CN112662680B - Tomato yellow-green-turning gene SlRHBDD2 and application thereof - Google Patents

Abstract

The invention discloses a cDNA sequence of a tomato yellow-green-turning gene SlRHBDD2 coding region, SEQ ID No.1 and an amino acid sequence of a coding protein thereof SEQ ID No. 2. The gene SlRHBDD2 for controlling the color of tomato leaves is cloned from early tomato leaf color yellowing mutant 'cely 403' by a map-based cloning technology, and a function complementation test proves that the SlRHBDD2 is a gene for controlling the color of tomato leaves to be yellow and green. The leaf color controlled by the gene changes from yellow to green, the cotyledon and the heart leaf are in yellow green at the early stage, the chlorophyll content is reduced, and the net photosynthetic rate is reduced; the leaves in the later stage are turned into green, the chlorophyll content and the net photosynthetic rate are recovered, so the method can be used as a seedling stage marker for early selection of filial generation in the breeding process of new tomato varieties, such as the seedling stage identification of a nuclear male sterile line, can also be applied to the aspects of seed production, purity identification and the like, and has good application prospect.

Description

Tomato yellow-green-turning gene SlRHBDD2 and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a tomato yellow-green-turning gene SlRHBDD2 and application thereof.

Background

Tomatoes (Solanum lycopersicum L.) belong to the genus of tomatoes in the family of Solanaceae, are one of the most popular vegetables cultivated all over the world, and play an important role in vegetable production and consumption in China. Leaf photosynthesis efficiency is a key factor in determining tomato yield and quality. The high efficiency of photosynthesis depends on the synthesis of chlorophyll and the normal development of chloroplast, so that the research on the chlorophyll metabolism and the chloroplast development process has profound significance.

Leaf color is an important agronomic trait, and under normal conditions, plant leaves are green. When the plant is affected by external environment or self inheritance, the development defect of chloroplast and the synthesis of chlorophyll are blocked, and leaf color mutation, such as albino, etiolation, light green, stripe and other phenotypes, can be generated. The leaf color mutant is an ideal material for researching the chloroplast structure and development, the chlorophyll synthesis and degradation, the photomorphogenetic establishment, the photosynthesis mechanism, the gene expression regulation and the like, and meanwhile, some leaf color mutants have excellent properties of reduced photosynthesis 'noon break', improved photosynthetic efficiency and the like, and can be used for genetic breeding. In addition, the variation of the leaf color has the characteristics of easy visual identification, no environmental influence and the like, the leaf color mutant can be used as a seedling stage marker character for rapidly identifying the purity of the variety, simplifying the fine variety breeding steps, and has good application prospect in the breeding of the tomato variety, such as the identification of a tomato male sterile plant. Tomato has obvious heterosis, the male sterile line is an important way for utilizing the heterosis, but most of cytoplasmic male sterile lines of the tomato reported at present can not find a maintainer line, and about 50 percent of fertile plants are required to be removed for preparing a nuclear male sterile dual-purpose line, so that time and labor are wasted. The rapid and effective identification of sterile plants is the key for improving productivity, and the method for using the leaf color mutation in the seedling stage as a gene marker is an effective method. However, most of the currently cloned leaf color mutant control genes are limited to model plants such as arabidopsis thaliana or rice, and for example, a plurality of genes for controlling chlorophyll synthesis and chloroplast development are separated and identified from pigment-deficient leaf color mutants. Such as the chlorophyllin-a oxidase gene of Arabidopsis thaliana (Oster et al 2000), the chloroplast ribosomes small subunit 17 gene (Schultes et al 2000), the magnesium-chelatase H subunit gene of rice, the RSA gene (Jung et al 2003; Su et al 2012), and the like.

At present, only a few genes of tomato leaf color controlling mutants are reported, especially the gradual turning green mutant of leaf color. The gradually turning green mutant refers to a mutant with chlorophyll content reduced in early leaf development stage and basically normal in mature stage (Archer and Bonnett, 1987). Different from albino and etiolated mutants, the mutation is non-lethal, does not influence the flowering result and the final yield of plants, is an ideal material serving as a morphological marker character in actual breeding, and has good application prospects in aspects of cross breeding, new variety breeding, seed purity identification and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a novel tomato yellow-green-turning gene SlRHBDD2 which has a tomato leaf color control function, and can be used for creating a mutant plant with a yellow-green-turning marker for identifying a male sterile plant or a plant with excellent characters in a seedling stage.

