WO2024108862A1 - 水稻白叶白穗基因wlp3及在水稻抗逆和增产中的应用 - Google Patents

水稻白叶白穗基因wlp3及在水稻抗逆和增产中的应用 Download PDF

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WO2024108862A1
WO2024108862A1 PCT/CN2023/086900 CN2023086900W WO2024108862A1 WO 2024108862 A1 WO2024108862 A1 WO 2024108862A1 CN 2023086900 W CN2023086900 W CN 2023086900W WO 2024108862 A1 WO2024108862 A1 WO 2024108862A1
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wlp3
rice
white
gene
leaf
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French (fr)
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饶玉春
芦涛
黄佳慧
殷文晶
钟芊芊
杨宇琪
陆天麒
孙静蕾
贾绮玮
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浙江师范大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2

Definitions

  • 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.
  • the chloroplasts in higher plants are about 6 ⁇ m long and 3 ⁇ m 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, namely chloroplast membrane, thylakoid, and matrix.
  • the membrane of chloroplasts is a double-layer membrane like the cell membrane.
  • the outer mold has good permeability, while the inner membrane has strong selectivity.
  • Thylakoids are flat bodies formed by a single-layer membrane.
  • the thylakoid 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 to form H and O 2 , and ADP is converted into ATP to provide energy for the subsequent dark reaction. Therefore, the thylakoid membrane is also called a photosynthetic membrane.
  • Many thylakoids are stacked together to form grana.
  • the substance between the chloroplast membrane and the thylakoid is the matrix, which is the site for carbon assimilation. It contains carbon assimilation enzymes, chloroplast DNA, proteins, starch and other substances. Its main function is to fix CO2 in organic matter and use ATP to reduce tricarbon sugars, providing sufficient raw materials for the synthesis of light reaction substances.
  • 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 its conversion directly determines the final yield and quality of rice. Chloroplasts are a semi-autonomous organelle that has its own genome and coordinates 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 will 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 is maintained at a relatively low level. 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), and the plastid transcription activity increases.
  • the third step is the increase in the expression of genes encoding light systems.
  • genes are mainly transcribed by the PEP expressed in the second stage, which also includes the two subunits encoding the light system: psaA, psbA and the two subunits of diphosphate carboxylase: rcL, rbcS, etc.
  • rice is a good carrier for studying the chloroplast development of monocots. Current research believes that the development of leaves will go through the p0-p6 stages, and each stage also has different gene-specific expressions.
  • the transcription levels of POLP1 and FtsZ involved in DNA replication are extremely high in the p0-p3 stage, and gradually decrease to almost zero at p4.
  • RpoTP the expression levels of RpoTP, subunits of PEP, and some ribosomal proteins such as RPS7 and RPS15 gradually increased, while the expression levels of photosynthesis-related genes such as Lhcb, rbcs, and rbcl were the highest at P5 and P6.
  • 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 damage to 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.
  • 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.
  • the present invention adopts the following technical solution:
  • the rice white leaf and white panicle gene wlp3 the cDNA sequence of the white leaf and white panicle gene wlp3 is shown in SEQ ID NO.1.
  • 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 with a single base substitution, i.e., the 380th base of SEQ ID NO.3.
  • the nucleotide T at position 127 was changed to C.
  • the amino acid at position 127 was changed from isoleucine to threonine.
  • Bioinformatics analysis showed that wlp3 encodes the large ribosomal subunit L18.
  • the protein sequence encoded by the wild-type wlp3 gene is shown in SEQ ID NO.4.
  • 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.
  • 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.
  • 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.
  • 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.
  • the newly grown leaves of wlp3 gradually turn green until the phenotype is restored to the same as the wild type.
  • 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.
  • 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.
  • Figure 1 shows the phenotypes of wild-type Zhonghua11 and mutant wlp3;
  • A leaf phenotypes of wild-type and wlp3 at seedling stage, scale bar is 1 cm;
  • B phenotypes of wild-type and wlp3 at tillering stage, scale bar is 10 cm;
  • C Phenotypes of wild type and wlp3 at maturity, scale bar is 20 cm;
  • D Phenotypes of wild type and wlp3 ear, scale bar is 5 cm;
  • Figure 2 shows the leaf phenotypes of the wild type and wlp3 at different temperatures and the chlorophyll content of the second and third leaves;
  • Figure 3 is a map of the location of the wlp3 gene; (A) initial location of wlp3; (B-C) fine location of WLP3; (D) gene structure of wlp3; (E-F) mutation sites of wlp3;
  • Figure 4 shows the response of wild type and wlp3 to cold stress;
  • A phenotype of wild type and wlp3 before cold treatment, scale is 5 cm;
  • B expression of cold-resistance related genes of wild type and wlp3 before cold treatment;
