CN111793635B - Application of rice gene LJS5-1 in controlling growth of leaf pillow and leaf angle of rice - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and in particular relates to the application of a rice gene LJS5-1 in controlling the development of rice cusps and the size of leaf angle. The present invention obtains the gene LJS5-1 (GENE ID: Os06g0166400 ). Using gene knockout technology to knock out LJS5-1 , the mutant showed a significantly smaller angle than the wild type, and the plant type was upright. By observing the cytological structure of the lamina, it was found that the paraxial parenchyma cells of the mutant were smaller than those of the wild type (Nihonbare). Therefore, knocking out the LJS5-1 gene through genetic engineering technology can change the cytological structure of the plant leaf pillow and the formation of leaf angle, thereby improving the plant type and planting density, and increasing the yield.

Description

Application of Rice Gene LJS5-1 in Controlling Leaf Pillow Development and Leaf Angle Size in Rice

technical field

The invention belongs to the field of plant genetic engineering, and in particular relates to the application of rice gene LJS5-1 in controlling the development of rice cusps and the size of leaf angle.

Background technique

Rice leaves are divided into three categories, including coleoptiles, incomplete leaves and complete leaves. Coleoptiles are the first white coleoptiles that appear during germination, which are deformations of leaves without chlorophyll. Incomplete leaf is the first green leaf that grows from the coleoptile, with only leaf sheath and no leaf and cuspid structure. A complete leaf starts from the second green leaf and has a leaf blade, a leaf sheath, and a leaf pillow. The main function of leaves is as an organ of photosynthesis. The leaf sheath wraps the main stem and can enhance the support of the stem. The leaf pillow is a kind of mechanical tissue unique to monocotyledonous grasses, which is the mechanical tissue connecting the leaf and the leaf sheath, including the leaf pillow belt, the leaf ear, and the ligule. As a mechanical tissue, the lamina can deviate from the main stem to form an angle when the leaves and sheaths grow (Hoshikawa and Ichii, 1989). Therefore, the presence or absence of the leaf pillow and the degree of development directly determine the size of the leaf angle, thereby affecting the plant type and yield of rice.

The growing population has an increasing demand for food. Improving the yield of rice 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, selection for traits related to plant photosynthesis, growth and grain yield (Donald, 1968). The traits that affect the plant type of rice mainly include plant height, number of tillers, panicle type, grain type, leaf shape, leaf angle and tiller angle, among which the leaf angle is an important agronomic trait that determines the erectness of leaves (Feng Rongkun, 2006). . Erect leaves can enhance the light-harvesting capacity of photosynthesis, act as a nitrogen source for grain filling, and can also increase planting density, thereby improving leaf area index and rice yield (Sakamoto, et al., 2006).

SUMMARY OF THE INVENTION

The purpose of the present invention is to obtain the gene LJS5-1 that regulates the development of the rice leaf pillow and the size of the leaf angle.

Another object of the present invention is to provide the application of the LJS5-1 gene in controlling the development of the lamina and the size of the leaf angle in rice.

In order to achieve the above purpose, the present invention clones the phyllocus developmental regulation gene LJS1-1 and its homologous gene LJS1-1L from the rice lamellar tissue by means of reverse genetics.

Specifically, the primer sequences used to clone the LJS5-1 gene are as follows:

LJS5-1-F CGGGATCCATGGATAGGAGGGAGGCCACC

LJS5-1-R GCGTCGACCTCGTCGTCCGGAGGTGTCCG

Specifically, the PCR conditions used to clone the LJS5-1 gene were: denaturation at 94°C for 3 min, 94°C for 30 s, 55°C for 1 min, 68°C for 2 min for 35 cycles, and extension at 68°C for 10 min.

Specifically, the total volume of the PCR reaction system used to clone the LJS5-1 gene was 50 μl, the template was 1 μl of Nipponbare cDNA (about 50 ng), 5 μl of 10×KOD enzyme reaction buffer, 2 μl of 25 mM MgCl ₂ , 5 μl of 5 mM dNTP, and 5 uM primers 5 μl (2.5 μl for each primer), 1 μl KOD enzyme, add ddH2O (sterile deionized water) to 50 μl.

