Rice White Leaf and White Panicle Gene wlp3 and Its Application in Rice Stress Resistance and Yield Increase

Abstract

The invention discloses a rice white leaf and white panicle gene wlp3, wherein the cDNA sequence of the white leaf and white panicle gene wlp3 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2; the rice white leaf and white panicle gene wlp3 is applied to improving rice stress resistance and increasing rice yield, wherein the white leaf and white panicle gene wlp3 improves plant cold resistance, enhances photosynthetic rate, increases plant height, whitens leaves at seedling stage, whitens panicles at heading stage, and increases panicle length at low temperature. The invention obtains the rice white leaf and white panicle gene wlp3 by screening and mutagenesis, and the gene is related to rice stress resistance and chlorophyll synthesis, thus providing a basis for rice breeding.

Description

Technical Field

[0001] The invention relates to the technical field of rice white leaf and white ear gene screening and breeding, and more specifically to the rice white leaf and white ear gene wlp3 and its application in rice breeding. Background Art

[0002] The chloroplasts in higher plants are about 6 μm long and 3 μm wide. A normal mesophyll cell contains 100-150 chloroplasts. The content of chloroplasts varies in different species and different parts. The morphological structure of chloroplasts consists of three parts: chloroplast membrane, thylakoid, and matrix. The membrane of chloroplasts is a double-layer membrane like the cell membrane. The outer membrane has good permeability, while the inner membrane has strong selectivity. Thylakoids are flat bodies formed by a single-layer membrane. There are many photosynthetic pigments and proteins involved in the electron transfer system on the thylakoid membrane, such as: cytochrome complex, plastoquinone, plastocyanin, ferredoxin, flavoprotein, etc. Water can be photolyzed in this part to form H and O 2 At the same time, ADP is converted into ATP to provide energy for the subsequent dark reaction. Therefore, the thylakoid membrane is also called the photosynthetic membrane. Many thylakoids are stacked together to form grana. The substance between the chloroplast membrane and the thylakoid is the matrix. The matrix is ​​the site for carbon assimilation. It contains carbon assimilation enzymes, chloroplast DNA, proteins, starch and other substances. Its main function is to convert CO 2 It is fixed in organic matter and uses ATP to reduce three-carbon sugars, providing sufficient raw materials for the synthesis of light reaction substances.

[0003] Chloroplasts, as important organelles in rice leaves, play an important role in the growth and development of rice. Chloroplasts can absorb light energy and convert it into chemical energy that can be used by the plant for storage. The efficiency of their conversion directly determines the final yield and quality of rice. Chloroplasts are semi-autonomous organelles that have their own genome and coordinate the development of plant chloroplasts with the nucleus. Chloroplasts develop from protoplasts, and their development is determined by external factors such as temperature and light, and internal factors such as hormone genes. Among these factors, the most important condition for chloroplast development is light. Only under sufficient light can protoplasts develop into chloroplasts, otherwise they will develop into xanthoplasts. The process of chloroplast development is mainly divided into three parts: the first is the synthesis of the plastid's own genomic DNA and the replication of the plastid, but in this step, the transcription level of the plastid's own genes remains at a relatively low level; the second step is the establishment of the chloroplast genetic system, at this time the nuclear-encoded RNA polymerase (NEP) will first be transcribed and expressed, such as RpoTP will be expressed in large quantities at this stage, then NEP will be responsible for the housekeeping genes of chloroplast development and the plastid-encoded RNA polymerase (PEP), the plastid transcription activity increases, the third step is the increase in the expression of genes encoding light systems, these genes are mainly transcribed by the PEP expressed in the second stage, which also include the two subunits of the light system encoding: psaA, psbA and the two subunits of diphosphate carboxylase: rcL, rbcS, etc. As a model plant, rice is a good carrier for studying the chloroplast development of monocots. Current research shows that leaf development goes through several stages from p0 to p6, and each stage also has different gene-specific expressions. The transcription levels of POLP1 and FtsZ involved in DNA replication are extremely high in p0-p3, and gradually decrease to almost zero in p4. In p4, the expression levels of RpoTP, PEP subunits, and some ribosomal proteins such as RPS7 and RPS15 gradually increase, while the expression levels of photosynthesis-related genes such as Lhcb, rbcs, and rbcl are the highest at P5 and P6.

