Application of transcription factor GmHdz4 gene in regulating soybean flower and pod traits

Abstract

The present invention provides the use of the transcription factor GmHdz4 gene in regulating soybean flower and pod traits. Transgenic soybean plants with overexpression and knockout of the transcription factor GmHdz4 gene were constructed. By observing soybean yield components, it was found that the GmHdz4 knockout plants had longer inflorescences and significantly increased the number of flowers and pods per plant.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to application of transcription factor GmHdz4 gene in regulating soybean flower and pod traits. Background Art

[0002] Soybean yield is determined by the number of plants per unit area, the number of pods per plant, the number of grains per pod, and grain weight. Scientists analyzed the correlation between agronomic traits and yield in approved soybean varieties in China between 1950 and 2000, and found that the number of pods per plant has the greatest contribution to yield. The number of pods per plant varies from 30 to 200 across different soybean varieties. The number of pods per plant is significantly positively correlated with the number of main stem nodes and the number of flowers. Although populations constructed by hybridizing high- and low-pod varieties have identified some QTLs associated with pod number, these QTLs are distributed across nearly all 20 soybean chromosomes (except for H), and the vast majority have a contribution rate below 20%. Researchers found that the expression of the GmCYP78A10 gene affects the number of pods per plant. Besides GmCYP78A10, no other genes associated with pod number have been reported in soybean, likely due to the complexity of pod number and its environmental influence.

[0003] The number of pods per plant is a highly complex quantitative trait regulated by multiple factors, including the number of flower buds that differentiate, the rate of successful ovule fertilization, and the rate of normal pod development of embryos after successful fertilization. Quantitative trait loci mapping and genome-wide association analysis have identified genes controlling pod number as candidate genes for meristem size. Pod number per plant is determined during the development of the shoot apical meristem and the differentiation and formation of the inflorescence meristem. The SHOOTMERISTEMLESS (STM) gene, encoding a KNOTTED class I Homeobox protein, is essential for embryonic development and formation of the shoot apical meristem in Arabidopsis. The shoot meristem develops from pluripotent stem cells, and the CLAVATA (CLV)-WUSCHEL (WUS) feedback signaling pathway regulates SAM size by controlling pluripotent stem cells. Studies have shown that the HD-ZipIII transcription factor interacts with type-BRRs and activates the WUS promoter, forming a CLV-independent positive feedback regulatory pathway.

[0004] Homeodomain-leucine zipper (HD-Zip) proteins are a class of transcription factors unique to higher plants. They consist of a conserved domain consisting of a 60-amino acid homeodomain (HD) and a tightly associated leucine zipper (LZ). Plant HD-Zip transcription factors can be divided into four subclasses (HD-Zip I–IV). HD-Zip I has been shown in studies of Arabidopsis, rice, and sesame to play a crucial role in regulating plant growth, development, morphogenesis, signaling networks, and environmental stress.

[0005] The rice OsSLI1 gene is induced by various abiotic stresses and exogenous abscisic acid and has been shown to be a transcriptional activator that regulates the expression of stress-responsive genes and panicle development in rice (Huang X, Duan M, Liao J et al. (2014) OsSLI1, a homeodomain containing transcription activator, involves abscisic acid-related stress response in rice (Oryza sativa L.). Sci. World J. 2014: 809353.). Barley SIX-ROWED SPIKE 1 (VRS1) is the main regulatory gene of spikelet number. Its wild allele Vrs1.b encodes the HD-Zip I transcriptional repressor, which specifically controls the cell division and fertility of the lateral spikelets (Komatsuda T, Pourkheirandish M, He C et al. (2007) Six-rowed barley originated from amutation in a homeodomain-leucine zipper I-class homeobox gene. Proc Natl Acad Sci USA 104: 1424–1429; Nadolska-Orczyk, A, Rajchel IK, Orczyk W et al. (2017) Majorgenes determining yield-related traits in wheat and barley. Theor Appl Genet 130: 1081–1098). Ectopic expression of the LeHB-1 gene in tomato (Solanum lycopersicum) interferes with the normal flowering process of transgenic plants, resulting in multiple flowers, altered flower morphology, abnormal sepal-to-carpel transformation, and induced fruit ripening (Lin Z, Hong Y, Yin M (2008) A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening. Plant J. 55: 301–310).Tomatoes overexpress the grape HD-Zip I gene VvHB58, and the transgenic tomatoes regulate fruit size, reduce seed number and hinder peel expansion through multiple hormonal signaling pathways (Li Y, Zhang S, Dong R et al. (2019) The grapevine homeoboxgene VvHB58 influences seed and fruit development through multiple hormonal signaling pathways. BMC Plant Biol. 19, 523). Genome-wide DNA methylation analysis showed that the HD-Zip I transcription factor GmHDZ20 gene is associated with soybean cotyledon curling. Constitutive expression of GmHDZ20 in Arabidopsis manifests as changes in rosette leaf morphology, shortening of siliques, and fewer seeds per silique (Yang H, Yang Z, Mao Z et al. (2021) Genome-wide DNA methylation analysis of soybean curled-cotyledons mutant and functional evaluation of a homeodomain-leucine zipper (HD-Zip) I gene GmHDZ20. Front Plant Sci. 11: 593999). Summary of the Invention

