CN110903370B - TCP transcription factor, gene for encoding TCP transcription factor and application - Google Patents

Abstract

The invention provides application of a gene BDI1 for encoding a TCP transcription factor, and relates to the technical field of genetic engineering; the amino acid sequence of the TCP transcription factor is shown as SEQ ID NO. 1. The TCP transcription factor of the invention has the function of a first reporter gene in barley, and mRNA expression analysis shows that the TCP transcription factor is only expressed in young spikes of barley, and the expression quantity is highest from the division period of the glume protecting primordia to the division period of the external reference primordia; the gene localization information shows that the gene BDI1 encoding the TCP transcription factor is localized on the 5H chromosome of barley; mutant experiments and gene silencing prove that the gene BDI1 is deleted or silenced, so that the middle and lower small ears of the barley ears can be converted into branched or double-grain small ears, and the number of ears is increased. The gene BDI1 can be used as a target gene for knocking out and is used for regulating and controlling the branch number and/or the seed number of the ears of the barley, so that the crop yield is increased.

Description

A TCP transcription factor, a gene encoding a TCP transcription factor and its application

Technical field

The present invention relates to the technical field of genetic engineering, and in particular to a TCP transcription factor, a gene encoding the TCP transcription factor and its application.

Background technique

Barley (scientific name: Hordeum vulgare L.) is an annual herbaceous plant of the Gramineae family and Hordeum genus. The characteristic of barley ears is that there are three parallel spikelets on each spike node, each spikelet only bears one small flower, and each spikelet can bear at most one grain. Therefore, the spikelets of barley are of limited growth type.

During the development of young barley ears, the spikelets at the base begin to differentiate first. As the cob grows, the spikelets at the upper part gradually form. One spikelet primordium of barley only differentiates into one floret and stops growing. At present, the genes that control the limited growth of barley spikelets are not yet clear, and they cannot effectively regulate the number of spike branches or seeds in barley or closely related species.

Contents of the invention

The purpose of the present invention is to provide a TCP transcription factor, a gene encoding a TCP transcription factor, and an application. The gene encoding the TCP transcription factor can be introduced or knocked out as a target gene for regulating panicle division of barley and its closely related species. number of branches and/or seeds per ear, thereby increasing crop yield.

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

The present invention provides a TCP transcription factor. The amino acid sequence of the TCP transcription factor is shown in SEQ ID NO. 1.

The present invention also provides a gene BDI1 encoding the TCP transcription factor described in the above scheme. The nucleotide sequence of the gene BDI1 is shown in SEQ ID NO. 2.

The present invention also provides the application of the TCP transcription factor or the gene BDI1 described in the above scheme in the breeding of barley and its related species.

Preferably, the application includes regulating the number of panicle branches and/or the number of panicle seeds of barley and its closely related species.

Beneficial effects of the present invention: The present invention provides a TCP transcription factor, the amino acid sequence of which is shown in SEQ ID NO. 1. The TCP transcription factor of the present invention is reported in barley for the first time. The mRNA expression analysis shows that the TCP transcription factor is only expressed in the young ears of barley, and the expression level is the highest from the glume primordium differentiation stage to the lemma primordium differentiation stage; gene positioning information It was shown that the gene BDI1 encoding this TCP transcription factor is located on the 5H chromosome of barley; mutant experiments have shown that the deletion of the gene BDI1 can transform the middle and lower spikelets of barley ears into branched or double-grained spikelets, increasing the number of grains per spike. The gene BDI1 can be introduced or knocked out as a target gene to regulate the number of panicle branches and/or panicle seeds in barley and closely related species, thereby increasing crop yield.

Description of the drawings

Figure 1 The panicle of wild type 08-49;

Figure 2. Spike of mutant bdi1;

Figure 3 Electron microscope photo of wild type 08-49 young panicle;

Figure 4 Electron micrograph of young panicles of mutant bdi1;

Figure 5 Expression of HORVU5Hr1G061270 gene in different tissues of wild type 08-49.

Detailed ways

The present invention also provides a TCP transcription factor, the amino acid sequence of which is shown in SEQ ID NO.1.