[0004] Rice is the main food for more than half of the world's population, and rice production plays a vital role in human development. Leaves are the main site of photosynthesis, and leaf whitening can cause the destruction of chloroplasts, leading to a decrease in photosynthetic rate, and ultimately causing a decrease in rice yield. Therefore, exploring the mechanism of rice leaf whitening and studying the chloroplast development process have far-reaching significance for understanding the molecular mechanism of rice photosynthesis.

[0005] Therefore, screening a gene related to white leaves and white panicles in rice and applying it to rice breeding is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention screened out a rice white leaf and white panicle gene wlp3, which is related to the color of rice leaves. Further research on the gene found that it can improve the cold resistance of rice, increase yield, and lay the foundation for the breeding of excellent rice varieties.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The rice white leaf and white panicle gene wlp3, the white leaf and white panicle gene wlp3 cDNA sequence is shown as SEQ ID NO.1.

[0009] The white-leaf and white-ear mutant gene wlp3 was cloned by the map-based cloning method. The wlp3 gene was derived from the LOC_Os03g61620 gene by a single base substitution, that is, the nucleotide T at position 380 of SEQ ID NO.3 was changed to C. As a result, the amino acid at position 127 was changed from isoleucine to threonine. Bioinformatics analysis showed that wlp3 encodes the large ribosomal subunit L18, and the protein sequence encoded by the wild-type wlp3 gene is shown in SEQ ID NO.4.

[0010] As an inventive concept identical to the above technical solution, the present invention also requests protection for a protein encoded by the sequence shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0011] As an inventive concept identical to the above-mentioned technical scheme, the present invention also requests protection for the application of the rice white leaf and white panicle gene wlp3 in improving rice stress resistance and increasing rice yield, wherein the white leaf and white panicle gene wlp3 improves the plant's cold resistance, enhances the photosynthetic rate, increases plant height, whitens leaves in the seedling stage, whitens panicles in the heading stage, and increases panicle length under low temperatures.

[0012] Preferably, the white leaf and white panicle gene wlp3 controls the rice plants to alleviate leaf whitening at low temperatures and cause leaf whitening at high temperatures.

[0013] The rice white-leaf and white-ear mutant wlp3 of the present invention is screened from an EMS mutagenesis library of japonica rice variety Zhonghua 11. The mutant wlp3 shows a white-striped phenotype at the two-leaf stage and lasts until the tillering stage, and the white stripes are distributed along the veins on the entire leaf. After the four-leaf stage, as the rice grows and develops, the newly grown leaves of wlp3 gradually turn green until the phenotype is restored to the same as the wild type.

[0014] The rice white-leaf and white-ear mutant wlp3 gene of the present invention can be used to enhance the resistance of crops to adverse environments by genetic engineering or genetic engineering methods, such as improving the photosynthesis of crops and increasing yields.

[0015] In summary, in order to explore new leaf color-related genes, the present invention screened a white leaf and panicle mutant from the EMS mutagenesis library of japonica rice variety Zhonghua 11 as the background, and named it wlp3 (white leaf and panicle 3). Phenotypic analysis and hormone response of wlp3 have broad application prospects in affecting rice leaf color. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 The attached figures show the phenotypes of wild-type Zhonghua11 and mutant wlp3; (A) leaf phenotypes of wild-type and wlp3 at seedling stage, with a scale of 1 cm; (B) phenotypes of wild-type and wlp3 at tillering stage, with a scale of 10 cm; (C) phenotypes of wild-type and wlp3 at maturity stage, with a scale of 20 cm; (D) phenotypes of wild-type and wlp3 ear, with a scale of 5 cm;