  • C expression of cold-resistance related genes of wild type and wlp3 after cold treatment;
  • D phenotype of wild type and wlp3 after cold treatment, scale is 5 cm;
  • E chlorophyll content of wild type and wlp3 after cold treatment;
  • Figure 5 is an analysis of the expression of genes related to leaf color in the wild type and wlp3; (A) the expression of genes related to chlorophyll synthesis in the wild type and wlp3; (B) the expression of genes related to ribosomal genes in the wild type and wlp3; (C) the expression of genes related to chloroplast development in the wild type and wlp3;
  • Figure 6 is a functional complementation diagram of wlp3.
  • 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°C, 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.
  • 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.
  • the newly grown leaves of wlp3 gradually turned green until they restored the same phenotype as the wild type.
  • the phenotype of wlp3 was basically the same as the wild type, but the plant height was higher than the wild type at maturity.
  • the panicle of wlp3 showed the albino phenotype again and the panicle length was longer than that of the wild type. (As shown in Figure 1)
  • 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.
  • the SSR primers evenly distributed on 12 rice chromosomes preserved in our laboratory were used to screen the mutants and Zhonghua 11 and 9311 for polymorphism. Then, linkage analysis was performed using 21 F 2 white-leaf and white-ear plants to preliminarily confirm the chromosome location of the target gene. Genomic DNA was extracted using the CTAB method. The specific steps are as follows:
  • 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.
  • the PCR reaction system used a 10 ⁇ L system: 1 ⁇ L DNA template, 1 ⁇ L 10 ⁇ PCR buffer, 0.5 ⁇ L each of forward and reverse primers (10 ⁇ mol/L), 1 ⁇ L dNTPs, 0.2 ⁇ L rTaq enzyme, and ddH 2 O to make up to 10 ⁇ L.
  • 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 to 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.
  • the PCR products were electrophoresed on a 4% agarose gel, and the gel was photographed and read using a gel imager after the electrophoresis.
  • the linkage analysis of the wlp3 gene using the 120 pairs of SSR primers screened above revealed a linkage phenomenon near the end of chromosome 3.
  • New Indel markers were designed upstream and downstream of the linkage markers, and these 21 strains were used to lock the target gene interval between the molecular markers M1 and M6.
  • New molecular markers were designed again in this interval, and 138 F2 strains were used to finally locate the gene in an interval of approximately 56kb between M1 and M2.
  • the primer sequences are shown in Table 2.
  • 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 380th position of the coding region of the third exon of the WLP3 gene, which caused the protein to change from isoleucine to threonine (as shown in Figure 3).
  • the cDNA sequence of the rice white leaf and white panicle gene wlp3 is as described in SEQ ID NO.1, and the amino acid sequence is as described in SEQ ID NO.2.
  • the cDNA sequence of WLP3 in Zhonghua 11 is as described in SEQ ID NO.3, and the amino acid sequence is as described in SEQ ID NO.4.
  • the base substitution on the WLP3 gene obtained by the present invention causes the phenotype of white leaves and white ears of rice plants.
  • results are shown in FIG4 , and it can be seen from FIG4 that: the results show that after cold treatment, the leaves of the wild type turn yellow, while the leaves of wlp3 remain 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.
  • the present invention used the qRT-PCR method to perform expression analysis on genes related to these pathways.
  • RNA extraction was performed using the RNeasy Plant Mini Kit (QIAGEN) to isolate total RNA from leaf samples of the mutant and wild type at the tillering stage.
  • the instructions of qPCR RT Kit (TOYOBO) were used to reverse transcribe into cDNA.
  • 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 three parallel wells were made for each reaction.
  • the relative quantitative analysis was performed by the 2 - ⁇ Ct method, and the independent reactions were 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 by Excel and SPSS19.0 software, and the differences between different data were compared by t-test.
  • the qRT-PCR reaction system (10 ⁇ L) was: 1 ⁇ L cDNA template, 6 ⁇ L SYBR qPCR Mix (TOYOBO), 1 ⁇ L each of forward and reverse primers (10 ⁇ mol/L), and ddH 2 O was added to 10 ⁇ L.
  • the qRT-PCR amplification program was as follows: 95°C30s; 95°C5s; 55°C10s; 72°C15s, 40 cycles.
  • the required primers are shown in Table 3.
  • the results are shown in Figure 5.
  • the genes involved in chlorophyll synthesis and chloroplast development in wlp3 were down-regulated, indicating that the mutation of wlp3 affected the synthesis of chlorophyll and the development of chloroplasts.
  • the gene described in SEQ ID NO.1 was subjected to a transgenic experiment.
  • the 700bp promoter upstream of wlp3 and the entire genomic DNA of wlp3 were amplified using primer pairs to construct a complementary vector, and then transformed into the callus tissue 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 at 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 this gene would cause the rice to show albino phenotype at the seedling stage, and turn green later (as shown in Figure 6).