The invention firstly provides a cDNA coding sequence of a separated tomato yellow-green-turning gene SlRHBDD 2. The tomato leaf color control gene SlRHBDD2 has the CDS region nucleotide sequence shown in SEQ ID No. 1.

Further, the gene nucleotide sequence is a mutant or allele nucleotide sequence produced by adding, replacing or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID No. 1.

The invention also provides an amino acid sequence coded by the gene as shown in SEQ ID No. 2.

Furthermore, the protein amino acid sequence coded by the tomato yellow-green leaf transfer gene RHBDD2 is an amino acid sequence or a derivative which is generated by adding, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID No.2 and has the same function.

The invention also provides an application of the tomato yellow-green leaf transfer gene RHBDD2 in creating yellow-green leaf transfer germplasm, which specifically comprises the following steps: and reducing the expression level of the SlRHBDD2 gene in the tomato germplasm by using RNAi, Crispr/Cas9 gene knockout or silencing and other methods to create and obtain the yellow and green-transformed leaf germplasm.

Furthermore, the invention provides the application of the DNA molecule, namely the mutant gene, in identifying whether a plant to be detected is a yellow-to-green plant or not in a seedling stage.

The gene SlRHBDD2 for controlling the tomato leaf color is cloned from an early tomato leaf color yellowing mutant "cel y 403" by a map-based cloning technology, and a function complementation test proves that SlRHBDD2 is a gene for controlling the tomato leaf color yellowing to green. Morphological and cytological observation shows that the cloned gene has the functions of regulating and controlling chloroplast development, influencing chloroplast number and further controlling leaf color. The yellowing-to-green leaf color mutant obtained by the mutation of the gene coding protein amino acid has the advantages of cotyledon yellowing, heart leaf yellow-green, gradual green turning of mature leaves and normal restoration of leaf color in the middle and later growth stages, so that the gene can be used as a seedling stage selection marker to be applied to progeny screening, hybrid purity identification and the like.

The invention has the advantages that: the gene can be used for researching and controlling the functions of related genes of chlorophyll metabolism or chloroplast development. The characteristic of cotyledon yellowing green-turning created by the gene can be used as a seedling stage marker for tomato variety breeding, particularly for breeding of a male sterile line, and the identification and screening steps of a target single plant are simplified. Especially the characteristic that the leaves turn green in the later period, does not influence the normal fruiting and seed collection of the tomatoes, and is an excellent material for tomato breeding.

Drawings

FIG. 1 shows the leaf color phenotype of tomato yellow-to-green leaf color mutant cel y403, where 403 is the mutant and 404 is the wild type.

FIG. 2 shows the growth characteristics of tomato mutant cely403 compared with wild type in different growth periods, such as plant height (A), leaf length (B), leaf width (C) and the like.

FIG. 3 is a photo-chrome content analysis of tomato mutant cel y403 in different growth periods from wild type, wherein A is chlorophyll a, B is chlorophyll B, C is carotenoid content, and D is total chlorophyll content.

Fig. 4 is a comparison of photosynthetic properties of tomato mutant cely403 with wild type, wherein: a is net photosynthetic rate (P)_n) And B is the conductance of the stoma (G)₂) C is intercellular CO₂Concentration (C)_i) D is the transpiration rate (T)_r)。

FIG. 5 shows the seedling morphology comparison of tomato wild type (A), mutant cely403(B) and SlRhbdd2 transgenic positive (C) plants.

FIG. 6 shows the expression characteristics of the tomato SlRhbd 2 gene in different tissues and organs.

Detailed Description

The invention is further described below with reference to the following figures and examples.

