Soybean GmHCF244 gene as well as encoding protein and application thereof

Abstract

The invention discloses a soybean GmHCF244 gene as well as an encoding protein and application thereof, and relates to the field of soybean gene engineering. The CDS sequence of the soybean GmHCF244 gene is as shown in SEQ ID NO: 1 in a sequence table. According to the application of the protein coded by the soybean GmHCF244 gene in the activity of the two reaction centers of the photosystem, the protein coded by the soybean GmHCF244 gene interacts with GmOHP1 and GmOHP2 to form a protein complex, and the activity of the two reaction centers of the photosystem is jointly regulated and controlled. The invention provides a GmHCF244 gene which is separated from soybeans and is used for coding high chlorophyll fluorescent 244 protein, and the protein coded by the gene can interact with GmOHP1 and GmOHP2 to form a protein complex which jointly influences the activity of two reaction centers of a light system.

Description

Technical Field

[0001] The invention relates to the field of soybean genetic engineering, and in particular to a soybean GmHCF244 gene and its encoded protein and application. Background Art

[0002] Photosystem II (PS II) is one of the multi-subunit protein complexes in the photosynthetic electron transport chain. It is located in the thylakoid membrane of photosynthetically active organisms and is essential for the initiation of photosynthesis and the promotion of electron transport.

[0003] The psbA gene encodes D1 protein, and the regulation of D1 protein synthesis is important for the correct biogenesis of PS II during chloroplast development and the maintenance of photosystem function. In higher plants, two key factors regulating psbA mRNA translation have been reported, HCF173 and HCF244. Summary of the invention

[0004] The invention discloses a soybean GmHCF244 gene and its encoded protein and application.

[0005] The CDS sequence of the soybean GmHCF244 gene of the present invention is shown as SEQ ID NO: 1 in the sequence listing.

[0006] The amino acid sequence of the protein encoded by the soybean GmHCF244 gene of the present invention is shown in SEQ ID NO:2.

[0007] The nucleotide sequence of the soybean GmHCF244 gene of the present invention is shown as SEQ ID NO: 3 in the sequence listing.

[0008] The application of the protein encoded by the soybean GmHCF244 gene in the activity of the photosystem II reaction center of the present invention is to utilize the protein encoded by the soybean GmHCF244 gene to interact with GmOHP1 and GmOHP2 to form a protein complex to jointly regulate the activity of the photosystem II reaction center.

[0009] The present invention provides a GmHCF244 gene encoding a high chlorophyll fluorescence 244 protein isolated from soybean, wherein the protein encoded by the gene can interact with GmOHP1 and GmOHP2 to form a protein complex, and jointly affect the activity of the reaction center of photosystem 2. The GmHCF244 gene of the present invention can be used to further explore how they affect the correct biogenesis and function of photosystem 2 during the development of soybean chloroplasts, and understand its function in the initiation of photosynthesis and the electron transfer process. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The phenotypes of the wild type and mutants on the 9th, 10th, 11th and 12th days after germination in Example 2;

[0011] Figure 2 Comparison of chloroplast morphology between the wild type and the Gmhcf244 mutant in Example 2;

[0012] Figure 3 Comparison of chlorophyll content between the wild type and the Gmhcf244 mutant in Example 2;

[0013] Figure 4 Comparison of chlorophyll fluorescence parameters between the wild type and the Gmhcf244 mutant after 0 to 96 hours of high light treatment in Example 2;

[0014] Figure 5 The trypan blue staining results of the wild type and Gmhcf244 mutant in Example 2;

[0015] Figure 6 The results of H2DCF fluorescent probe staining of wild type and Gmhcf244 mutant in Example 2;

[0016] Figure 7 The subcellular localization results of GmHCF244 protein in Example 3;

[0017] Figure 8 The interaction between GmHCF244 protein and GmOHP1 and GmOHP2 proteins in Example 3;

[0018] Fig. 9 This is to detect the distribution of core subunits of each protein complex on the chloroplast thylakoid membrane of the wild type and the Gmhcf244 mutant in Example 3. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0021] Specific implementation method 1: In this implementation method, the CDS sequence of soybean GmHCF244 gene is shown in SEQ ID NO: 1 in the sequence list.

