CN116144674A

CN116144674A - Rice albino-transgenic green leaf mutant gene TWINKLE and application thereof

Info

Publication number: CN116144674A
Application number: CN202310238934.8A
Authority: CN
Inventors: 冯萍; 王楠; 何光华; 王通明
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2023-03-14
Filing date: 2023-03-14
Publication date: 2023-05-23
Anticipated expiration: 2043-03-14
Also published as: CN116144674B

Abstract

The invention relates to a rice albino-transgenic green leaf mutant gene TWINKLE and application thereof, wherein the CDS sequence of the rice albino-transgenic green leaf mutant gene TWINKLE is shown as SEQ ID No.1, the amino acid sequence of a coding protein is shown as SEQ ID No.2, the phenotype is albino in seedling stage, and leaves start to turn green partially in 4-5 leaf stage in the field, and the character is continued until the mature stage. Can be used for breeding new rice varieties and identifying the purity of seeds, and provides a new gene and method for breeding pure sterile lines.

Description

Rice albino-transgenic green leaf mutant gene TWINKLE and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering and rice molecular breeding, in particular to a rice albino green leaf mutant gene TWINKLE and application thereof.

Background

Leaves are main organs for photosynthesis of rice, and have close relation with biological yield and economic yield of rice. Leaf color mutation is a common mutation property of rice, and the mutation types are more. The previous researches on the leaf color mutant materials of the rice are more, and the leaf color mutant mechanism of the rice is discussed through the positioning, cloning and functional researches on the leaf color mutant genes of the rice, and the method has important significance for promoting genetic improvement of the rice, high-yield breeding of the rice and the like.

At present, the following safety problems mainly exist in the production of hybrid rice: firstly, the sterility instability phenomenon caused by the influence of low temperature in the seed production process of the rice photo-thermo-sensitive sterile line affects the purity and the yield of the rice photo-thermo-sensitive sterile line and the two-line hybrid rice. Secondly, in the three-line cross breeding, part of excellent sterile lines are easy to produce fertile pollen during seed production and propagation at a higher temperature, so that a small amount of selfing and maturing are caused, and the purity of the three-line sterile lines and the purity of the hybrid rice are affected.

In breeding practice, rice leaf albino is an ideal marker trait that can be used in breeding. The marker traits currently considered to be applied to hybrid rice production should meet the following conditions: 1. the marking character is obvious, and the identification can be carried out in the seedling stage; 2. the marker character is recessive nuclear gene control; 3. the stable expression of the marker character is not easily influenced by environmental factors; 4. the marker trait expression has no negative effect on the agronomic traits of the sterile line and the hybrid progeny thereof. The leaf color mutant controlled by recessive nuclear genes is utilized, the leaf color mutation characters are led into a sterile line of three lines or two lines by means of hybridization, backcrossing and the like, false seeds can be accurately identified and removed in a seedling stage, and the purity and quality of improved seeds are ensured; the yield per unit area is improved.

Disclosure of Invention

The invention aims to provide a rice albino-transgenic green leaf mutant gene TWINKLE and application thereof, wherein the gene phenotype is albino in a seedling stage, the leaves start to turn green partially in a 4-5 leaf stage in the field, the character can be used as a rice breeding mark until the mature stage, false seeds can be accurately identified and removed in the seedling stage, and the purity and quality of improved seeds are further ensured, so that the yield per unit area is improved.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a rice albino-transgenic green leaf mutant gene TWINKLE, wherein the CDS sequence is shown as SEQ ID No.1, the amino acid sequence of the coding protein is shown as SEQ ID No.2, the phenotype is albino in the seedling stage, and when the leaf stage is 4-5 in the field, the leaf starts to turn green partially, and the property is continued until the mature stage.

The invention also provides application of the rice albino green leaf mutant gene TWINKLE in rice breeding.

Furthermore, the application is that the rice albino-transgenic green leaf mutant gene TWINKLE is used as an early breeding marker, false seed identification and elimination are carried out in the seedling stage, and the purity and quality of improved seeds are ensured.

Furthermore, the application is that the rice albino green leaf mutant gene TWINKLE is introduced into a three-line or two-line sterile line by a hybridization and backcross method, and the albino character of the TWINKLE gene is utilized for identifying and eliminating false seeds in a seedling stage, so that the purity and quality of improved seeds are ensured.

Further, the application is that TWINKLE gene is introduced into a rice maintainer line and a sterile line, and three-line and/or two-line sterile lines with seedling stage albino leaf character are cultivated; after the hybrid F1 generation obtained by hybrid seed production is identified in the seedling stage, the plants with albino leaves are false hybrids of sterile line selfing, and are removed, so that the seed production risk and loss of three-line and/or two-line hybrid rice can be effectively reduced.

The invention also provides a method for breeding a pure-bred sterile line, which is to use the rice albino green leaf mutation gene TWINKLE as an early breeding marker, and perform false-breeding identification and elimination in a seedling stage to breed the pure-bred sterile line.