The present invention obtains the LJS5-1 gene sequence comprising the nucleotides described in SEQ ID NO. 1 by the above method.

The present invention also obtains the amino acid sequence encoded by the LJS5-1 gene, as shown in SEQ ID NO.2.

In the present invention, the obtained genes identified by the method of bioinformatics are all genes regulating lamina development, and the RNA-seq data of lamina in different periods are used to verify the expression specificity of the gene LJS5-1 in the process of lamina development.

Application of LJS5-1 gene in the improvement of grass crops.

Application of LJS5-1 gene in improving rice plant type and increasing rice yield.

The application of LJS5-1 gene in regulating the development of phylloxoccipital and leaf angle size in rice. During the application, a CRISPR/CAS9 vector containing LJS5-1 gene was constructed and transferred into Nipponbare to knock out the LJS5-1 gene. The obtained transgenic The line shows that the leaf angle is smaller than that of the wild type (Nihonbare), so this method can reduce the size of the parenaxial parenchymal cells of the leaf occipital surface to reduce the leaf angle, improve the plant type and planting density, and increase the yield. Regulation of leaf pillow development and leaf angle size in rice.

Compared with the prior art, the beneficial effects of the present invention are:

1. The LJS5-1 gene is a gene that is specifically expressed in the late stage of rice lamina development, which makes it possible to use this gene to only change the angle of the crop without affecting other traits.

2. The LJS5-1 gene is an effective gene for changing the leaf angle. It can change the leaf angle of crops by affecting the size of the lamellar cells.

3. At present, there are few genes that regulate the size of the leaf angle by changing the cytological structure of the lobe.

4. At present, there are few studies on the mechanism of plant erection to increase yield. After LJS5-1 was successfully knocked out by CRISPR/Cas9, rice yield was 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 phyllogenesis, Figure 1b shows the specific expression of LJS4-1 in the late stage of rice cusp development, and Figure 1c shows LJS3-1 and its homologues The gene LJS3-1L is specifically expressed at the S3 and S4 stages of rice phylloxyl development, respectively. Figure 1d shows the specific expression of LJS5-1 at the later stage of rice phylloxyl development, and Figure 1e shows the specific expression of LJS1S2-1 at the early stage of rice phylloxyl development. Figure 1f It is the specific expression of LJS4-2 in the late stage of rice phyllo-pillar development. Figure 1g shows the specific expression of LJS5-2 and its homologous gene LJS5-2L in the S4 and S5 stages of rice phylloxyl development;

Figure 2 shows the leaf angle phenotype and heading stage plant phenotype of transgenic rice seedlings with CRISPR/CAS9 knockout target genes, and the leaf angle size statistics of transgenic rice seedlings and wild type (Nihonbare) after knockout. Because LJS1-1 and its homologous gene LJS1-1L ; Fig. 2b target gene is LJS4-1 ; Fig. 2c target gene is LJS3-1 and its homologous gene LJS3-1L ; Fig. 2d target gene is LJS5-1 ; Fig. 2e The 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 shows the cytological structure changes of the flag leaf cuspid of the seedlings at heading stage of transgenic rice with CRISPR/CAS9 knockout target gene and wild type (Nihonbare), in which the target gene in Figure 3a is LJS1-1 and its homologous gene LJS1- 1L ; Fig. 3b target gene is LJS4-1 ; Fig. 3c target gene is LJS3-1 and its homologous gene LJS3-1L ; Fig. 3d target gene is LJS5-1 ; Fig. 3e target gene is LJS1S2-1 and its homologous gene LJS1S2-1L ; Fig. 3f target gene is LJS4-2 ; Fig. 3g target gene is LJS5-2 and its homologous gene LJS5-2L ;

Figure 4a shows the expression changes of the target genes that promote and inhibit lignin synthesis regulated by Real-time PCR in transgenic rice after CRISPR/CAS9 knockout of the target gene, in which the target gene in Figure 4a is LJS4-1 , 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 ;

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

Figure 6 shows the effect of CRISPR/CAS9 knockout of the target gene on the yield of transgenic rice. 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 LJS1-1. 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 , and the target gene in Figure 6f is LJS4-2 , 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 , the 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 is 71.59%), gene LJS4-2 (GENE ID: Os07g0674800 ), gene LJS5-2 (GENE ID: Os10g0536100 ) and its homologous gene LJS5-2L (GENE ID: Os03g0122600 , with a homology of 63.88%), the genes identified by bioinformatics methods are all genes that regulate phyllo-occipital development. The RNA-seq data of stage occipital lobules validated the expression specificity of the obtained genes during lobe occipital development. The present invention transfers the above gene into Nipponbare by 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 leaf angle size.