[0018] Figure 2 The attached figure shows the leaf phenotypes of the wild type and wlp3 at different temperatures and the chlorophyll content of the second and third leaves;

[0019] Figure 3 The attached figures are the localization maps of wlp3 gene; (A) initial localization of wlp3; (BC) fine localization of WLP3; (D) gene structure of wlp3; (EF) mutation sites of wlp3;

[0020] Figure 4 The attached figures show the response of wild type and wlp3 to cold stress; (A) Phenotype of wild type and wlp3 before cold treatment, scale bar is 5 cm; (B) Expression level of cold-resistance related genes of wild type and wlp3 before cold treatment; (C) Expression level of cold-resistance related genes of wild type and wlp3 after cold treatment; (D) Phenotype of wild type and wlp3 after cold treatment, scale bar is 5 cm; (E) Chlorophyll content of wild type and wlp3 after cold treatment;

[0021] Figure 5 The attached figures show the expression analysis of genes related to leaf color in wild type and wlp3; (A) the expression of genes related to chlorophyll synthesis in wild type and wlp3; (B) the expression of genes related to ribosomal genes in wild type and wlp3; (C) the expression of genes related to chloroplast development in wild type and wlp3;

[0022] Figure 6 The attached figure is a functional complementation diagram of wlp3. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1 Obtaining mutant materials

[0025] Chemical mutagenesis of japonica rice variety Zhonghua 11 by EMS: Soak seeds in water for 8 hours. Then drain the water and pour in a well-proportioned 1% EMS solution to soak for 8 hours. Stir with a small wooden stick several times to ensure uniform mixing. After mutagenesis, rinse the toxic EMS buffer with running water, sow after germination at 25℃, transplant and harvest single plants, and manage the growth period according to conventional field management. A white leaf and white panicle mutant wlp3 was screened.

[0026] The traits of the mutant have been stably inherited after multiple generations of self-pollination. All rice materials were planted in the experimental field of the College of Biochemistry of Zhejiang Normal University in Jinhua City, Zhejiang Province, and managed conventionally.

[0027] Example 2 Phenotypic analysis of plants

[0028] Under field conditions, the mutant wlp3 showed a white stripe phenotype at the two-leaf stage and continued to the tillering stage. The white stripes were distributed along the veins throughout the leaf. After the four-leaf stage, as the rice grew and developed, the newly grown leaves of wlp3 gradually turned green until they restored the same phenotype as the wild type. During the growth process, the phenotype of wlp3 was basically the same as that of the wild type, but the plant height was higher than that of the wild type at maturity. At the young panicle stage, the panicles of wlp3 showed the albino phenotype again and the panicle length was longer than that of the wild type. (e.g. Figure 1 )

[0029] Example 3 Population Construction and Genetic Analysis

[0030] The mutant wlp3 was reciprocally crossed with conventional indica rice 9311 and ZF802, and the F1 plants all showed normal wild-type phenotypes, indicating that wlp3 is controlled by a recessive nuclear gene. The segregation ratio of the F2 segregation population was statistically analyzed (Table 1). The results showed that the segregation ratio of plants with normal phenotypes and plants with mutant phenotypes was close to 3:1 after the chi-square test, indicating that the white leaf and white panicle phenotype of wlp3 is controlled by a pair of single recessive nuclear genes.

[0031] Table 1 Genetic analysis of the white-leaf and white-ear mutant wlp3

[0032]

[0033] Example 4 Fine positioning of wlp3 gene

[0034] The mutants and Zhonghua 11 and 9311 were screened for polymorphism using SSR primers uniformly distributed on 12 rice chromosomes preserved in our laboratory. 2 Linkage analysis was performed on the individual plants of white leaves and white spikes to preliminarily confirm the chromosome location of the target gene. Genomic DNA was extracted using the CTAB method. The specific steps are as follows:

[0035] ① Weigh 0.1g of rice leaves and grind them into powder with liquid nitrogen, then add 600μl of DNA extraction buffer prepared with CTAB solution (2% (m / V) CTAB, 100mmol / L Tris-HCl, 20mmol / L EDTA, 1.4mol / L NaCl; pH8.0), and place in a 65℃ water bath for 40 minutes. Add 600μl of chloroform:isoamyl alcohol (24:1 volume ratio) and mix well. Centrifuge at 10000rpm for 5 minutes and transfer the supernatant to a new centrifuge tube.