Furthermore, the method is to introduce the rice albino green leaf mutation gene TWINKLE into a three-line or two-line sterile line by means of hybridization and backcross, and to utilize the albino character of the TWINKLE gene to perform false seed identification and elimination in the seedling stage so as to breed a pure sterile line.

Further, the method comprises the steps of introducing TWINKLE genes into rice maintainer lines and sterile lines, and cultivating three-line and/or two-line sterile lines with albino leaf character in a seedling stage; after the hybrid F1 generation obtained by hybrid seed production is identified in the seedling stage, the plants with albino leaves are false hybrids of sterile line selfing, and are removed, so that the seed production risk and loss of three-line and/or two-line hybrid rice can be effectively reduced.

The invention utilizes a chemical mutagen Ethyl Methylsulfonate (EMS) to mutagenize indica rice western 1B to obtain a genetically stable albino-transgenic green leaf mutant named twinkle. Wild Type (WT) plants are green throughout the growth phase, except for the maturity phase, and compared to wild type, mutant twinkle leaves whiten during the seedling phase and begin to turn green partially during the 4-5 leaf phase in the field, the trait persisting until maturity. Through genetic analysis and gene location, it is determined that the albino transgenic green leaf of rice is controlled by TWINKLE recessive gene, single base substitution occurs to 13 th exon of LOC_Os06g45980 gene, so that coded amino acid Gly is changed into Asp, the CDS sequence is shown as SEQ ID No.1, and the amino acid sequence of coded protein is shown as SEQ ID No. 2. Further amplified genomic fragments (comprising the upstream of the TWINKLE promoter, the coding sequence, downstream of the terminator) from wild-type gDNA were transformed into TWINKLE mutants for complementation verification, and in transgenic plants the mutant phenotype was completely restored. Further, it was confirmed that albino green leaf mutation of rice was controlled by the mutant gene TWINKLE. Therefore, the albino green leaf mutant gene TWINKLE of rice can be used for rice breeding.

The invention has the beneficial effects that: the invention discloses a rice albino-transgenic green leaf mutant gene TWINKLE, wherein the CDS sequence is shown as SEQ ID No.1, the amino acid sequence of the coding protein is shown as SEQ ID No.2, the phenotype is albino in the seedling stage, and when the leaf stage is 4-5 in the field, the leaf starts to turn green partially, and the property is continued until the mature stage. The gene can be used for breeding new varieties of rice and identifying the purity of seeds, and provides a new gene and a new method for breeding pure sterile lines.

Drawings

FIG. 1 is a plot of the results of a positioning analysis and complementary verification of TWINKLE;

a: TWINKLE positioning analysis results;

B-D: sequencing a complementary positive transgenic plant;

E-G: phenotypic observations of wild type (E), mutant twinkle (F), com-twinkle plants (G).

FIG. 2 shows the results of pigment content analysis.

FIG. 3 shows the results of related gene expression analysis;

the expression condition of TWINKLE gene;

and B, C, chloroplast development, photosynthesis and pigment metabolism related gene expression.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention. It should be noted that, the experimental materials in the examples of the present invention are all commercially available, and the experimental methods without specific conditions are usually performed according to conventional conditions or according to the recommended conditions of the experimental material manufacturer.

EXAMPLE 1 obtaining and morphological observations of albino-transgenic Green leaf mutants of Rice

A genetic stable albino transgenic green leaf mutant named as twinkle is obtained by mutagenesis of indica rice western 1B with a chemical mutagen Ethyl Methylsulfonate (EMS). Wild Type (WT) plants are green throughout the growth phase, except for the maturity phase, and compared to wild type, mutant twinkle leaves whiten during the seedling phase and begin to turn green partially during the 4-5 leaf phase in the field, the trait persisting until maturity.

Example 2TWINKLE Gene localization

Taking a mutant TWINKLE as a female parent and Hui No. 10 as a male parent, hybridizing to obtain an F1 generation, selfing the F1 generation to obtain an F2 generation, separating the appearance characters of the F2 generation, and verifying that the normal phenotype and the mutation phenotype accord with a separation ratio of 3:1 by using a chi-square, so that the mutation characters are controlled by a pair of recessive nuclear genes and named TWINKLE.

Gene localization was performed using mutant plants in the F2 generation, and finally the TWINKLE gene was located between chromosome 6 primer markers Chr6-25.40 and Chr6-27.90 at a physical distance of 2.50M (FIG. 1A).

The whole genome sequencing of wild type and mutant TWINKLE leaf samples is carried out, the comparison is carried out through software IGV_2.13.2, finally single base substitution (G to A) of 13 th exon of LOC_Os06G45980 gene is found in the interval of Chr6-25.40 and Chr6-27.90, so that coded amino acid Gly to Asp (figure 1A) is caused, and the original proof that TWINKLE gene is a mutant gene of LOC_Os06G45980 is shown in SEQ ID No.1, and the CDS sequence of the coded protein is shown in SEQ ID No. 2.