[0006] The present invention provides a new application of the transcription factor GmHdz4 gene in increasing soybean flower and pod traits, and provides a reference basis for soybean germplasm resource innovation.

[0007] The specific technical solutions are as follows:

[0008] The invention discloses an application of the transcription factor GmHdz4 gene in regulating soybean flower and pod traits. The nucleotide sequence of the GmHdz4 gene is shown in SEQ ID NO.1. The flower and pod traits are inflorescence length and the number of flowers and pods per plant.

[0009] SEQ ID NO.1:.

[0010] The regulation method is: by knocking out the GmHdz4 gene, the length of the soybean inflorescence is extended, and the number of flowers and pods per plant is increased.

[0011] Furthermore, the genetically engineered bacteria contains a CRISPR / Cas9 vector for editing the GmHdz4 gene; the nucleotide sequence of the GmHdz4 gene is shown in SEQ ID NO.1.

[0012] Furthermore, the host cell of the genetically engineered bacteria is Agrobacterium tumefaciens EHA105, and the original vector of the CRISPR / Cas9 vector is pBGK041.

[0013] Furthermore, the method of regulation is:

[0014] (1) Design the target sequence sgRNA of the transcription factor GmHdz4 gene and construct the CRISPR / Cas9 vector of the transcription factor GmHdz4 gene;

[0015] (2) transferring the CRISPR / Cas9 vector into Agrobacterium tumefaciens competent cells to obtain Agrobacterium containing the CRISPR / Cas9 vector;

[0016] (3) Agrobacterium containing the GmHdz4 gene editing vector was used to infect the cotyledonary nodes of wild-type soybeans that had germinated for one day. Seedlings were then recovered through tissue culture, and mutant plants lacking the transcription factor GmHdz4 gene were screened.

[0017] Furthermore, the Agrobacterium tumefaciens is EHA105.

[0018] Furthermore, in step (1), the target sequence sgRNA is shown as SEQ ID NO. 2 (5′-GTCCGAAAGAAAGGATAGGC-3′).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention constructed transgenic soybean plants with overexpression and gene knockout of the transcription factor GmHdz4 gene. By observing the soybean yield components, it was found that the inflorescence length of the transcription factor GmHdz4 gene knockout plant was longer and the number of flowers and pods per plant was significantly increased.

[0021] (2) The present invention found that the expression levels of genes GmFT2a, GmFT5a, GmFUL, GmLFY2, and GmAP1 that regulate the development of inflorescence meristem and floral meristem in plants with the transcription factor GmHdz4 gene knockout were significantly increased, and the expression level of the GmKNT1 gene that negatively regulates the development of inflorescence tissue was significantly downregulated in the knockout strains, indicating that the transcription factor GmHdz4 is a key gene that regulates the development of soybean inflorescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The genetic transformation process of soybean.

[0023] Figure 2 This is the PCR identification result of the overexpression strain in Example 1.