The present invention also provides a gene BDI1 encoding the TCP transcription factor described in the above scheme; the nucleotide sequence of the gene BDI1 is shown in SEQ ID NO.2.

The gene BDI1 of the present invention can be used as a target gene to construct a transvector; in the process of using the gene BDI1 of the present invention as a target gene to construct a transvector, there is no special restriction on the promoter, and conventional promoters in this field can be used; specifically, the present invention During implementation, the promoter preferably includes cauliflower mosaic virus (CAMV) 35S promoter or Ubiquitin promoter. The present invention has no special restrictions on the method of cell transformation, and conventional methods in the field can be used; during the specific implementation of the present invention, the transformation method includes Agrobacterium transformation method or gene gun method. The present invention has no special limitations on the method for identifying transformed cells. During the specific implementation in this field, the method for identifying transformed cells includes selective markers, enzymes resistant to antibiotics, and the use of color changes (such as B-glucuronide). Enzymes that identify compounds such as acid glycosidase (GUS) or luminescence (e.g., luciferase), or use label-free selection.

The present invention also provides the application of the gene BDI1 or the protein or the recombinant vector described in the above scheme in the breeding of barley and its closely related species; the application preferably includes regulating the number of panicle branches of barley and its related species. and/or number of seeds per ear.

The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the present invention.

Example 1 BDI1 gene is involved in the regulation of limited growth of barley spikelets

The barley inflorescence morphology mutant bdi1 (branched and indeterminate spikelet 1) is a panicle branch mutant selected from the barley mutant library of barley line 08-49 mutated with ⁶⁰ Co-γ rays. The middle and lower parts of the panicle of the mutant bdi1 have multiple branches of varying lengths. The upper part of the panicle is similar to that of wild-type six-row barley 08-49. The leaves and plant height of the mutant are the same as those of wild-type 08-49. After harvesting from a single plant, After two consecutive years of planting, the mutant's traits were stable. Scanning electron microscope observation showed that (Figure 1) the wild-type 08-49 young spikelets were arranged neatly, the florets had differentiated, and the growth points of the spikelet primordia no longer grew and were pushed to the side by the florets, making them almost invisible. In the mutant bdi1 (Figure 2), the spikelets are not arranged neatly and the florets have differentiated, but the growth points of the spikelet primordia continue to grow. Some lower spikelet primordia even differentiate into two grains or grow into one. branch. The electron microscope observation results are shown in Figures 3 and 4. Figure 3 is an electron microscope photo of the wild type 08-49 young panicle; Figure 4 is an electron microscopy photo of the mutant bdi1 young panicle. The results show that the BDI1 gene is involved in the regulation of limited growth of barley spikelets and is a key gene for the morphological structure of barley inflorescences.

Example 2 Genetic studies show that BDI1 is a new barley inflorescence morphological structure gene

Mutant bdi1 was crossed with wild type 08-49. F ₁ was the normal six-sided panicle type, and F ₂ isolated two panicle types: six-sided panicle type and mutant panicle type. The ratio of the two phenotypes was consistent with 3:1 (χ ² _3:1 =0.3, P>0.05), indicating that the panicle mutation phenotype is controlled by a recessive single gene.

Mutant bdi1 and mutant prbs/vrs4 (provided by Chinese Academy of Agricultural Sciences, see [Shang Y, Yang F, Schulman AH, Zhu JH, Jia Y, Wang JM, Zhang XQ, Jia QJ, Hua W, Yang JM, Li CD.GeneDeletion in Barley Mediated by LTR-retrotransposon BARE.Scientific Reports, 2017, 7: 43766]) was crossed and tested for allelism, and F ₁ restored the two-row panicle type (the wild-type Pudamai No. 2 of the mutant prbs is the second Ribbed barley), indicating that the mutant bdi1 spike mutation gene is not the same allele as vrs4, and is a new inflorescence morphology mutation gene. F ₂ isolated normal spike types (two-sided and six-sided) and mutant spike types. After testing, the ratio of the two phenotypes was consistent with 9:7 (χ ² _9:7 = 0.05, P>0.05), indicating that the inflorescence morphology mutation gene bdi1 and vrs4 have complementary effects. Two-rowed branch progeny were isolated from F ₂ (the upper part of the mutant panicle is similar to two-rowed barley), showing that the panicle mutant gene bdi1 has different functions from vrs4 and does not inhibit the expression of VRS1.