The experimental procedures used in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 obtaining and characterization of tomato yellow-to-green leaf color mutant "cel y403

1. Obtaining of tomato yellow-green leaf color changing mutant' cel y403

The tomato yellowing green leaf color changing mutant is derived from a high-generation inbred line T12404 of a variety LA3320 of a genetic resource pool (TGRC) of a tomato, a natural leaf color mutation plant is found in the field planting process, cotyledons of the mutant are yellowed, heart leaves are yellow green in the seedling stage, leaves are gradually changed to green in the later stage, and the normal state is recovered. The mutant strain is obtained by selfing and reserving seeds of the mutant plants, the mutant strain is bred and selected by multi-generation selfing, the mutant is named as 'cel 403' (Cotyledon and Early Leaf yellow), and the mutant is preserved and provided by a tomato research group of the institute of agriculture and biotechnology of Zhejiang university.

2. Character identification of tomato yellow-green leaf color changing mutant cely403

After sowing mutant cely403 and wild type 404 seeds, culturing in an environment with a temperature cycle of 25 ℃/20 ℃ and a photoperiod of 16h/8h, and after germination, yellowing of mutant cotyledons can be seen, true leaves are yellow-green, and wild type cotyledons and true leaves are both normal green (figure 1). As the plants grew, the mutant mature leaves turned green gradually, and only the heart leaves remained yellow-green. Until about 50 days of growth, the mutant gradually returns to normal, the heart leaves turn green, and thereafter, the heart leaves grown by the mutant are all green, and the mutant flowers and fruits normally. The early growth dynamics of the mutant and the wild type are observed at different periods, and the weak growth vigor and the reduced plant height of the mutant are found (figure 2A); meanwhile, the leaf length and leaf width of the mutant leaf were both smaller than those of the wild type (FIGS. 2B and 2C).

Example 2 comparison of chlorophyll content of tomato yellow-to-green leaf color mutant with photosynthetic Properties

At different times after sowing, randomly selecting 12 plants of each single plant with consistent growth vigor from the mutant and the wild type respectively, selecting a second true leaf which is fully unfolded under a growth point, cutting the second true leaf into 2-3 cm, weighing 0.2g, soaking the second true leaf in 25mL of mixed solution of acetone and ethanol (the volume ratio of 95% acetone to absolute ethanol is 2: 1), sealing and keeping out of the sun, and performing dark culture at 26 ℃ for 24 hours. Measuring OD values of the extractive solution at 470nm, 645nm and 663nm 3 wavelengths with ultraviolet spectrophotometer, and repeating for 3 times. According to an improved Arnon calculation method of Livchtenthalter, the contents of chlorophyll a, chlorophyll b and carotenoid are respectively calculated, and the total chlorophyll content is the sum of the chlorophyll a and the chlorophyll b. The results show that the chlorophyll a and chlorophyll b contents of the mutant leaves are both significantly reduced in the early growth phase compared to the wild type, while the carotenoid contents are not significantly different (fig. 3). At 15 days after sowing, the chlorophyll a content of the wild type leaves is 1.13mg/g, and the mutant content is only 0.94 mg/g; the chlorophyll b content of the wild type is 0.53mg/g, and the content of the mutant is as low as 0.39 mg/g. The difference between the 35 d-growth mutant and the wild-type chlorophyll content is similar to 15d, and the chlorophyll a, chlorophyll b and total chlorophyll content of the mutant are obviously lower than that of the wild-type. And at the later growth stage of 55d, the chlorophyll a content of the mutant and the wild type is respectively 1.54mg/gFW and 1.57mg/gFW, the chlorophyll b content is respectively 1.36mg/gFW and 1.37mg/gFW, the carotenoid content is 0.17mg/gFW, no obvious difference exists between the two, and the mutant basically returns to the normal state (figure 3). The chlorophyll content measurement result is consistent with the leaf color phenotype.

