A protein zmdap1 for regulating plant growth and development, and an encoding gene and application thereof

Abstract

The application discloses a protein ZmDAP1 for regulating plant growth and development, a coding gene thereof and application, and belongs to the technical field of genetic engineering. The amino acid sequence of the protein DAP1 is: a. an amino acid sequence as shown in SEQ ID NO:1; or b. an amino acid sequence as shown in SEQ ID NO:1 which is substituted, deleted and / or added with one or more amino acids, and an amino acid sequence of a functional protein for expressing a plant growth and development regulating protein. The protein ZmDAP1 and the coding gene thereof disclosed by the application are related to the regulation of plant growth and development, and analyzing the function of the ZmDAP1 gene not only helps to analyze the molecular mechanism of corn ear development and plant type regulation, but also provides a new gene resource for ensuring high yield and stable yield of corn.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a protein ZmDAP1 that regulates plant growth and development, its encoding gene, and its applications. Background Technology

[0002] Maize (Zea mays L.) is one of the most important food crops, making a significant contribution to meeting the enormous human demand for food. In high-yield maize breeding, the number of kernels per ear is a crucial breeding indicator. This number depends on the normally developed female ear and the male ear capable of producing normal pollen grains; the normal development of the ear directly affects the final yield. In addition, maize plant architecture significantly influences ear size and yield. Genes YIGE1 and YIGE2, which control ear length, participate in inflorescence development, thus affecting yield. Maize glutamate reductase MSCA1 and its two homologs (ZmGRX2 and ZmGRX5) regulate ear development. These three homologs exhibit strong functional redundancy; the triple mutant shows inhibited spikelet development and differentiation, smaller plant architecture, and consequently, a significant decrease in maize yield. Currently, only a few genes related to ear length and plant architecture have been cloned, making the discovery of new genes regulating ear development and plant architecture development an urgent priority. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a protein ZmDAP1 that regulates plant growth and development, its encoding gene, and its application, so as to solve the technical problem that the plant type and ear development of maize cannot be regulated.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a protein ZmDAP1 that regulates plant growth and development, the amino acid sequence of which is as follows:

[0005] a. An amino acid sequence as shown in SEQ ID NO: 1; or

[0006] b. An amino acid sequence as shown in SEQ ID NO: 1, with one or more amino acids substituted, deleted, and / or added, and expressing an amino acid sequence of a functional protein that regulates plant growth and development.

[0007] Furthermore, the amino acid sequence of protein ZmDAP1 can also be: an amino acid sequence labeled in Table 1 linked to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1, and an amino acid sequence expressing a functional protein that regulates plant growth and development.

[0008] Table 1. Amino acid sequence of the tag

[0009] Label residues sequence Poly-Arg 5 RRRRR FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0010] This invention also discloses a gene, ZmDAP1, encoding the aforementioned protein that regulates plant growth and development. The nucleotide sequence of this gene is as follows:

[0011] a. A nucleotide sequence as shown in SEQ ID NO: 2; or

[0012] b. The nucleotide sequence from position 120 to 1340 of the 5′ end as shown in SEQ ID NO: 2; or

[0013] c. A nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 2 and encodes a functional protein that regulates plant growth and development; or

[0014] d. A nucleotide sequence as shown in SEQ ID NO: 2, wherein one or more nucleotides have been substituted, deleted, and / or added, and / or missense mutations of one or more base pairs have been performed, and which encodes a functional protein that regulates plant growth and development.

[0015] The ZmDAP1 protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0016] Based on the above technical solution, the present invention can be further improved as follows:

[0017] Furthermore, the stringent conditions were hybridization and washing of the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0018] The present invention also discloses a recombinant expression vector containing the above-mentioned ZmDAP1 gene.

[0019] The present invention also discloses a recombinant microorganism comprising the above-mentioned recombinant vector.

[0020] This invention also discloses the application of the gene ZmDAP1, recombinant expression vectors, or recombinant microorganisms in regulating plant growth and development.

[0021] Based on the above technical solution, the present invention can be further improved as follows:

[0022] Furthermore, by silencing or knocking out the ZmDAP1 gene, the regulation of plant growth and development can be achieved.

[0023] Furthermore, by silencing or knocking out the ZmDAP1 gene, the plant height was reduced, and the number of branches, length, size, and height of the tassels decreased.

[0024] Furthermore, the plants are monocotyledonous.

[0025] Furthermore, the plant is corn.