Example 3 Cloning of BDI1 gene

In order to map-site clone the mutant gene bdi1, two F ₂ populations (containing 187 and 226 individual plants) were constructed by crossing the mutant bdi1 with Morex and Bowman respectively, and the development of heterozygous individuals in the Morex/mutant bdi1 F ₂ population. _F3 group. (Morex and Bowman are two barley varieties used for international barley genome sequencing. Their genome sequences have been published and are publicly available)

Using the BSA method, first use the mutant individuals in the Bowman/mutant bdi1F ₂ population to construct a mixed pool, screen the linked primers in the mixed pool, and then identify the two F ₂ populations. Preliminary results show that the panicle mutant gene bdi1 is located between the 5H chromosome markers Bmag0337 and CBIC178, and the distances from the two markers are 0.8cM and 0.4cM respectively. The panicle mutant gene bdi1 co-segregates with marker CBIC175.

To finely map the BDI1 gene, we resequenced the genome of wild-type barley 08-49. Based on the alignment differences between the genome sequence of wild-type 08-49 and the Morex genome sequence, the target gene segment (marker CBIC175 is located in the assembled Morex Genome 51.5972cM) designed a large number of Indel markers. Using newly developed molecular markers, genotype analysis was performed on 1079 mutant individual plants in the F ₂ and F ₃ populations, and the map was encrypted.

Finally, the panicle mutant gene bdi1 was located between markers CBIC361 and CBIC412, and the distances from the two markers were 0.55cM and 0.09cM respectively. Transcriptome sequencing of the mutant bdi1 and wild-type 08-49 spikelets showed that a gene, HORVU5Hr1G061270, was found to be significantly differentially expressed in the target gene segment. The gene-specific primer CBIC355 did not amplify the product in the mutant bdi1. This gene is deleted in the mutant bdi1.

Example 4 Expression analysis confirms that HORVU5Hr1G061270 is the BDI1 gene

The expression of HORVU5Hr1G061270 gene was examined in different tissues of wild type 08-49, shoots, leaves, roots and spikelets.

The results showed that the HORVU5Hr1G061270 gene was only expressed in young panicles (see Figure 5), and the expression pattern of this gene was different in different developmental stages of young panicles, with the highest expression level in the glume primordium differentiation stage to the lemma primordium differentiation stage. Therefore, it was further determined that HORVU5Hr1G061270 is the BDI1 gene.

Example 5 BDI1 encodes a plant-specific TCP gene family transcription factor

Analysis of HORVU5Hr1G061270 through the NCBI website showed that the gene is a TCP gene family transcription factor. The full length of the BDI1 gene cDNA is 1435bp, with only one exon, the coding region length is 822bp, and the length of the encoded protein is 273 amino acids. The most similar homologous genes of BDI1 are rice REP1 gene, maize BAD1 gene, barley INT-C gene, maize TB1 gene and Arabidopsis BRC1 gene.

Through the full-length analysis of the BDI1 gene in 14 wild barley and 68 barley germplasms from around the world, a total of 4 haplotypes were examined, of which haplotype 1 and haplotype 2 are the two main types. In wild barley and Both were detected in common barley, Morex and Bowman belong to haplotype 1, while the mutant wild type 08-49 belongs to haplotype 2; haplotype 3 was only detected in 2 common barley, and haplotype 4 was detected. Detected in 1 serving of wild barley. This result shows that the natural mutation rate of the BDI1 gene is very low.

The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

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Claims (1)

1. Application of a gene BDI1 encoding a TCP transcription factor in barley breeding. The amino acid sequence encoded by the gene BDI1 is as shown in SEQ ID NO.1; the nucleotide sequence of the gene BDI1 is as shown in SEQ ID NO.2 As shown; the application includes regulating the number of spikes in barley.