The specific implementation method is as follows. It should be noted that the following gene synthesis process or rice application test, unless otherwise specified, are conventional test methods and technical means in the field; the reagents or biological materials involved, unless otherwise specified Description, 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 lamina and the size of leaf angle. The specific steps are as follows:

1. The acquisition of the gene LJS1-1 and its homologous gene LJS1-1L that regulates the development of the foliar pillow and the size of the leaf angle in rice

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

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

1.3 The primers used are as follows:

LJS1-1-F CGGGATCCATGGCGCGGAGGGGGAGA

LJS1-1-R GCGTCGACTGCACTTCCTTCCTCCTGCC

LJS1-1L-F CGGGATCCATGGAGGGAGGAGGGAGGAGG

LJS1-1L-R GCGTCGACAGAGCTCACTCCTGATCTTGGCT

2. Using RNA-seq data to verify that the gene LJS1-1 and its homologous gene LJS1-1L are specifically expressed in the rice leaf pillow

Rice Nipponbare seedlings that grow for 4, 5, 6, 7, and 9 days from seed soaking under normal conditions are defined as the first stage (S1), the second stage (S2), the third stage (S3), the first stage (S1), the second stage (S2), the third stage (S3), the first The fourth phase (S4) and the fifth phase (S5). The leaves and cusps of the first complete leaves were taken respectively, and 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 five stages and the corresponding GO terms for functional enrichment analysis were obtained, and the differential gene sets were selected, including the single-stage specific expression gene sets M01-M05 and phyllo-occipital development-related gene sets. GO terms gene collection, extract promoter sequences, use MEME software to analyze the enriched motifs (motifs) on promoters, 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 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 identifies them as belonging to the corresponding transcription factor family, and the second is that they belong to the corresponding transcription factor family. The second is that TFs and their corresponding target genes specifically expressed at the same time have a high correlation (PCC ≥ 0.9). The present invention obtains the gene LJS1-1 and its homologous gene LJS1-1L by performing the above analysis on the differential gene set M01 specifically expressed in the S1 phase . Finally, the expression levels of the gene LJS1-1 and its homologous gene LJS1-1L in leaves and cusps of five stages were obtained. The gene LJS1-1 was found to be specifically expressed at S1 in lamina development (Fig. 1a).

3. The regulation of LJS1-1 gene and its homologous gene LJS1-1L on rice leaf pillow

3.1 Construction of CRISPR/Cas9 vector

The fragment containing the CRISPR target site sequence of the gene LJS1-1 and its homologous gene LJS1-1L was used, and the fragment was cloned into the pCXUN-CAS9 vector using the KpnI restriction site. 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 choicesof promoters for CRISPR/Cas9 mediated genome editing. Journal of genetics andgenomics = 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 adopted by the LJS1-1 gene is 1 μl (about 50 ng) of the U6 vector; the LJS1-1L gene The templates used were 1 μl of U3 carrier (about 50 ng), 5 μl of 1×KOD enzyme reaction buffer, 2 μl of 25 mM MgCL2, 5 μl of 5 mM dNTP, 5 μl of 5 μM primer (2.5 μl of forward and reverse primers respectively), and 1 μl of KOD enzyme. Add ddH2O (sterile deionized water) to 50 μl.

