Rice leaf color control gene SEL, mutant gene thereof and application thereof in rice leaf color improvement

文档序号：729371 发布日期：2021-04-20 浏览：4次中文

阅读说明：本技术 水稻叶色控制基因sel、其突变基因及其在水稻叶色改良中的应用 (Rice leaf color control gene SEL, mutant gene thereof and application thereof in rice leaf color improvement ) 是由叶靖张小明叶胜海翟荣荣朱国富巫明明于 2021-02-02 设计创作，主要内容包括：本发明公开了一种水稻叶色控制基因SEL、其突变基因及其在水稻叶色改良中的应用。水稻叶色控制基因编码的氨基酸序列如SEQ ID NO：2所示,其编码基因的cDNA序列如SEQ ID NO.1所示,全长1092bp。该基因发生纯合突变后可导致叶绿素合成受阻,叶绿体发育不正常,苗期黄化致死,在苗期可以作为一种标记性状,对目标性状进行选择。本发明还公开了一种对水稻叶色改良具有重要价值的黄化致死突变体sel,其杂合型植株SEL/sel,在生育前期表现出正常叶色表型,抽穗后叶色开始变淡,后期叶片枯萎慢。可将其应用于水稻叶色改良工作中,特别是针对叶色较深杂交稻,能够使其叶色变淡,并延迟叶片衰老,对进一步提高水稻产量具有重要的意义。(The invention discloses a rice leaf color control gene SEL, a mutant gene thereof and application thereof in rice leaf color improvement. The amino acid sequence of the rice leaf color control gene code is shown as SEQ ID NO: 2, the cDNA sequence of the coding gene is shown as SEQ ID NO.1, and the total length is 1092 bp. The homozygous mutation of the gene can cause chlorophyll synthesis obstruction, chloroplast abnormal development and yellowing death in the seedling stage, and the gene can be used as a marker character to select target characters in the seedling stage. The invention also discloses a yellowing lethal mutant SEL with important value for improving the leaf color of rice, wherein a heterozygous plant SEL/SEL shows a normal leaf color phenotype in the early growth stage, the leaf color becomes light after heading, and the leaf withering is slow in the later stage. The method can be applied to the rice leaf color improvement work, particularly for hybrid rice with darker leaf color, can lighten the leaf color and delay the leaf senescence, and has important significance for further improving the rice yield.)

1. The coded amino acid sequence of the separated rice leaf color control gene is shown as SEQ ID NO: 2, respectively.

2. The rice leaf color control gene according to claim 1, having a nucleotide sequence as set forth in SEQ ID NO: 1 is shown.

3. An isolated rice leaf color mutant gene which has one or more nucleotides substituted, inserted or deleted in the sequence shown in SEQ ID No.1, resulting in a mutant gene which does not encode the nucleotide sequence shown in SEQ ID No. 1: 2, or a pharmaceutically acceptable salt thereof.

4. The rice leaf color mutant gene according to claim 3, which has a nucleotide sequence shown as SEQ ID NO: 3, respectively.

5. A vector comprising the rice leaf color control gene or rice leaf color mutant gene according to any one of claims 1 to 4.

6. A cell comprising the rice leaf color control gene or rice leaf color mutant gene according to any one of claims 1 to 4.

7. The use of the rice leaf color mutant gene according to claim 3 or 4 for improving rice leaf color.

8. The application of claim 7, wherein the application comprises: the rice leaf color mutant gene is transferred into a rice variety to be improved.

Technical Field

The invention belongs to the field of rice genetic breeding, and particularly relates to a rice leaf color control gene and application of a mutant gene thereof in rice leaf color improvement.

Technical Field

The rice is one of the most important grain crops in China, however, the phenomena of dark leaf color and early leaf senescence in the late growth period exist in a plurality of rice varieties, particularly the indica rice varieties have short growth period, fast leaf senescence and short grain filling time, and the further improvement of the yield of the indica rice varieties is seriously influenced. How to continuously improve the yield per unit of rice and meet the increasing grain requirements becomes one of the most main directions of research, and the cultivation of the 'ideal plant type' with high light efficiency and strong green-keeping property is an important way.

The traditional breeding method is utilized to improve the color of the hybrid rice leaves, the method is time-consuming, labor-consuming and low in efficiency, the leaf color mutant is an ideal material for researching the chlorophyll metabolism and the chloroplast development of the rice, the research on the leaf color mutant is helpful for understanding the mechanism of the chlorophyll metabolism and the chloroplast development, the photosynthetic efficiency and the photosynthetic time of the rice are improved, and the method has important value for improving the color of the rice leaves. In the reported rice leaf color mutants, only a few will die, and most of the die mutants are albino die, and the reports on yellowing die are very few.

Disclosure of Invention

The invention provides a rice leaf color control gene and a mutant gene thereof, and also provides application of the gene.

The invention provides a separated rice leaf color control gene, and an encoded amino acid sequence of the gene is shown as SEQ ID NO: 2, respectively.

Wherein, the nucleotide sequence of the rice leaf color control gene is shown as SEQ ID NO: 1 is shown.

The invention provides an isolated rice leaf color mutant gene, which substitutes, inserts or deletes one or more nucleotides in a sequence shown in SEQ ID NO.1, so that the mutant gene does not code for the nucleotide sequence shown in SEQ ID NO. 1: 2, or a pharmaceutically acceptable salt thereof.

Wherein, the nucleotide sequence of the rice leaf color mutant gene is shown as SEQ ID NO: 3, respectively.

The invention provides a carrier containing the rice leaf color control gene or the rice leaf color mutant gene.