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Abstract

提供一种水稻白叶白穗基因wlp3，其cDNA序列如SEQ ID NO.1所示，编码的氨基酸序列如SEQ ID NO.2所示。将水稻白叶白穗基因wlp3应用于提高水稻抗逆和水稻增产中，其中，白叶白穗基因wlp3在低温下提高植株耐寒能力、增强光合速率、增加株高、苗期叶片白化、抽穗期穗白化、穗长增加。通过筛选诱变获得水稻白叶白穗基因wlp3，该基因与水稻的抗逆性胁迫和叶绿素合成量相关，为水稻育种提供了基础。

Description

水稻白叶白穗基因wlp3及在水稻抗逆和增产中的应用 技术领域
本发明涉及水稻白叶白穗基因筛选及育种技术领域，更具体地说是涉及水稻白叶白穗基因wlp3及在水稻育种的应用。
背景技术
高等植物中的叶绿体长约6μm，宽3μm，正常的叶肉细胞中含有100-150个的叶绿体，在不同物种和不同部位叶绿体的含量也会有所差异。叶绿体的形态结构由3部分组成，分别为叶绿体膜，类囊体，基质。叶绿体的膜与细胞膜一样均为双层膜，外模具有良好的渗透性，而内膜具有较强的选择性。类囊体是由单层膜包裹形成的扁平小体，在类囊体薄膜上存在许多的光合色素和参与电子传递系统的蛋白质，如：细胞色素复合体，质体醌，质体蓝素，铁氧还原蛋白，黄素蛋白等，在这个部位可以将水进行光解，形成H和O2，同时ADP转变为ATP，为后面的暗反应提供能量，因此类囊体薄膜也称为光合膜。许多类囊体堆叠在一起形成基粒。叶绿体膜和类囊体之间的物质为基质，基质是进行碳同化的部位，当中含有碳同化的酶叶绿体DNA，蛋白质淀粉等物质，主要功能是将CO2固定在有机物中，并利用ATP将三碳糖还原，为光反应物质合成提供充足的原料。
叶绿体作为水稻叶片重要的细胞器，在水稻的生长发育过程中起到重要作用。叶绿体可以吸收光能并转化成植株可以利用的化学能贮藏起来，其转化效率高低直接决定了水稻最终产量及品质。叶绿体是一种半自主性的细胞器，它拥有自身的基因组，并且与细胞核共同协调植物叶绿体的发育。叶绿体是由原质体发育而来，其发育受到温度光照等外部因素和激素基因等内部因素共同决定，在这些因素中对叶绿体发育最为重要的条件即为光照，只有在足够的光照下原质体才会发育成叶绿体，反之则会发育形成黄质体。叶绿体发育的过程主要分为三个部分：第一个是质体自身基因组DNA的合成及质体的复制，但在这一步中，质体自身基因的转录水平却保持在一个比较低的 水平；第二步是叶绿体遗传系统的建立，这时核编码的RNA聚合酶(NEP)会首先转录表达如RpoTP会在这个阶段大量表达，接着NEP会负责叶绿体发育的管家基因以及质体编码的RNA聚合酶(PEP)，质体转录活性增加，第三步是编码光系统相关基因的表达增加，这类基因主要由第二阶段表达的PEP转录，这之中也包括编码光系统的两个亚基：psaA，psbA以及二磷酸羧化酶的两个亚基：rcL，rbcS等。水稻作为模式植物，是研究单子叶植物叶绿体发育的良好载体，现在的研究认为，叶片的发育会经历p0-p6这几个阶段，每个阶段也有不同的基因特异性表达，p0-p3期中参与DNA复制的POLP1和FtsZ转录水平极高，到p4开始逐渐下降直到几乎为零。在p4期RpoTP，PEP的亚基，以及部分核糖体蛋白如RPS7和RPS15表达量逐渐升高，而Lhcb，rbcs，rbcl等与光合作用相关的基因在P5和P6表达水平最高。
水稻是全球一半以上人口的主要粮食，水稻的产量对于人类发展有着至关重要的作用。叶片时光合作用的主要场所，叶片的白化会引起叶绿体的破坏，导致光合速率的下降，最终引起水稻产量的下降，因此探究水稻叶片白化的机制，研究叶绿体发育过程，对于理解水稻光合作用的分子机制有着深远的意义。
因此，筛选一种与水稻白叶白穗相关的基因，并将其应用于水稻育种中是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明筛选到一种水稻白叶白穗基因wlp3，该基因与水稻叶片颜色有关，对该基因进行进一步研究，发现其可以提高水稻的抗寒能力，增加产量，为水稻优良品种的选育奠定基础。
为了实现上述目的，本发明采用如下技术方案：
水稻白叶白穗基因wlp3，白叶白穗基因wlp3 cDNA序列如SEQ ID NO.1所示。
采用图位克隆的方法克隆了白叶白穗突变体基因wlp3。wlp3基因是由LOC_Os03g61620基因发生了单碱基替换而来的，即SEQ ID NO.3的第380 位核苷酸T转变为C。导致了127位氨基酸由异亮氨酸转变为苏氨酸，生物信息学分析显示wlp3编码核糖体大亚基L18，野生型wlp3基因的编码的蛋白序列如SEQ ID NO.4所示。
作为与上述技术方案相同的发明构思，本发明还请求保护一种由SEQ ID NO.1所示的序列编码的蛋白质，氨基酸序列如SEQ ID NO.2所示。
作为与上述技术方案相同的发明构思，本发明还请求保护水稻白叶白穗基因wlp3在提高水稻抗逆和水稻增产中的应用，其中，白叶白穗基因wlp3在低温下提高植株耐寒能力、增强光合速率、增加株高、苗期叶片白化、抽穗期穗白化、穗长增加。
优选地，白叶白穗基因wlp3控制水稻植株在低温下叶片白化缓解，高温出现白化。
本发明的水稻白叶白穗突变体wlp3是从粳稻品种中花11的EMS诱变体库中筛选得到的。突变体wlp3在二叶期时出现白条表型并持续到分蘖期，白条沿着叶脉分布于分布于整片叶子，在四叶期后随着水稻生长发育wlp3新生长出来的叶片逐渐转绿直到恢复与野生型相同的表型。