[0036] ② Add 2 / 3 to 1 volume of pre-cooled (to 4°C) isopropanol to the supernatant obtained after centrifugation in step ① above, and gently mix until DNA is precipitated. Centrifuge at 13,000 rpm for 8 minutes and pour out the supernatant.

[0037] ③ Wash the DNA precipitate obtained in step ② above with 200 μl of 70% (volume concentration) ethanol.

[0038] ④ Dry the washed DNA and dissolve it in 100 μl TE buffer or pure water.

[0039] ⑤Ultraviolet spectrophotometry was used to detect the concentration of the DNA sample obtained in step ④ above, and 0.7% agarose gel electrophoresis was used to detect the integrity of the DNA. The complete and suitable DNA was used for PCR amplification, and the incomplete DNA was re-extracted until the complete DNA was obtained.

[0040] The PCR reaction system used a 10 μL system: 1 μL DNA template, 1 μL 10× PCR buffer, 0.5 μL each of forward and reverse primers (10 μmol / L), 1 μL dNTPs, 0.2 μL rTaq enzyme, and ddH 2 The PCR amplification program was as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55°C-60°C for 30 s (temperature varies with primers), extension at 72°C for 30 s, 40 cycles; and finally extension at 72°C for 10 min.

[0041] The PCR products were electrophoresed on a 4% agarose gel, and the gel was photographed and read using a gel imager. The 120 pairs of SSR primers screened above were used to perform linkage analysis on the wlp3 gene, and linkage was found near the end of chromosome 3. New Indel markers were designed upstream and downstream of the linkage markers, and the target gene interval was locked between molecular markers M1 and M6 using these 21 strains. New molecular markers were designed again in this interval, and 138 F 2 The gene was finally located in the interval of about 56 kb between M1 and M2. The primer sequences are shown in Table 2.

[0042] Table 2 Molecular markers used for fine mapping

[0043]

[0044]

[0045] According to the data information of the rice genome database (http: / / rice.plantbiology.msu.edu / ), the accession number is LOC_Os03g61620, indicating that this gene is a candidate gene. The DNA of the wild type and mutant was amplified using primers covering the gene region. The sequencing results showed that there was a T to C mutation in the third exon of the WLP3 gene, at position 380 of the coding region, which caused the protein to change from isoleucine to threonine (e.g. Figure 3 ).

[0046] The cDNA sequence of the rice white leaf and white panicle gene wlp3 is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2.

[0047] The cDNA sequence of WLP3 in Zhonghua 11 is as shown in SEQ ID NO.3, and the amino acid sequence is as shown in SEQ ID NO.4.

[0048] The base substitution on the WLP3 gene obtained by the present invention causes the phenotype of white leaves and white ears of rice plants.

[0049] Example 5 Response of wlp3 to cold stress

[0050] Low temperature stress: Wild-type and wlp3 plants at the seedling stage were selected and transferred to incubators at 4°C and 28°C, respectively. Other culture conditions were the same and the treatment was carried out for 7 days. The chlorophyll content of the wild-type and wlp3 at different temperatures was then measured, and RNA was extracted to analyze the expression levels of genes related to cold stress.

[0051] The results are as follows Figure 4 According to Figure 4It can be learned that: the results showed that after cold treatment, the leaves of the wild type turned yellow, while the leaves of wlp3 remained green. The expression levels of cold-resistance-related genes DREB1A, DREB1B, and DREB2B were detected by qRT-PCR, and it was found that the expression levels of these genes in wlp3 increased sharply after cold treatment, indicating that the mutation of wlp3 will lead to an improvement in the cold tolerance of the plant.