The invention provides a cell containing the rice leaf color control gene or the rice leaf color mutant gene.

The invention provides application of the rice leaf color mutant gene in rice leaf color improvement.

Wherein the application comprises: the rice leaf color mutant gene is transferred into a rice variety to be improved.

The invention identifies a new gene SEL for controlling the leaf color of rice, and researches show that 2 bases of the mutant SEL are deleted to cause frame shift mutation.

Because the rice mutant sel is lethal in the seedling stage, the mutant can only be used as a marker character in the seedling stage, and the target character is selected, for example, in the hybrid seed production process, the mutant is yellow and lethal, so that impurities can be simply removed automatically, and the purity of the hybrid is improved. In addition, the heterozygous plant SEL/SEL with the mutant gene shows a normal leaf color phenotype in the early growth stage, the leaf color becomes lighter after heading, and the leaf green time is long in the later stage. F can be reduced due to apomixis₁The heterosis is fixed, so the heterosis gene can be applied to improve hybrid rice with darker leaf color, delay leaf senescence, still maintain better photosynthetic function in the late stage of grain filling, have obvious advantages in photosynthetic time and be beneficial to further improving the rice yield.

Drawings

FIG. 1 shows the phenotype of wild-type rice and the yellowing lethal mutant sel in comparison with photosynthetic pigments; wherein A-D are respectively: A. wild type and sel germinating 9d plant phenotypes; B. measuring the chlorophyll content of 9d germinated plants; C. germinating 12d plant phenotype; D. germinating 15d plant phenotype; bar is 1 cm;

FIG. 2 shows the comparison of the traits of the wild type and heterozygous plants SEL/SEL at 20 days of heading;

FIG. 3 is a schematic illustration of gene mapping;

FIG. 4 shows the functional complementation test T0 transgenic shoots (left SEL/1390: mutant SEL transformed empty pCUbi 1390; right SEL/1390: SEL: mutant SEL transformed complementation vector pCUbi1390: SEL);

FIG. 5 is a transmission electron micrograph of chloroplast ultrastructures of wild type and mutant sel at the seedling stage.

Detailed Description

Example 1: isolation and genetic analysis of sel mutants

The rice yellowing lethal mutant sel is obtained by natural mutation of japonica rice variety Nipponbare. Compared to the wild type, mutant sel showed yellow color in all leaves from shoot stage to four-leaf stage, and the chlorophyll a (chla), chlorophyll B (chlb) and carotenoid (car) contents were significantly reduced (a-B in fig. 1). Four leaves later the mutant sel senesced first in the fourth leaf, followed by withering of the third leaf and finally death of the whole plant (C-D in fig. 1). These results indicate that the mutant sel chlorophyll synthesis is hindered and does not grow normally. Homozygous mutant SEL is yellow-lethal, however, heterozygous plant SEL/SEL with the mutant gene has light leaf color after heading, wild type leaf tip begins to wither after 20 days of heading, while heterozygous plant SEL/SEL still maintains normal phenotype and is not senescent (fig. 2)

Because homozygous mutant sel yellows and dies, the seed is obtained by selfing mutant heterozygous strains, and the single plant is harvested and sown respectively to obtain 5 phenotypically separated groups. 1726 green seedlings and 527 yellow dead seedlings were found in the 5 segregating populations, and the segregation ratio (chi) was 3:1²＝3.11<χ² _0.053.84) indicates that the mutant trait is controlled by a single recessive nuclear gene.

Example 2: fine localization and complementation validation of SEL genes

To clone the SEL gene, we crossed mutant heterozygous (SEL/SEL) lines with indica rice variety 9311, F₁Harvesting and sowing the single plants, and selectingF capable of isolating the yellow leaf phenotype₂The population was used for gene mapping. Through mixed pool screening and individual plant verification, the SEL gene is initially located between RM5794 and RM12272 on chromosome 1. Based on the difference of the sequences of 9311 of Nippon Qinghai and indica rice varieties, a new marker was developed in the interval RM5794 and RM12272, the sequences of the primers are shown in Table 1, the SEL is located between the markers B3 and B5 by the chromosome walking method, and the physical distance is about 43.3kb (FIG. 3).

TABLE 1 Fine positioning primer information of SEL

There are 6 Open Reading Frames (ORFs) in this interval and sequencing results show that mutant sel has a two base deletion within ORF4, resulting in a frameshift mutation. To further confirm the candidate genes, we performed genetic complementation experiments by ligating the promoter of wild-type ORF4 to the vector pCUbi1390 after ligation to cDNA, constructing the complementation vector pCUbi1390: SEL, and transforming into mutant SEL, which transformed empty vector pCUbi1390 as a control. All positive transgenic lines restored normal leaf color phenotype (fig. 4). These results demonstrate that ORF4 is the gene controlling the sel phenotype. The nucleotide sequence of the SEL gene is shown as SEQ ID NO: 1, and the coded amino acid sequence is shown as SEQ ID NO: 2, deletion of the mutant sel at bases "ca" at positions 136-137 of the gene results in a frame shift mutation.

Example 3: observation of chloroplast transmission electron microscope

The observation of the chloroplast ultrastructure of the wild type and the mutant sel in the seedling stage by a transmission electron microscope shows that: the wild type chloroplast has orderly arranged and clear thylakoid lamellar structure inside, while the mutant sel has disordered, less quantity and unclear layers of chloroplast arrangement (figure 5). The products of photosynthesis are stored in the form of starch, which is often present in granular form, called starch granules. The number of starch granules in chloroplasts in the wild type body is large and the volume is large, while the mutant body has many empty vesicles and no obvious starch granules.

Sequence listing

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