of occipital development; from Figure 1f, it can be seen that LJS4-2 is specifically expressed in the S4 stage of leaf occipital development; from Figure 1g, it can be seen that LJS5-2 and its homologous gene LJS5-2L are expressed in S4 and S4 stages of leaf occipital development. S5 specific expression.

2. It can be seen from Figure 2a that after the successful knockout, the homozygous mutant plants have a smaller seedling angle and an upright plant at the heading stage, and the homozygous mutant plant leaf angle at seedling stage is significantly smaller than that of the wild type (Nipponbare) ; As can be seen from Figure 2b, after the successful knockout, the homozygous mutant plants have a smaller heading angle, and the plant type is upright; as can be seen from Figure 2c, after the successful knockout, the homozygous mutant plants have a smaller heading angle , the plant type is upright; it can be seen from Figure 2d that after the successful knockout, the angle of the homozygous mutant plant becomes smaller; it can be seen from Figure 2e that after the successful knockout, the leaf angle of the homozygous mutant plant is smaller than that of the wild type ( Nipponbare) is small; it can be seen from Figure 2f that after the successful knockout, the angle at the heading stage of the homozygous mutant plants becomes smaller and the plant type is erect; it can be seen from Figure 2g that after the successful knockout, the angle between the homozygous mutant plants becomes smaller Small, erect plant.

3. It can be seen from Figure 3a that after the successful knockout, the structure of the seedling leaf pillow of the homozygous mutant plants changes, and the number of leaf pillow cell layers is more than that of the wild type (Nipponbare); it can be seen from Figure 3b that the successful knockout After that, the lignin accumulation in the sclerenchyma tissue of the flag leaf pillow of the homozygous mutant plants at the heading stage was higher than that of the wild type (Nipponbare), and the mechanical strength increased; it can be seen from Figure 3c that after the successful knockout, the homozygous mutant plants at the heading stage Compared with the wild type (Nipponbare), the accumulation of lignin in the sclerenchyma tissue of the flag leaf pillow increased, and the mechanical strength of the leaf pillow increased; it can be seen from Figure 3d that after the successful knockout, the seedling leaf pillow structure of the homozygous mutant plants changed , the parenchyma cells on the adaxial surface of the leaf pillow are smaller than those of the wild type (Nipponbare); it can be seen from Figure 3e that after the successful knockout, the structure of the leaf pillow of the homozygous mutant plants changes at the seedling stage, and the sclerenchyma cells on the abaxial surface are smaller than those of the wild type type (Nipponbare) increased; it can be seen from Figure 3f that after the successful knockout, the homozygous mutant plants accumulated more lignin in the sclerenchyma tissue of the flag leaf pillow at the heading stage than the wild type (Nipponbare), and the mechanical strength increased; In 3g, it can be seen that the adaxial parenchyma cells of the leaf pillow of the homozygous mutant plant are smaller than those of the wild type (Nipponbare) after the successful knockout at the same time.

4. From Figure 4a, it can be seen that the expression level of LJS4-1 target genes that promote and inhibit lignin accumulation detected by qRT-PCR in LJS4-1-cri transgenic lines indicates that LJS4-1 develops in the S4 region of the leaf pillow. expression, which is related to lignin accumulation in the sclerenchyma of the leaf pillow; it can be seen from Figure 4b that the target genes that promote and inhibit lignin accumulation detected by qRT-PCR are in the CRISPR transgenic lines of LJS3-1 and its homologous gene LJS3-1L The expression level changes in the line indicate that the expression of LJS3-1 and its homologous gene LJS3-1L in the S3 and S4 of the leaf pillow development is related to the accumulation of lignin in the leaf pillow sclerenchyma; it can be seen from Figure 4c that through qRT-PCR detection of the expression of cell cycle-related genes in LJS1S2-1-cri/LJS1S2-1L-cri transgenic lines showed that the expression of LJS1S2-1 in the early stage of occipital development was related to the proliferation of occipital cells; It can be seen from Figure 4d that the expression level of LJS4-2 target genes that promote and inhibit lignin accumulation detected by qRT-PCR in LJS4-2-cri transgenic lines indicates that the expression of LJS4-2 in S4 of leaf pillow development is consistent with Associated with lignin accumulation in the sclerenchyma of the leaf occipital.