[0026] The present invention also discloses an agent for regulating plant growth and development, which includes a reagent for inhibiting the expression of the ZmDAP1 gene.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention constructs a KO-ZmDAP1 knockout vector and transforms it into the maize inbred line B73-329 (hereinafter referred to as wild-type WT). Compared with B73-329, the T2 generation transgenic plants exhibit a change in ZmDAP1 amino acids due to the deletion of ZmDAP1 nucleotides, resulting in the loss of ZmDAP1 protein function. This leads to reduced plant height and abnormal tassel development in the knockout transgenic plants. Other related agronomic traits, such as the number of tassel branches, tassel length, ear size, and ear height, are also reduced to varying degrees. This indicates that the ZmDAP1 gene and its encoded protein are related to the regulation of maize ear development and plant architecture. This not only helps to elucidate the molecular mechanisms of maize ear development and plant architecture regulation but also provides new gene resources to ensure high and stable maize yields. Attached Figure Description

[0029] Figure 1 ZmDAP1 is a homolog of rice DAP1;

[0030] Figure 2 Comparison of the amino acid sequences of ZmDAP1 and DAP1;

[0031] Figure 3 Genotyping comparison between wild-type WT and knockout transgenic plants;

[0032] Figure 4 Comparison of plant morphology between wild-type WT and knockout transgenic plants;

[0033] Figure 5 Statistics on plant height of wild-type WT and knockout transgenic plants;

[0034] Figure 6 The ear height was statistically analyzed for wild-type WT and knockout transgenic plants;

[0035] Figure 7 The tassels and ears of wild-type WT plants were compared with those of knockout transgenic plants; among them, Figure 7 (a) Comparison of male spike phenotypes Figure 7 (b) Comparison of ear phenotypes;

[0036] Figure 8 Statistics on the length of male spikelets of wild-type WT and knockout transgenic plants;

[0037] Figure 9 Statistics on the number of male spike branches in wild-type WT and knockout transgenic plants;

[0038] Figure 10 Statistics on ear length of wild-type WT and knockout transgenic plants. Detailed Implementation

[0039] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0040] Recombinant expression vectors containing the ZmDAP1 gene can be constructed using existing plant expression vectors. When constructing plant expression vectors using the ZmDAP1 gene, any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene promoter (pUbi), etc., which can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the gene of this invention, enhancers can also be used, including translational enhancers or transcriptional enhancers. These enhancer regions can be ATG start codons or adjacent start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. Plant expression vectors carrying the ZmDAP1 gene, which encodes proteins related to maize ear development and plant architecture regulation, can be transformed into plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, Agrobacterium-mediated transformation, and gene guns. The plant host that is transformed can be either a monocotyledonous plant such as maize or a dicotyledonous plant such as Arabidopsis thaliana.

[0041] To improve screening efficiency when identifying and screening transgenic plants or cells, the plant expression vectors used can be processed, such as by adding antibiotic resistance markers (kanamycin, etc.) or chemical reagent resistance marker genes (herbicide resistance genes), or genes that express color-changing enzymes or luminescent compounds in plants (GUS genes, luciferase genes, etc.).

[0042] The ZmDAP1 protein disclosed in this invention consists of 406 amino acid residues, and its amino acid sequence is as follows:

[0043] MSRPTGAWRLGMATCTIPAAPRAHVGRLPGIRQAARPALLLPSFSSTLLCHRLPPILPLVLQLLRVRANHAMAASAGTVYEADAEAVVRRITPALDRARHKGQAGKIAVIGGCREYTGAPYFAAISALKVGADLSHVFCTKDAATVIKSYSPELIVHPILEESYSVRVDERASVSSKILTEVAKWMERFDCIVVGPGLGRDPFL LECVSNIMRHARQANIPTVVDGDGLFLVNNNLNLVEGNPLAILTPNVYEYKRLVQKVLNCDVDEESASEQLIALCQKIGDVTIMQKGKADVISDGKTVTQVST FGSPRRCGGQGDILSGSVAVFASWARHFVLTNEEPTEKRVNPMTLGCIAASLLLRKAASHAFEKNKRSTVTSDIIEFLGKRSVCLHSVLNDLCSSSSF*(SEQ ID NO: 1).

[0044] The full-length cDNA sequence of the ZmDAP1 gene consists of 1467 nucleotides, and its nucleotide sequence is as follows:

[0045]

[0046] The maize inbred line B73-329 used below was obtained from China Agricultural University. The primers and sequence information used are shown in Table 2.