本发明的水稻白叶白穗突变体wlp3基因可以用基因工程或遗传工程方法增强作物对不良环境的抗性，如提高作物的光合作用提高产量等。
综上，为了挖掘新的叶色相关基因，本发明从粳稻品种中花11为背景的EMS诱变体库中筛选到一份白叶白穗的突变体，将其命名为wlp3(white leaf and panicle3)。对wlp3进行表型分析以及激素响应，在影响水稻叶色方面具有广阔的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为野生型中花11与突变体wlp3表型；(A)野生型和wlp3苗期叶片表型，标尺为1cm；(B)野生型和wlp3分蘖期表型，标尺为10cm；(C) 野生型和wlp3成熟期表型图，标尺为20cm；(D)野生型和wlp3穗子表型图，标尺为5cm；
图2附图为不同温度下的野生型和wlp3叶片表型及第二叶和第三叶的叶绿素含量图；
图3附图为wlp3基因的定位图；(A)wlp3的初定位；(B-C)WLP3的精细定位；(D)wlp3的基因结构；(E-F)wlp3的突变位点；
图4附图为野生型和wlp3对冷胁迫响应情况；(A)野生型和wlp3在冷处理前的表型图，标尺为5cm；(B)野生型和wlp3在冷处理前耐冷相关基因的表达量；(C)野生型和wlp3在冷处理后耐冷相关基因的表达量；(D)野生型和wlp3在冷处理后的表型图，标尺为5cm；(E)野生型和wlp3在冷处理后叶绿素含量；
图5附图为野生型和wlp3中与叶色相关基因表达量分析；(A)野生型和wlp3中与叶绿素合成相关基因的表达量；(B)野生型和wlp3中核糖体基因相关基因的表达量；(C)野生型和wlp3中与叶绿体发育相关基因的表达量；
图6附图为wlp3的功能性互补图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1突变体材料的获得
通过EMS化学诱变粳稻品种中花11：清水浸种8h。然后沥出水分，倒入已经配比好的1％EMS溶液浸泡诱变8h，期间用小木棍搅拌多次确保混合均匀。诱变完成后用流动水冲洗净有毒的EMS缓冲液，25℃催芽后播种，单株插秧单株收种，生长时期按常规大田管理。通过筛选到一份白叶白穗突变体wlp3。
该突变体的性状经过多代自交已稳定遗传，所有水稻材料种植于浙江省金华市浙江师范大学生化学院试验田，常规管理。
实施例2植株的表型分析
大田条件下，突变体wlp3在二叶期时出现白条表型并持续到分蘖期，白条沿着叶脉分布于分布于整片叶子，在四叶期后随着水稻生长发育wlp3新生长出来的叶片逐渐转绿直到恢复与野生型相同的表型，在生长过程中，wlp3的表型与野生型基本相同，而株高在成熟时高于野生型。幼穗期时wlp3的穗再次表现出白化的表型且穗长要长于野生型。(如图1)
实施例3群体构建和遗传分析
将突变体wlp3和常规籼稻9311和ZF802进行正反交，F1植株均表现出正常野生型表型，说明wlp3受隐性核基因控制。统计F2分离群体分离比(表1)，结果表明，正常表型的植株和突变体表型的植株的分离比经过卡方检验接近3:1分离，这表明wlp3的白叶白穗表型是由一对单隐性核基因控制。
表1白叶白穗突变体wlp3的遗传分析
实施例4wlp3基因的精细定位
利用本实验室保存的均匀分布于水稻12条染色体的SSR引物对突变体与中花11、9311进行多态性筛选。然后用21个F2中白叶白穗单株进行连锁分析，初步确认目标基因所在的染色体位置。基因组DNA采用CTAB法提取。具体步骤如下：
①称取0.1g的水稻叶片用液氮研磨成粉状，然后加入600μl的CTAB溶液(2％(m/V)CTAB，100mmol/L Tris-HCl，20mmol/L EDTA，1.4mol/L NaCl；pH8.0)配制的DNA提取缓冲液，65℃水浴40分钟。再加600μl的氯仿:异戊醇(24:1的体积比)，并混匀。10000rpm离心5分钟，将上清液转移到新的离心管中。
②在上述步骤①离心后所得的上清液中加2/3～1倍体积预冷(至4℃)的异丙醇，轻轻混匀至DNA沉淀。13,000rpm离心8分钟，倒出上清液。
③再用70％(体积浓度)的乙醇200μl洗涤上述步骤②所得的DNA沉淀物。
④将上述洗涤后的DNA晾干并溶于100μl TE缓冲液或纯水中。
⑤紫外分光光度法检测上述步骤④所得的DNA样品的浓度，0.7％的琼脂糖凝胶电泳检测DNA的完整性。完整合适的DNA用于PCR扩增，不完整的DNA则重新提取，直至获得完整的DNA。
PCR反应体系采用10μL体系：DNA模板1μL，10×PCR缓冲液1μL，正反向引物(10μmol/L)各0.5Μl，dNTPs 1μL，rTaq酶0.2μL，加ddH2O补足10μL。PCR扩增程序如下：94℃下预变性4min；94℃下变性30s，55℃～60℃下退火30s(温度因引物不同而异)，72℃下延伸30s，40个循环；最后72℃下延伸10min。
PCR产物用4％琼脂糖凝胶电泳，电泳结束后在凝胶成像仪拍照并读胶。利用上述筛选的120对SSR引物进行wlp3基因连锁分析发现在第3号染色体的末尾附近处表现出连锁现象。在连锁标记上下游设计新的Indel标记，用这21个单株将目的基因区间锁定在分子标记M1与M6之间。在此区间再次设计新的分子标记，用138个F2单株最终将该基因定位在M1和M2之间大约56kb的区间内。引物序列见表2。
表2精细定位所用分子标记