[0024] Figure 3 The effects of the wild type, GmHdz4 overexpression, and GmHdz4 gene-edited mutant strains on inflorescence length and floret number in Example 2;

[0025] Among them, Figure A is a photo; Figure B is the inflorescence length; Figure C is the number of florets; GmHdz4-oe represents the GmHdz4 gene overexpression line, WT represents the wild-type non-transgenic plant, and gmhdz4 represents the gene-edited GmHdz4 mutant.

[0026] Figure 4 This is the result of GmHdz4 in situ hybridization in Example 2;

[0027] Figure A shows the in situ hybridization results of GmHdz4 in soybean inflorescence tissue; Figure B shows a magnified view of the area within the box in Figure A.

[0028] Figure 5 To investigate the agronomic traits of wild-type, GmHdz4 overexpression and GmHdz4 gene-edited mutant lines;

[0029] Among them, Figure A is a photo, and Figure B is a bar chart of agronomic trait indicators; GmHdz4-oe represents the GmHdz4 gene overexpression line, WT represents the wild-type non-transgenic plant, and gmhdz4 represents the gene-edited GmHdz4 mutant.

[0030] Figure 6 The expression levels of different genes (GmFT2a, GmFT5a, GmSOC1, GmFUL, GmKNT1, GmWUS, GmAP1 and GmLFY2) in the wild type, GmHdz4 overexpression and GmHdz4 gene-edited mutant strains. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

[0032] Example 1: Obtaining overexpression and mutant strains using Agrobacterium-mediated method

[0033] The GmHdz4 CDS sequence was amplified using primers GmHdz4_plus_F: 5'-CAGTGAATTCCTGGACGTCCGTACGTTCGA-3' and GmHdz4_plus_R: 5'-CGATGAATTCCGGCGCAAAAATCACCAGTC-3' and inserted into the EcoRI site of the pTF102 vector. Transcription was controlled by the CaMV35S promoter and transformed into Agrobacterium tumefaciens EHA105. The sgRNA sequence was amplified using probes Oligo_UP: 5'-GGGTTGGTCCGAAAGAAAGGATAGGC-3' and Oligo_DOWN: 5'-AAACGTCCGAAAGAAAGGATAGGCCA-3'. The amplified dimer was ligated into the pBGK041 empty vector to construct a knockout vector containing the Cas9 protein and sgRNA expression cassettes driven by the S35 and GmU6 promoters, respectively, and transformed into Agrobacterium tumefaciens EHA105.

[0034] The method described by Yang et al. (Yang XF, Yu XQ, Zhou Z et al. (2016) A highefficiency Agrobacterium tumefaciens mediated transformation system using cotyledonary node as explants in soybean (Glycine max L.). Acta Physiol Plant. 38 (3): 1-10.) was used to optimize the Agrobacterium tumefaciens-mediated transformation system. The constructed overexpression vector and CRISPR / Cas9 vector were used, and the cotyledonary nodes of Tianlong No. 1 soybean that had germinated for one day were used as explants. After seed sterilization, seed germination, explant separation, Agrobacterium tumefaciens infection and co-cultivation, bud induction, bud elongation and bud rooting, T0 generation GmHdz4 overexpression strains and mutants (such as Figure 1 shown).

[0035] Results: The soybean genetic transformation efficiency in this experiment was stable at around 4.5%.

[0036] The bar test strips and glufosinate smear tests identified three T0 generation GmHdz4 overexpression independent transformant lines and 11 mutant lines. The PCR results of the bar gene and target gene in the overexpression lines were all positive ( Figure 2 ), and the status of each strain is shown in Table 1. After T0 generation propagation, T1 generation mutants were further identified, and five single peak strains were screened (Table 2). Among them, Ko-171 strain had two mutations, and Ko-334 and Ko-342 strains had three consecutive base deletions that did not affect the frameshift mutation.