[0022] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that the nucleotide sequence of soybean GmHCF244 gene is shown in SEQ ID NO: 3 in the sequence list. Other aspects are the same as those of specific embodiment 1.

[0023] Specific implementation method three: As described in specific implementation method one, the amino acid sequence of the protein encoded by the soybean GmHCF244 gene is shown in SEQ ID NO:2.

[0024] Specific embodiment 4: Application of the protein encoded by the soybean GmHCF244 gene as described in specific embodiment 1 in the activity of the reaction center of photosystem II.

[0025] Specific embodiment 5: This embodiment is different from specific embodiment 4 in that the application method of the protein encoded by soybean GmHCF244 gene is to utilize the protein encoded by soybean GmHCF244 gene to interact with GmOHP1 and GmOHP2 to form a protein complex to jointly regulate the activity of the second reaction center of the photosystem.

[0026] Example 1: Isolation and structural analysis of the GmHCF244 gene in soybean

[0027] (1) Gene isolation

[0028] mRNA was extracted from young leaves of wild soybean variety Henong 85, and used as a template to synthesize the first strand of cDNA using oligo(dT)17 as a primer. Then, the first strand of cDNA was used as a template to perform PCR amplification using upstream primer F (5'-GGGTTCGAAATCGATGGATCCATGAAGATGGCGCTGCTC-3') and downstream primer R (5'-GTCCTAGGCTACGTAGGATCCTCAGAAGATAATATCTGTCTGCTTT-3'), and a 1191 bp cDNA fragment was obtained, which was the CDS sequence of the GmHCF244 gene and cloned into the myc-pBA vector and named myc-pBA-GmHCF244.

[0029] The CDS sequence of the GmHCF244 gene is shown in SEQ ID NO: 1 in the sequence listing, which is 1191 bp in total. It encodes a protein of 397 amino acids, and its amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing.

[0030] (2) Gene structure analysis

[0031] The young leaves of soybean wild-type variety Henong 85 were used as materials to extract DNA, which was used as a template to amplify the GmHCF244 genomic fragment. The DNA sequence of the GmHCF244 gene is shown in SEQ ID As shown in NO:3, it is 5033bp in total, containing 5 introns and 6 exons, among which exon 1 is located at 1-390bp, exon 2 is located at 456-554bp, exon 3 is located at 634-921bp, exon 4 is located at 998-1093bp, exon 5 is located at 1169-1309bp, and exon 6 is located at 4424-4600bp; intron 1 is located at 391-455bp, intron 2 is located at 555-633bp, intron 3 is located at 922-997bp, intron 4 is located at 1094-1168bp, and intron 5 is located at 1310-4423bp.

[0032] Example 2: Soybean GmHCF244 gene mutation causes soybean yellowing and death

[0033] Through γ-ray mutagenesis of Henong 85, a related mutant library was obtained, from which a yellow lethal plant was screened. After that, the wild-type plants were self-pollinated for multiple generations, and the isolated strains were selected for further experiments to purify the mutant background. The wild-type plants after multiple generations of self-pollination were hybridized with the distant variety Hefeng 55 for multiple times to screen out the F2 plants that can isolate the yellow lethal mutant phenotype. The ratio of wild-type phenotype plants to yellow lethal mutant phenotype plants in the F2 generation after hybridization met the segregation ratio of 3:1 by the chi-square test, so it is believed that a single recessive nuclear gene controls the yellow lethal phenotype. Mutants and wild-type plants were further collected for whole genome sequencing and mapping verification, confirming that the GmHCF244 gene was a candidate gene, and the yellow lethal mutant was named Gmhcf244 mutant.