EXAMPLE 3 complementation verification of the twinkle mutant

Amplifying genome fragment (comprising upstream of TWINKLE promoter, downstream of coding sequence and terminator) from wild gDNA, recombining the fragment into pCAMBIA1301 vector, extracting plasmid after positive cloning, transforming recombinant vector and pCAMBIA1301 empty vector into TWINKLE mutant by agrobacterium transformation method to obtain Com-TWINKLE and Ve-TWINKLE positive transgenic plant respectively.

After obtaining the transgenic plant, carrying out GUS staining preliminary identification on the transgenic plant by using a GUS reporter gene on a pCAMBIA1301 vector, then extracting DNA of the preliminary identified transgenic plant for amplification and sequencing analysis, and identifying positive transgenic plant Com-twinkle (figure 1) by comparing the sequencing result: (1) determining whether the wild-type gene is transferred, and performing reverse sequencing due to poor forward sequencing effect (FIG. 1B); (2) determining whether the wild-type vector is ligated (FIG. 1C); (3) it was determined whether the infected mutant calli were reverse sequenced (FIG. 1D).

Phenotype observation of the positive transgenic plants revealed that the Com-TWINKLE plants restored the overall plant height and leaf phenotype (FIGS. 1E-G) compared with the mutant phenotype, further demonstrating that the TWINKLE gene was the mutant gene of the LOC_Os06G45980 gene.

Example 4 functional analysis

1. Pigment content and photosynthesis analysis

Pigment content determination is carried out on wild type and mutant leaves at the tillering stage, and the result shows that compared with the wild type, chlorophyll in green and white leaves of the mutanta. Chlorophyll b, carotenoids and total chlorophyll content were all very significantly reduced (figure 2); meanwhile, compared with a wild type, the photosynthetic data analysis shows that the net photosynthetic rate Pn and the transpiration rate Tr of the green leaf of the mutant are obviously reduced, and the net photosynthetic rate Pn, the stomatal conductance Gs and the intercellular CO of the white leaf of the mutant ₂ The concentration Ci, the transpiration rate Tr, were extremely significantly reduced (table 1). It is shown that mutation of the TWINKLE gene affects leaf pigment content and photosynthesis of plants.

TABLE 1 photosynthesis analysis results

2. Expression analysis

Extracting wild-type root, stem, leaf, sheath and spike RNA, reversely transcribing into cDNA, analyzing the expression condition of TWINKLE gene, finding that the gene is expressed in root, stem, leaf, sheath and spike, belonging to constitutive expression (figure 3A); meanwhile, the analysis of the expression of some chloroplast development-related, photosynthesis-related and pigment metabolism-related genes was carried out, and these genes were affected to different extents in the leaves of the mutants as compared with the wild type (FIGS. 3B, C).

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. The CDS sequence of the rice albino-transgenic green leaf mutant gene TWINKLE is shown as SEQ ID No.1, the amino acid sequence of the coding protein is shown as SEQ ID No.2, the phenotype is albino in the seedling stage, and when the leaf stage is 4-5 in the field, the leaf begins to turn green partially, and the character is continued until the mature stage.

2. The use of the rice albino green leaf mutant gene TWINKLE of claim 1 in rice breeding.

3. The use according to claim 2, characterized in that the rice albino green leaf mutant gene, twin kle, is used as an early breeding marker for false seed identification and elimination during the seedling stage.

4. The use according to claim 3, wherein the use is to introduce the albino green leaf mutant gene of rice, twinkme, into a sterile line of three lines or two lines by means of hybridization and backcrossing, and to perform false seed identification and elimination in seedling stage by utilizing albino trait of the twinkme gene.

5. The use according to claim 4, wherein the use is to introduce the twin gene into rice maintainer line and sterile line, and to cultivate three-line and/or two-line sterile line with seedling albino leaf trait; after the hybrid F1 generation obtained by hybrid seed production is identified in the seedling stage, the plants with albino leaves are false hybrids of sterile line selfing, and are removed, so that the seed production risk and loss of three-line and/or two-line hybrid rice can be effectively reduced.

6. A method for breeding a pure sterile line is characterized in that the rice albino green leaf mutant gene TWINKLE of claim 1 is used as an early breeding marker, false seed identification and elimination are carried out in a seedling stage, and pure sterile line is bred.

7. The method for breeding pure-bred sterile line according to claim 6, characterized in that the method is to introduce the rice albino green leaf mutation gene TWINKLE of claim 1 into a sterile line of three lines or two lines by means of hybridization and backcross, and to utilize the albino character of the TWINKLE gene to perform false-breeding identification and elimination in seedling stage, so as to breed pure-bred sterile line.

8. The method for breeding a pure sterile line according to claim 7, wherein the method is to introduce the twin gene into a rice maintainer line and a sterile line, and cultivate a three-line and/or two-line sterile line with seedling albino leaf trait; after the hybrid F1 generation obtained by hybrid seed production is identified in the seedling stage, the plants with albino leaves are false hybrids of sterile line selfing, and are removed, so that the seed production risk and loss of three-line and/or two-line hybrid rice can be effectively reduced.