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

The primers used were:

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 pCXUN-CAS9 vector containing LJS1-1 and LJS1-1L targets, add it to 50 μl of EHA105 competent, and mix well. Add to the pre-cooled electric shock cup for electric shock conversion. Electrostimulator parameter settings: 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

The rice seeds were hulled, and the plump and clear grains were soaked in 70% ethanol for 1 min, and rinsed 1-2 times with sterile water; Add 60 ml of water to the solution), add 1-3 drops of Tween 20, soak for more than 30 minutes (usually 40 minutes, up to 1 hour). Shake from time to time, then rinse with sterile water 4-5 times. Pour it onto a sterilized plate and filter paper and blot dry for about 1 hour; put it on N6D solid medium (10 grains/25 ml/bottle), with the embryos facing up or touching the medium, 28°C, dark culture for 25 ~30d. N6D medium: N6 salts 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 spoon to scrape the Agrobacterium, use the back of the spoon to stick the bacteria to the tube wall and pat lightly to disperse, OD ₆₀₀ = 0.8-1.0; dry the pre-cultured callus on sterile filter paper, and then concentrate to a The plate is transferred into the bacterial liquid at one time, and the centrifuge tube is gently rotated to distribute the bacterial liquid evenly, and the standing time is about 15-20 min; the bacterial liquid is poured out, and the callus is placed on sterile filter paper for about 1.5 hours to ensure that the bacterial liquid Blot dry, connect it to 1/2 N6D AS, and cultivate at 20°C for 2-3 days in the dark. If you see that there is a biofilm in the contact part between the callus and the medium, the bacteria 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 calli into a 50 ml centrifuge tube, wash 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 dry for about 2h, as the case may be; transfer the dried callus into N6D-AS, add 250 mg of cephalosporin Cn /L, 28 °C, dark culture for 7-10 d.

3.2.4 Screening of callus

Pick out the callus that is not contaminated by Agrobacterium, add Cn 250 mg/L and Hn (50 mg/L) for 15-20 days for the first time; the second time is the same as above, without adding Cn, add hygromycin Hn, put all the All calluses are transferred again, 15 to 20d. The new callus is selected for the third time and screened with Hn for 15-20 days; the number of times must be arranged according to the above, but it should be ensured that the callus is screened on Hn for at least 45 days, and the new callus selected for the third time The best callus is screened for 20 days; N6D screening medium: N6D+Cn250 mg/L+Hn50mg/L, pH=5.8～5.9.

3.2.5 Differentiation and rooting

All the callus from the fourth screening were transferred into MS, Hn 50 mg/L, dark cultured, and pre-differentiated (pH 5.9) for 12-15 d. Select the fresh callus with good growth, transfer it into MS (PH 6.0), cultivate in light for 15-20 d, green buds can be seen to grow, generally change the medium every 15 d; select the green buds that grow more than 1cm , peel off the excess callus around, cut off the root (about 0.5cm long) and transfer it into a test tube, 1/2 MS rooting culture. MS differentiation medium: MS salts and vitamins, 2g/l casein hydrolysate, 30g/l sucrose, 25g/l sorbitol, 2mg/l 6-BA, 0.5mg/l NAA, 0.2mg/l zeatin (Zeatin ), 0.5 mg/l KT, 3.0 g/l Phytagel, pH 5.8, 50 mg/l hygromycin B, 200 mg/l cephalosporin. 1/2 MS rooting medium: 1/2 MS salt, MS vitamins, 30 g/l sucrose, 1 mg/l paclobutrazol, 0.5 mg/l NAA, 50 mg/l hygromycin, 2.5 g/l Phytagel, pH 5.8.

4. Transplantation, expression level identification and phenotypic analysis

Each of the rooted transgenic plants was genetically constructed into 20 lines, transplanted into the greenhouse, and the CRISPR transgenic plants of the gene LJS1-1 and its homologous gene LJS1-1L were taken from leaves to extract DNA, and amplified and edited fragments 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

Yield measurements at different planting densities were performed on LJS1-1-cri/LJS1-1L-cri and wild-type Ni in the field. Normal density (Normal, N) was planted with 30cm row spacing and 15cm plant spacing, with an average of 22.2 rice plants per square meter. High density (Dense, D) was planted with 15cm row spacing and 15cm plant spacing, with an average of 44.4 rice plants per square meter. Each treatment was replicated three times in randomized blocks. Each cell is 2m long and 2m wide. For character investigation, select intermediate plants except surrounding plants for statistics. Data processing was performed using SPSS17.0. Multiple comparisons were performed using Tukey's Honest SignificantDifference test (P<0.05).