[0052] Example 6 Expression analysis of genes related to chloroplast development

[0053] In order to analyze whether the mutant wlp3 affects the expression of genes related to chlorophyll synthesis and chloroplast development, the present invention used the qRT-PCR method to perform expression analysis on genes related to these pathways.

[0054] RNA extraction was performed using the RNeasy PlantMini Kit (QIAGEN) to isolate total RNA from leaf samples of the mutant and wild type at the tillering stage. The expression of each gene in the wild type and mutant was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). OsActin was used as the internal reference gene, and 3 parallel wells were made for each reaction. -ΔΔCt Methods Relative quantitative analysis was performed and the reaction was repeated three times. The real-time PCR instrument was 7500 real-time PCR system (Applied Biosystems, Life Technologies). The experimental data obtained were statistically analyzed using Excel and SPSS19.0 software, and the differences between different data were compared using t-test. The qRT-PCR reaction system (10 μL) was: cDNA template 1 μL, SYBR qPCR Mix (TOYOBO) 6 μL, forward and reverse primers (10 μmol / L) 1 μL each, ddH 2 O was added to 10 μL. The qRT-PCR amplification program was as follows: 95°C for 30 s; 95°C for 5 s; 55°C for 10 s; 72°C for 15 s, 40 cycles. The required primers are shown in Table 3. The results are shown in Table 3. Figure 5 In wlp3, genes involved in chlorophyll synthesis and chloroplast development were downregulated, indicating that the mutation of wlp3 affected the synthesis of chlorophyll and the development of chloroplasts.

[0055] Table 3 Primer sequences for real-time fluorescence quantitative PCR

[0056]

[0057]

[0058] Example 7 Transgenic Experiment

[0059] The gene described in SEQ ID NO.1 was subjected to a transgenic experiment. The promoter of 700 bp upstream of wlp3 and the entire genome of wlp3 were amplified using primer pairs to construct a complementary vector, and the vector was transformed into the callus of wlp3 using Agrobacterium. The result was that 21 transgenic plants were obtained after transformation. The phenotype was observed and it was found that 2 plants still showed albino phenotype in the seedling stage, while 19 transgenic plants recovered the green phenotype. Through identification, it was found that the 19 transgenic plants were positive plants and all showed normal phenotypes. The genetic complementation of wlp3 verified that LOC_Os03g61260 was the WLP3 gene. The single base mutation of the gene would cause the rice to show albino phenotype in the seedling stage, and turn green later (such as Figure 6 ).

[0060] The agronomic traits of the wild type and wlp3 mutant are compared in Table 4 ;

[0061] Table 4 Agronomic traits of wild type and wlp3

[0062] Trait ZH11 wlp3 Plant height Plantheight / cm 106.20±2.17 129.73±6.64** Panicle length / cm 20.32±1.32 26.90±1.97** Effective number of panicle 10.60±3.21 8.40±1.52 Flagleaflength / cm 36.00±8.19 26.93±2.54 Primary branch number 12.00±1.00 14.40±1.52* Secondary branch number 23.80±6.46 33.00±2.65* Tiller number 9.80±2.39 9.20±1.30 Filledgrainnumberperpanicle 121.00±23.97 150.00±11.00** Seed-setting rate / % 41.65±1.74 48.75±2.20** 1000-grainweight / g 18.90±0.46 15.90±0.78*

[0063] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0064] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Rice white leaf and white panicle gene wlp3, characterized in that: The cDNA sequence of the white leaf and white ear gene wlp3 is shown in SEQ ID NO.

1.

2. A protein encoded by the sequence shown in SEQ ID NO.1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. Use of the rice white leaf and white panicle gene wlp3 according to claim 1 in improving rice stress resistance and increasing rice yield, wherein: The white leaf and white ear gene wlp3 improves the plant's cold resistance, increases plant height and ear length under low temperatures.

4. The use according to claim 3, characterized in that: The white leaf and white panicle gene wlp3 controls the alleviation of leaf whitening in rice plants under low temperature.

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