[0037] Table 1 Identification results of T0 generation overexpression lines and mutant lines

[0038]

[0039]

[0040] Table 2 Identification results of T0 generation gene-edited strains

[0041]

[0042] Example 2 GmHdz4 affects soybean inflorescence structure and grain size

[0043] Wild-type, GmHdz4, and knockout strains were planted in gallon pots and cultured in a controlled greenhouse at 28°C with a 10h / 14h light / dark cycle. Before the initial flowering stage (R1), inflorescence length was observed and inflorescence samples were collected for in situ hybridization. Floret number was counted at the peak flowering stage (R2). Observation of inflorescence structure revealed that both floret number and inflorescence length in the mutant were significantly higher than those in the wild-type ( Figure 3 In situ hybridization results also showed that the GmHdz4 gene was abundantly expressed in the apical meristem of the inflorescence ( Figure 4 ).

[0044] The resulting Gmhdz4 overexpression lines #5, #6, and #16 and gene-edited mutant lines Ko-165, Ko-166, and Ko-171 were self-pollinated and propagated to the T2 generation. Agronomic traits were examined after maturity. There were no significant differences in the number of main stem nodes and branches between the wild-type, overexpression, and gene-edited lines, but the mutant lines were shorter in height. The number of pods per plant in the mutant lines was significantly higher than that in the overexpression and wild-type lines ( Figure 5 ).

[0045] Example 3

[0046] The transition from the soybean apical meristem to the inflorescence meristem has been shown to be determined by GmFT2a and GmFT5a, two genes that regulate the formation of the primary inflorescence meristem. The expression levels of both genes, particularly GmFT5a, were significantly increased in the knockout line gmhdz4. Their downstream genes, GmSOC1 and GmFUL, work together with GmFT2a and GmFT5a to regulate the development of the secondary inflorescence meristem. In the knockout line gmhdz4, GmSOC1 expression levels were not significantly different from those in the wild type and overexpression lines, while GmFUL expression levels were significantly increased. Studies have shown that the GmKNT1 gene, which causes floral atrophy, is specifically expressed in the apical meristem and inflorescence tissues, and its expression is significantly downregulated in the knockout line gmhdz4. The genes GmLFY2, GmAP1 and GmWUS, which activate floral organ function and determine floral meristem, showed significantly increased expression levels of GmLFY2 and GmAP1 in the knockout line gmhdz4, while the expression level of GmWUS only increased slightly in the knockout line Ko-171.

Claims

1. Application of the transcription factor GmHdz4 gene in regulating soybean flower and pod traits, characterized in that: The nucleotide sequence of the GmHdz4 gene is shown in SEQ ID NO.1; the flower and pod traits are inflorescence length and the number of flowers and pods per plant; the regulation method is: by knocking out the GmHdz4 gene, the inflorescence length is extended and the number of flowers and pods per plant of soybean is increased.

2. The application of genetically engineered bacteria in improving soybean flower and pod traits is characterized by: The genetically engineered bacteria contain a CRISPR / Cas9 vector for editing the GmHdz4 gene; the nucleotide sequence of the GmHdz4 gene is shown in SEQ ID NO.1; and the improvement method is: by knocking out the GmHdz4 gene, the inflorescence length is extended, and the number of flowers and pods per soybean plant is increased.

3. The use according to claim 2, characterized in that The host cell of the genetically engineered bacteria is Agrobacterium tumefaciens EHA105, and the original vector of the CRISPR / Cas9 vector is pBGK041.

4. The use according to any one of claims 1 to 3, characterized in that The method of said regulation is: (1) Design the target sequence sgRNA of the transcription factor GmHdz4 gene and construct the CRISPR / Cas9 vector of the transcription factor GmHdz4 gene; (2) transferring the CRISPR / Cas9 vector into Agrobacterium tumefaciens competent cells to obtain Agrobacterium containing the CRISPR / Cas9 vector; (3) Agrobacterium containing the GmHdz4 gene editing vector was used to infect the cotyledonary nodes of wild-type soybeans that had germinated for one day. Seedlings were obtained through tissue culture, and mutant plants lacking the transcription factor GmHdz4 gene were screened.

5. The use according to claim 4, characterized in that The Agrobacterium tumefaciens is EHA105.

6. The use according to claim 5, characterized in that In step (1), the target sequence sgRNA is shown as SEQ ID NO.2.

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