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

The primers used in step 1.3 are:

LJS4-1-F CGGGATCCATGTGCGGCGGGTGCAATCCTC

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 expressing differential genes specific for S4 phase as above. Finally, the expression levels of LJS4-1 in the occipital lobe of five stages were obtained. The gene LJS4-1 was found to be specifically expressed at S4 in lamina development (Fig. 1b).

The gene LJS4-1 in step 3.1 uses the U6 vector as the template, and the primers used are:

LJS4-1-U6F GGGGGACGACACACATGACAgttttagagctagaaatagcaagtta

LJS4-1-U6R TGTCATGTGTGTGCTCCCCAACCTGAGCCTCAGCGCAGC

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 cusp and 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 obtained by performing the above analysis on the differential gene sets M03 and M04 specifically expressed in the S3 phase and the S4 phase. Finally, the expression levels of LJS3-1 and its homologous gene LJS3-1L in the lobules of five stages were obtained. The gene LJS3-1 and its homolog LJS3-1L were found to be specifically expressed at S3 and S4 of lamina development, respectively (Fig. 1c).

In step 3.1, the template used for LJS3-1 gene is 1ul (about 50ng) of U6 vector; the template used for LJS3-1L gene is 1ul (about 50ng) of U3 vector, 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 the lamina and the size of the leaf angle in rice. The test method is the same as above.

The primers used in step 1.3 are:

LJS5-1-F CGGGATCCATGGATAGGAGGGAGGCCACC

LJS5-1-R GCGTCGACCTCGTCGTCCGGAGGTGTCCG

The LJS5-1 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., LJS5-1 was obtained by performing the above analysis on the S5 phase-specific differential gene set M05 . The expression levels of LJS5-1 in the occipital lobe of five stages were obtained. The gene LJS5-1 was found to be specifically expressed at S5 in lamina development (Fig. 1d).

The primers used in step 3.1 are:

LJS5-1-U6F GCGAGCCGAACAAGCGGTCGgttttagagctagaaatagcaagtta

LJS5-1-U6R CGACCGCTTGTTCGGCTCGCAACCTGAGCCTCAGCGCAGC

The resulting sequence fragment of the LJS5-1 target site gcgagccga acaagcggtc g .

The primers used in step 4. are:

LJS5-1-genomeF GCGAGGATGGATAGGAGGGA

LJS5-1-genomeR TAGAACACGGCGGTGTCGTA

(5) The application test of LJS1S2-1 gene and its homologous gene LJS1S2L-1L in controlling the development of rice cusp and the size of 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 cusps of five stages were obtained. The genes LJS1S2-1 and LJS1S2L-1L were found to be specifically expressed in S1 and S2 of lamina development (Fig. 1e).

In step 3.1, the gene LJS1S2-1 adopts the template U6 vector, the gene LJS1S2L-1 adopts the template 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:

The LJS1S2-1 promoter (the method for obtaining the LJS1S2-1 promoter sequence is the same as above) was first connected to the pCAMBIA1300 vector backbone (LJS1S2-1-proF and LJS1S2-1- proR) to obtain pCAMBIA1300-pLJS1S2-1 , and then the full-length cDNA of LJS1S2-1 was cloned into pCAMBIA1300-pLJS1S2-1 using BamHI/SalI (LJS1S2-1-OE-F and LJS1S2-1-OE-R) to obtain The plant expression vector pLJS1S2-1::LJS1S2-1 was transferred 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 CCGCTGCGTGGGGTTggtaccTGCCGACGTCCTCGAGCTCG

Step 4. Each of the rooted transgenic plants was genetically constructed into 20 lines, transplanted into the greenhouse, the CRISPR transgenic plants of LJS1S2-1 were taken from leaves to extract DNA, and the edited fragments were amplified to identify homozygous single plants (LJS1S2-1-genomeF and LJS1S2- 1-genomeR). pLJS1S2-1::LJS1S2-1 transgenic plants were extracted from leaves by extracting plant protein, 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 the lamina and the size of the leaf angle in rice. The test method is the same as above.