And (3) selecting the second fully unfolded true leaves under the growing points of the mutant and the wild type at different periods after sowing, measuring the net photosynthetic rate of the second fully unfolded true leaves by using an LI-6400 portable photosynthetic instrument, and performing statistical analysis. Measuring its net photosynthetic rate (P) using an LI-6400 Portable photosynthesis apparatus_n) Air hole conductivity (G)₂) Intercellular CO₂Concentration (C)_i) Transpiration rate (T)_r) And the like. The results show thatAt 30 days, the net photosynthetic rate of the mutant is significantly reduced compared with that of the wild type, and the net photosynthetic rate of the wild type is 14.28 mu mol CO₂ m^-2s^-1The mutant has only 9.45 mu mol CO₂ m^-2s^-1. However, when grown to 50d, the mutants had returned to normal with no difference in net photosynthetic rate from the wild type (FIG. 4). The change in net photosynthetic rate is consistent with the differential change in chlorophyll content. In addition, intercellular CO of the mutant₂The concentration was significantly higher than that of the wild type, but the conductance of the pores and the transpiration rate were not significantly different from those of the wild type (FIG. 4).

Example 3 genetic analysis and Gene mapping of tomato yellow-to-green leaf color mutation traits

1. Genetic analysis of tomato yellow-to-green leaf color mutation character

F is obtained by hybridizing the mutant cel y403 with the wild type 404₁Inbred to obtain F₂Instead, utilize F₂Genetic analysis was performed on the segregating population. The results show that F₁The leaf color of the generation plant is green, and the generation plant is of a wild type phenotype; sowing F₂And totally counting 483 plant leaf colors, wherein the number of plants with normal leaf color and yellow leaf color is 378 and 105, and the chi-square test result (X)²＝2.57<X² _0.05,13.84) meets the separation ratio of 3:1, the above results are repeated three times, and the experimental results all show that the mutant character is controlled by a recessive single gene.

2. Gene location of tomato yellow-to-green leaf color mutation character

Using F₂Generating separated normal leaf color and yellow leaf color groups, randomly selecting 20 normal leaf color plants and 20 yellow leaf color plants, extracting genome DNA to respectively construct a DNA mixed pool, taking 403 and 404 as parents, and F₂BSA sequencing was performed in the etiolated yellow pool (405-Y) and the leaf color normal pool (405-G). Sequencing, aligning, annotating by ANNOVAR, detecting the marker of homozygous difference between two parents, and screening 20961 SNP loci and 3576 InDel loci in total. Then, using the parent 404 as a reference parent, respectively calculating All-index (SNP-index and InDel-index) of the two progeny pools at the homozygous difference site. To visualize the area of difference between two child poolsAnd calculating the difference value of the two child All-index: Δ (All-index) — allndex (S2) -allndex (S1). The distribution of Δ (All-index) was reflected by choosing 1Mb as the window and 1kb as the step size and calculating the average of Δ (All-index) in each window. And (4) selecting a window which is obviously different in the two filial pools in the whole genome range according to the calculated All-index and the delta (All-index) and is larger than a threshold value at a certain confidence level as a candidate interval. The results show that the difference sites are mainly concentrated on chromosome 3, only a few sites on other chromosomes have differences, and the chromosome 3 is positioned beyond the threshold region to form a primary candidate interval.

3. Candidate gene prediction and sequence alignment analysis

And (3) selecting SNP or InDel locus with obvious difference in the whole genome range, wherein All-index in a progeny green pool is less than or equal to 0.5, and selecting the locus with All-index being more than or equal to 0.8 in a progeny yellowing pool as the SNP or InDel candidate locus. And analyzing all the predicted genes in the candidate interval according to the positioning result, and combining the expression characteristics of the candidate genes to obtain a coded diamond domain-containing protein (SlRhbdd 2). It is localized in the Golgi apparatus and in the mutant the A → T mutation occurs in the 5' UTR region of the gene.