根据水稻基因组数据库(http://rice.plantbiology.msu.edu/)数据信息，登陆号为LOC_Os03g61620，预示该基因为候选基因。利用覆盖该基因区域的引物分别扩增野生型和突变体的DNA，测序结果表明，WLP3基因第三个外显子，编码区第380有一个T到C的突变，导致蛋白质由异亮氨酸转变为苏氨酸(如图3)。
水稻白叶白穗基因wlp3的cDNA序列如SEQ ID NO.1所述，氨基酸序列如SEQ ID NO.2所述。
中花11中WLP3的cDNA序列如SEQ ID NO.3所述，氨基酸序列如SEQ ID NO.4所述。
本发明所获得的WLP3基因上的碱基替换造成了水稻植株叶片白化，穗子白化的表型。
实施例5wlp3对冷胁迫的响应
低温胁迫：选取苗期的野生型和wlp3植株，分别转移至4℃和28℃的培养箱中，其他培养条件相同，处理7d，随后测量不同温度下野生型和wlp3的叶绿素含量，并提取RNA，对与冷胁迫相关基因的表达量进行分析。
所得结果如图4所述，根据图4可得知：结果发现在冷处理之后，野生型的叶片发黄，而wlp3的叶片仍然保持绿色。通过qRT-PCR检测耐冷相关基因DREB1A、DREB1B、DREB2B的表达量，发现在冷处理后wlp3中这些基因的表达量有剧烈上升，由此表明，wlp3的突变会引起植株耐冷能力的提高。
实施例6与叶绿体发育相关基因的表达分析
为了分析突变体wlp3是否影响叶绿素合成及叶绿体发育相关基因的表达，本发明利用了qRT-PCR方法对这些途径的相关基因进行了表达分析。
RNA的提取按照总RNA提取试剂盒RNeasy Plant Mini Kit(QIAGEN)的方法步骤分离突变体和野生型分蘖期叶片样本总RNA，再按照逆转录试剂盒ReverTraqPCR RT Kit(TOYOBO)的说明反转录成cDNA。利用实时荧光定量PCR(qRT-PCR)方法，分析各基因在野生型和突变体中的表达量。以基因OsActin作为内参基因，每个反应做3个平行复孔，采用2-ΔΔCt方法进行相对定量分析，重复做3次独立反应。实时PCR仪器为7500实时PCR体系(Applied Biosystems，Life Technologies)，对所得到的实验数据通过Excel和SPSS19.0软件进行统计分析，采用t检验比较不同数据间的差异。qRT-PCR反应体系(10μL)是：cDNA模板1μL，SYBR qPCR Mix(TOYOBO)6μL，正反引物(10μmol/L)各1μL，ddH2O补足至10μL。qRT-PCR扩增程序如下：95℃30s；95℃ 5s；55℃ 10s；72℃ 15s，40个循环。所需引物见表3。结果如图5，在wlp3中参与叶绿素合成及叶绿体发育相关基因出现了下调表达，说明wlp3的突变影响了叶绿素的合成及叶绿体的发育。
表3实时荧光定量PCR的引物序列