The primers used in step 1.3 are:

LJS4-2-F CGGGATCCATGTGTGGCGGCGCGATCATTT

LJS4-2-R GCGTCGACCATCGGCACGGCCGTGTGGAT

In step 2., LJS4-2 is obtained by performing the above analysis on the set of cell wall-related GO term genes that express differential genes specific for S4 phase as above. Finally, the expression levels of LJS4-2 in the occipital lobe of five stages were obtained. The gene LJS4-2 was found to be specifically expressed at S4 in lamina (Fig. 1f)

The gene LJS4-2 in step 3.1 uses the U3 vector as the template, and the primers used are:

LJS4-2-U3F ACGGCCGCCGCCTGATGCCAgttttagagctagaaatagcaagtta

LJS4-2-U3R TGGCATCAGGCGGCGGCCGTgccacggatcatctgcacaactc

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 lamina 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 performing the above analysis on the differentially expressed gene set M05 specific for the S5 stage. Finally, the expression levels of LJS5-2 in the occipital lobe of the five stages. The gene LJS5-2 was found to be specifically expressed in the S5 stage of phyllo-occipital development (shown in Figure 1g).

In step 3.1, the template used by the gene LJS5-2 gene 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 GCTCGTCGGGTTCTCAATCCAACCTGAGCCTCAGCGCAGC

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

Test Results and Conclusions

1. It can be seen from Figure 1a that LJS1-1 is specifically expressed in the early stage of lamina development; it can be seen from Figure 1b that LJS4-1 is specifically expressed in the S4 stage of lamina development; it can be seen from Figure 1c that LJS3-1 and Its homologous gene LJS3-1L is specifically expressed in the S3 and S4 stages of lamina development, respectively; it can be seen from Figure 1d that LJS5-1 is specifically expressed at the later stage of lamellar development; it can be seen from Figure 1e that LJS1S2-1 is expressed in the It is specifically expressed in the early stage of occipital development; it can be seen from Figure 1f that LJS4-2 is specifically expressed in the S4 stage of lamina S5 phase specific expression.

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

3. It can be seen from Figure 3a that after the successful knockout, the structure of the lamina of the homozygous mutant plants at the seedling stage changes, and the number of lamellae cells is increased compared with that of the wild type (Nihonbare); it can be seen from Figure 3b that the successful knockout Compared with the wild type (Nihonbare), the lignin accumulation in the sclerenchyma of the leaf pillows of the flag leaves of the homozygous mutant plants increased at the heading stage, and the mechanical strength increased; it can be seen from Fig. The lignin accumulation in the sclerenchyma of the leaf pillow of the flag leaf was higher than that of the wild type (Nipponbare), and the mechanical strength of the leaf pillow increased; it can be seen from Figure 3d that after the successful knockout, the structure of the leaf pillow at the seedling stage of the homozygous mutant plants changed. , the parenaxial parenchyma cells of the lamina are smaller than those of the wild type (Nihonbare); it can be seen from Figure 3e that after successful knockout, the structure of the lamina in the seedling stage of the homozygous mutant plants changed, and the abaxial parenchyma cells were smaller than those of the wild type. type (Nihonbare) increased; it can be seen from Figure 3f that after successful knockout, the lignin accumulation in the sclerenchyma of the flag leaf cusps of the homozygous mutant plants at heading stage was higher than that of the wild type (Nihonbare), and the mechanical strength was increased; from the figure It can be seen in 3g that after successful knockout at the same time, the paraxial parenchyma cells of the lamina of the homozygous mutant plant are smaller than those of the wild type (Nihonbare).

4. From Figure 4a, it can be seen that qRT-PCR detects the expression changes of LJS4-1 target genes that promote and inhibit lignin accumulation in LJS4-1-cri transgenic lines, indicating that LJS4-1 plays an important role in the development of S4 in the lamina. The expression is related to the accumulation of lignin in the lamina sclerenchyma; it can be seen from Figure 4b that the target genes that promote and inhibit lignin accumulation by qRT-PCR detection are in the CRISPR transgenic lines of LJS3-1 and its homologous gene LJS3-1L The changes in the expression levels in the lamina indicate that the expression of LJS3-1 and its homologous gene LJS3-1L in S3 and S4 of lamina development is related to the accumulation of lignin in lamellar sclerenchyma; it can be seen from Figure 4c that by The expression analysis of cell cycle-related genes in LJS1S2-1-cri/LJS1S2-1L-cri transgenic lines detected by qRT-PCR indicated that the expression of LJS1S2-1 in the early stage of lamina development was related to the proliferation of lamellar cells; In Figure 4d, it can be seen that qRT-PCR detects the expression changes of LJS4-2 target genes that promote and inhibit lignin accumulation in LJS4-2-cri transgenic lines, indicating that the expression of LJS4-2 in S4 of phyllocus development is similar to that of LJS4-2. Lignin accumulation in lamina sclerenchyma.