Example 4 functional complementation experiments prove that SlRhbdd2 is a target gene for controlling leaf color yellowing transcription

1. Expression vector construction

According to the full-length cDNA sequence (SEQ ID No.1) of the coding region of the SlRhbdd2 gene, a specific primer pair is synthesized: the upstream primer SlRhbdd 2F: 5' -CCGGGATCCATGAGAGGAGGAGATATAGAAAGC-3' (SEQ ID No.3) and a downstream primer SlRhbdd 2R: 5' -CGCTCTAGAGGTTAATTGCCATCACCG-3' (SEQ ID No.4) is underlined as a cleavage site). PCR amplification is carried out by using high fidelity enzyme, the PCR product is purified and then is subjected to double enzyme digestion by using BamH I and Xba I together with pCAMBIA1301 vector, and the purified product is connected for 12h at 16 ℃. The ligation product was transformed into E.coli DH 5. alpha. competent cells, and the transformed bacteria were plated with a medium containing 50 mg. multidot.L^-1Spec and 50 mg. L^-1Str screening plate, selecting plaque and shake bacteria, PCR screening positive clone of bacterial liquid, and sending to companyAfter the sequencing verification is correct, an over-expression vector is obtained, bacteria shaking is carried out, and a plasmid is extracted for later use, wherein the vector is named as pCAMBIA1301-35S, Sl Rhbdd 2.

2. Transgenic tomato acquisition and validation

The target vector is transformed into agrobacterium GV1301, and the plasmid of the recombinant agrobacterium is extracted and sequenced to prove that the recombinant agrobacterium is positive. Sl Rhbdd2 is transformed into a tomato mutant inbred line 'cel 403' by using a 'leaf disc method', meanwhile, a pCAMBIA1301 blank vector is transformed to be used as a control, 21 positive transgenic SlRhbdd2 plants are obtained through PCR and GUS detection, and the single-copy inserted homozygous overexpression transgenic tomato is identified.

3. Phenotype observation of transgenic tomato plant with SlRhbdd2 gene

Sowing and raising the seeds of the SlRhbdd2 transgenic positive tomatoes, the wild type 404 and the mutant cely403, observing after the seeds grow for 15 days under normal growth conditions, continuously observing the dynamic change of the color of the leaves of the tomatoes with different genotypes, and detecting the chlorophyll content and the photosynthetic property once every 5 days. As a result, the mutant cely403 seedling is yellow in cotyledon, the heart leaves are yellow-green at the stage 30 days before (4 leaves and 1 heart), and the normal green is gradually changed at the later stage. And the positive transgenic plant tomato leaf color does not show yellowing phenomenon in the seedling stage, and is consistent with the leaf color of a wild plant. The complementation vector was shown to be able to fully restore the phenotype of early leaf yellowing of the mutation of "cely 403" (FIG. 5).

The chlorophyll content of the leaves of the over-expression plants, the wild plants and the mutant plants is analyzed by a colorimetric method to find that: the average content of total chlorophyll of wild type leaves at 20 days after sowing is 1.78mg/g, the average content of total chlorophyll of mutant "cely 403" leaves is only 1.08mg/g, and the average content of total chlorophyll of SlRhbdd2 gene overexpression plants of the "cely 403" leaves is 1.67mg/g, and the wild type level is recovered.

Example 5 expression Properties of tomato SlRhbdd2 Gene

The expression of the SlRhbdd2 gene in each tissue and leaf of tomato at different growth stages was compared by using qRT-PCR technology, and as can be seen from fig. 6, the expression level in leaf was significantly higher than that in other organs, and the expression level in young leaf was also higher than that in mature and senescent leaf, which also confirms that it mainly plays a role in the leaf development process from one side.

The above description is only a few specific embodiments of the present invention, and it should be noted that all modifications that can be derived or suggested from the disclosure of the present invention by those skilled in the art are considered to be within the scope of the present invention.

Sequence listing

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WUXI DIMODE BIOLOGICAL SEED INDUSTRY TECHNOLOGY Co.,Ltd.

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Claims (3)

1. The application of the tomato yellow-green leaf-turning gene SlRHBDD2 in creating yellow-green leaf-turning germplasm is disclosed in the specification, wherein the nucleotide sequence of the SlRHBDD2 gene is shown in SEQ ID No. 1.

2. The application according to claim 1, characterized in that it is in particular: and reducing the expression level of the SlRHBDD2 gene in the germplasm by RNAi, Crispr/Cas9 gene knockout or knock-down method to create and obtain the etiolated and greening leaf germplasm.

3. Use according to claim 1 or 2, wherein the germplasm is tomato.

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