实施例7转基因实验
将SEQ ID NO.1所述基因进行转基因实验，利用引物对分别扩增出wlp3上游700bp的启动子及整个wlp3整个基因组的DNA用于构建互补载体，并利用农杆菌转入wlp3的愈伤组织中，所得结果为转化后共得到21株转基因植株，观察表型发现有2株在苗期仍然出现白化的表型，而19株转基因植株恢复了绿色的表型，通过鉴定，发现该19株转基因植株为阳性植株，均表现出正常的表型。通过wlp3的遗传互补验证了LOC_Os03g61260即为WLP3基因，该基因的单碱基突变会导致水稻在苗期出现白化表型，而后期转绿(如图6)。
对比野生型和wlp3突变体的农艺性状见表4；
表4野生型与wlp3农艺性状
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (4)

  1. 水稻白叶白穗基因wlp3，其特征在于，白叶白穗基因wlp3 cDNA序列如SEQ ID NO.1所示。
  2. 一种由SEQ ID NO.1所示的序列编码的蛋白质，其特征在于，氨基酸序列如SEQ ID NO.2所示。
  3. 水稻白叶白穗基因wlp3在提高水稻抗逆和水稻增产中的应用，其中，白叶白穗基因wlp3在低温下提高植株耐寒能力、增强光合速率、增加株高、苗期叶片白化、抽穗期穗白化、穗长增加。
  4. 根据权利要求3所述的应用，其特征在于，白叶白穗基因wlp3控制水稻植株在低温下叶片白化缓解，高温出现白化。
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