[0034] The phenotypes of wild-type Henong 85 and Gmhcf244 mutant were compared. The whole-plant phenotypes of wild-type and mutant were recorded on the 9th, 10th, 11th and 12th days after seed germination. Figure 1 The phenotypes of wild type and mutants on the 9th, 10th, 11th and 12th days after germination (e.g. Figure 1 , bar = 2cm). The true leaf samples of wild-type Henong 85 and Gmhcf244 mutant were taken 14 days after seed planting, and the differences in chloroplast morphology between mutant and wild-type were observed by transmission electron microscopy. The results showed that compared with the wild-type, the chloroplast thylakoid membrane of Gmhcf244 mutant was reduced and the boundary was blurred, and the number of osmophilic granules increased; among them, Figure 2 Comparison of chloroplast morphology between wild type and Gmhcf244 mutant (e.g. Figure 2, bar = 500nm). By comparing the chlorophyll content of the wild type and the Gmhcf244 mutant at 0 hours and 24 hours of high light treatment, it was shown that even under weak light, the total chlorophyll content, chlorophyll A content, chlorophyll B content and carotenoid content of the wild type were significantly higher than those of the mutant. After 24 hours of high light treatment, this difference was more obvious. At the same time, compared with before high light treatment, the total chlorophyll content, chlorophyll A content, chlorophyll B content and carotenoid content of the wild type were significantly increased compared with those before treatment, while the mutant was the opposite, showing a significant downward trend. Figure 3 Comparison of chlorophyll content between wild type and Gmhcf244 mutant ( Figure 3 In addition, the chlorophyll fluorescence parameters of the wild type and Gmhcf244 mutant were detected after 0 to 96 hours of high light treatment. The changes in Fv' / Fm', PiPS2, ETR and NPQ values ​​all reflected that the mutant's photosystem II was severely damaged. Figure 4 Comparison of chlorophyll fluorescence parameters between wild type and Gmhcf244 mutant after 0 to 96 hours of high light treatment (e.g. Figure 4 ). Trypan blue staining results showed that there was a large amount of cell death in the leaves of the Gmhcf244 mutant compared with the wild type, among which, Figure 5 Trypan blue staining results of wild type and Gmhcf244 mutant (eg Figure 5 ). The results of H2DCF fluorescent probe staining showed that a large amount of ROS accumulated in the leaves of the Gmhcf244 mutant compared with the wild type. Figure 6 The results of H2DCF fluorescent probe staining of wild type and Gmhcf244 mutant (such as Figure 6 ).

[0035] Example 3: Soybean GmHCF244 interacts with GmOHP1 and GmOHP2 and affects the activity of PS II reaction center

[0036] Subcellular localization results showed that the protein encoded by soybean GmHCF244 gene was localized in chloroplasts. Figure 7 The subcellular localization results of GmHCF244 protein (such as Figure 7 ). According to the luciferase complementation experiment, GmHCF244 protein can interact with GmOHP2 protein, and GmOHP2 protein can interact with GmOHP1 protein, among which, Figure 8 The interaction between GmHCF244 protein and GmOHP1 and GmOHP2 protein ( Figure 8). Therefore, it is speculated that the GmHCF244 protein can interact with the GmOHP2 protein and the GmOHP1 protein to form a protein complex to function. According to the results of the western experiment on the distribution of the core subunits of each protein complex (PS I, PS II, Cytb6 f and ATP synthase) on the chloroplast thylakoid membrane, it can be found that the core subunits PsaA and PsaB of PSI; the core subunit Cytf of Cytb6 f and the core subunit ATP b of ATP synthase can all be detected in the mutant and are almost the same as those in the wild type, while the PS II core subunits D1 and D2 can hardly be detected in the mutant. Fig. 9 To detect the distribution of core subunits of each protein complex on the chloroplast thylakoid membrane of wild type and Gmhcf244 mutant ( Fig. 9 ), reflecting that the PS II reaction center of the Gmhcf244 mutant was severely damaged.

Claims

1. A soybean GmHCF244 gene, characterized in that: The CDS sequence of soybean GmHCF244 gene is shown as SEQ ID NO: 1 in the sequence listing.

2. The soybean GmHCF244 gene according to claim 1, characterized in that: The nucleotide sequence of the soybean GmHCF244 gene is shown in SEQ ID NO: 3 in the sequence listing.

3. The protein encoded by the soybean GmHCF244 gene as claimed in claim 1, characterized in that: The amino acid sequence of the protein encoded by the soybean GmHCF244 gene is shown in SEQ ID NO:

2.

4. Use of the protein encoded by the soybean GmHCF244 gene as claimed in claim 1 in the activity of the second reaction center of the photosystem.

5. The use of the protein encoded by the soybean GmHCF244 gene according to claim 4, characterized in that: The protein encoded by the soybean GmHCF244 gene interacts with GmOHP1 and GmOHP2 to form a protein complex, which jointly regulates the activity of the second reaction center of the photosystem.

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