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 obtained transgenic line was larger than that of the wild type (Nihonbare), indicating that LJS1S2-1 It has the function of controlling the development of the rice leaf pillow and the size of the leaf angle.

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

sequence listing

<120> Application of the rice gene LJS5-1 in the control of the development of the leaf pillow and the size of the leaf angle in rice

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 555

<212> DNA

<213> Rice (Oryza sativa)

<400> 1

atggatagga gggaggccac cgtcttcttg cccccgccgc cgccaccgca gccgacgcag 60

ccgcagcagc agcctgcggc ggcggcggtg agggctccgg taggcgggag gggtggggga 120

ggagggaggc agtatcgcgg ggtgcggatg cggaagtggg ggaagtgggt ggcggagatc 180

cgcgagccga acaagcggtc gcggatctgg ctcggctcct actccaccgc cgtcgccgcc 240

gcgcgggcct acgacaccgc cgtgttctac ctccgcggcc gctccgcgcg cctcaacttc 300

cccgaccagc tcgacggcgc gggtggtggt ggcgcgggcg ccggcggcgc cgaggaccac 360

agggagctca ccgccgccgt catccgcaag aaggccgccg aggtcggcgc ccgcgtcgac 420

gcgcagcact ccgtcgtcgg cgccgcggcg cccgtgccgc tccagccgcc gcagcccccg 480

ccgccccagc gccgccggac caagaacccc gacctcaacc gggagcccac cccggacacc 540

tccgacgacg agtag 555

<210> 2

<211> 184

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Asp Arg Arg Glu Ala Thr Val Phe Leu Pro Pro Pro Pro Pro Pro

1 5 10 15

Gln Pro Thr Gln Pro Gln Gln Gln Pro Ala Ala Ala Ala Val Arg Ala

20 25 30

Pro Val Gly Gly Arg Gly Gly Gly Gly Gly Arg Gln Tyr Arg Gly Val

35 40 45

Arg Met Arg Lys Trp Gly Lys Trp Val Ala Glu Ile Arg Glu Pro Asn

50 55 60

Lys Arg Ser Arg Ile Trp Leu Gly Ser Tyr Ser Thr Ala Val Ala Ala

65 70 75 80

Ala Arg Ala Tyr Asp Thr Ala Val Phe Tyr Leu Arg Gly Arg Ser Ala

85 90 95

Arg Leu Asn Phe Pro Asp Gln Leu Asp Gly Ala Gly Gly Gly Gly Ala

100 105 110

Gly Ala Gly Gly Ala Glu Asp His Arg Glu Leu Thr Ala Ala Val Ile

115 120 125

Arg Lys Lys Ala Ala Glu Val Gly Ala Arg Val Asp Ala Gln His Ser

130 135 140

Val Val Gly Ala Ala Ala Pro Val Pro Leu Gln Pro Pro Gln Pro Pro

145 150 155 160

Pro Pro Gln Arg Arg Arg Thr Lys Asn Pro Asp Leu Asn Arg Glu Pro

165 170 175

Thr Pro Asp Thr Ser Asp Asp Glu

180

Claims (2)

1. the application of rice gene LJS5-1 in regulating the development of phyllocus and leaf angle, it is characterized in that, the aminoacid sequence of described rice gene LJS5-1 encoded protein is as shown in SEQ ID NO.2, when applying, constructs The CRISPR/CAS9 vector containing the LJS5-1 gene was transferred into Nipponbare, and the LJS5-1 knockout could reduce the leaf angle by reducing the size of the parenaxial parenchyma cells of the occipital lobe, and promote the erection of the plant. Increase rice yield by increasing the number of fertile ears per unit area at high densities. 2. The application according to claim 1, wherein the nucleotide sequence of the rice gene LJS5-1 is shown in SEQ ID NO.1.

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