5. It can be seen from Figure 5 that the promoter and full-length CDS of LJS1S2-1 were cloned into pCAMBIA1300 and transferred into Nipponbare. The leaf angle of the transgenic line obtained was larger than that of the wild type (Nipponbare), indicating that LJS1S2-1 It has the function of controlling the development of rice leaf pillow and the size of leaf angle.

6. It can be seen from Figure 6 that after the target gene is successfully knocked out, the rice yield is increased by increasing the number of fertile panicles per unit area under high density.

sequence listing

<120> Application of rice gene LJS4-2 in controlling rice leaf pillow development and leaf angle

<160> 2

<170> SIP Sequence Listing 1.0

<210> 1

<211> 651

<212>DNA

<213> Rice (Oryza sativa)

<400> 1

atgtgtggcg gcgcgatcat ttccgacttc atcccgcagc gggaagccca ccgcgcggcc 60

accggcagca agcgtgccct ctgcgcctcc gacttctggc cgtcggcgtc gcaggaagcc 120

gccgacttcg accacctcac cgccccctgc accttcaccc ccgaccaagc ggcagaggag 180

ccgaccaaga agcggggagcg gaagacgctg taccgtggca tcaggcggcg gccgtgggggg 240

aagtgggcgg cggagatccg cgacccggcg aagggcgcgc gcgtctggct cggcaccttc 300

gccaccgccg aggcggcggc ccgcgcctac gactgcgccg cccgccgcat ccgcggggcc 360

aaggccaagg tcaacttccc caacgaggac ccgccactcg acgacccggc cgccgacggc 420

cacagccacg gcggcgccgc catcccgtgc agggagttca tggactacga cgccgtcatg 480

gcgggcttct tccaccagcc ctacgtcgtc gccgacggcg tgccggccgt gccggcggag 540

gaggcgccca cggtggcgta cgtgcaccac cacctgccgc cgcagccgca gcaggacgcg 600

gggctggagc tctggagctt tgataacatc cacacggccg tgccgatgtg a 651

<210> 2

<211> 216

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Cys Gly Gly Ala Ile Ile Ser Asp Phe Ile Pro Gln Arg Glu Ala

1 5 10 15

His Arg Ala Ala Thr Gly Ser Lys Arg Ala Leu Cys Ala Ser Asp Phe

20 25 30

Trp Pro Ser Ala Ser Gln Glu Ala Ala Asp Phe Asp His Leu Thr Ala

35 40 45

Pro Cys Thr Phe Thr Pro Asp Gln Ala Ala Glu Glu Pro Thr Lys Lys

50 55 60

Arg Glu Arg Lys Thr Leu Tyr Arg Gly Ile Arg Arg Arg Pro Trp Gly

65 70 75 80

Lys Trp Ala Ala Glu Ile Arg Asp Pro Ala Lys Gly Ala Arg Val Trp

85 90 95

Leu Gly Thr Phe Ala Thr Ala Glu Ala Ala Ala Arg Ala Tyr Asp Arg

100 105 110

Ala Ala Arg Arg Ile Arg Gly Ala Lys Ala Lys Val Asn Phe Pro Asn

115 120 125

Glu Asp Pro Pro Leu Asp Asp Pro Ala Ala Asp Gly His Ser His Gly

130 135 140

Gly Ala Ala Ile Pro Cys Arg Glu Phe Met Asp Tyr Asp Ala Val Met

145 150 155 160

Ala Gly Phe Phe His Gln Pro Tyr Val Val Ala Asp Gly Val Pro Ala

165 170 175

Val Pro Ala Glu Glu Ala Pro Thr Val Ala Tyr Val His His His Leu

180 185 190

Pro Pro Gln Pro Gln Gln Asp Ala Gly Leu Glu Leu Trp Ser Phe Asp

195 200 205

Asn Ile His Thr Ala Val Pro Met

210 215

Claims (2)

1. Knock-out rice geneLJS4-2The application of the gene in regulating and controlling the development of leaf pillows and the size of leaf included angles is characterized in that the rice geneLJS4-2The amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and when in application, the encoded protein is constructed to containLJS4-2CRISPR/CAS9 vector of gene, and transformation into Nipponbare, knock-outLJS4-2The gene can reduce the leaf angle, promote the plant type to be upright and increase the rice yield by improving the fertile ear number per unit area under high density by regulating and controlling the lignin accumulation and the mechanical strength of the thick-wall tissue of the leaf pillow.

2. The use of claim 1, wherein the rice gene is a rice geneLJS4-2Nucleotide sequence of (a)Is shown as SEQ ID NO. 1.

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