[0047] Table 2 Primer sequence list

[0048] Primer name sequence serial number YM-1F 5'-GGAACAACTGAACGGCCGGCA-3' SEQ ID NO: 3 YM-1R 5'-GCCCGGACCCGGAGGAGCT-3' SEQ ID NO: 4 YM-2F 5'-ATTCGGCAGGCAGCTCGTC-3' SEQ ID NO: 5 YM-2R 5'-ACTAGGCATGTCCCCAATT-3' SEQ ID NO: 6

[0049] Example 1: Discovery of ZmDAP1, a protein that regulates ear development and plant type in maize.

[0050] To identify proteins regulating ear development and plant architecture in maize, homologous proteins of the DAP1 protein, which controls ear development and plant architecture in rice, were analyzed. The results showed that the protein encoded by the maize GRMZM5G840982 gene is a direct homolog of DAP1, and this encoded protein was then named ZmDAP1. Figure 1 Gene annotation indicates that the ZmDAP1 gene encodes an ATP-dependent (S)-NAD(P)H hydrate dehydratase. Amino acid sequence alignment revealed that the rice DAP1 protein shares a 73.65% amino acid sequence similarity with the maize ZmDAP1 protein. Figure 2 The above results indicate that ZmDAP1, as a homolog of DAP1, has a very high sequence similarity to DAP1 and is likely to have a similar function to the rice DAP1 protein, participating in the genetic regulatory network of maize ear development and plant architecture.

[0051] Example 2: Obtaining and identifying ZmDAP1 transgenic maize.

[0052] I. Construction of KO-ZmDAP1 knockout vector and obtaining transgenic maize material

[0053] Knockout target sequences were designed using the CRISPR-P2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Two knockout target sites were designed on the first exon of maize ZmDAP1. The first target site (target 1) sequence is 5'-GCAGCACCGCGTGCGCATGT-3', located at positions 174-193 of SEQ ID NO: 2; the second target site (target 2) sequence is 5'-GGTGTACGAGGCGGACGCGGAGG-3', located at positions 356-375 of SEQ ID NO: 2. The construction of the CRISPR / Cas9 vector for knocking out the maize ZmDAP1 gene and the genetic transformation of the CRISPR / Cas9 vector were both completed by the transformation platform of the Maize Research Center of China Agricultural University.

[0054] II. PCR Identification of Genetically Modified Maize

[0055] Specific primers YM-1F (SEQ ID NO: 3), YM-1R (SEQ ID NO: 4), YM-2F (SEQ ID NO: 5), and YM-2R (SEQ ID NO: 6) were designed near the target site to amplify the genomic fragment containing the target site and sequence it. The sequencing results were compared with the sequence of the transformed recipient material B73-329 to confirm whether it was a knockout transgenic plant and to determine the knockout type. Two knockout transgenic plants were obtained and named KO-zmdap1-1 and KO-zmdap1-2, respectively.

[0056] Figure 3 The genotypes of B73-329, KO-zmdap1-1, and KO-zmdap1-2 were determined using the following method: A 325 bp genome sequence containing target 1 was amplified using primer pairs YM-1F and YM-1R, and a 507 bp genome sequence containing target 2 was amplified using primer pairs YM-2F and YM-2R. The amplified results were then analyzed and compared with the sequence of B73-329. The comparison results showed that in the knockout transgenic plant KO-zmdap1-1, the nucleotide sequence "T" corresponding to position 191 of SEQ ID NO: 2 in the ZmDAP1 gene was deleted, and the nucleotide sequence "G" was inserted between positions 371 and 372 of SEQ ID NO: 2. In the knockout transgenic plant KO-zmdap1-2, the nucleotide sequence "T" corresponding to position 191 of SEQ ID NO: 2 in the ZmDAP1 gene was deleted, and the nucleotide sequence "5'-GGACGCG-3'" between positions 368 and 374 of SEQ ID NO: 2 was replaced with "5'-CGTCGTGCGCCGGATCACGCCGGCACTCGACCGCCGG GACGGTGTAC-3'". These changes in nucleotide sequence lead to alterations in the ZmDAP1 amino acid sequence, resulting in the loss of ZmDAP1 protein function and affecting the plant phenotype.

[0057] III. Phenotypic Identification of Genetically Modified Maize

[0058] The genomic sequence containing the target site was amplified using specific primers and sequenced. The sequencing results were compared with the transformed recipient material B73-329 to determine the mutation type of the knockout transgenic plant. The KO-ZmDAP1 knockout vector was introduced into B73-329, resulting in a loss of ZmDAP1 protein function due to changes in the ZmDAP1 nucleotide sequence. Phenotypic observation showed that the T2 generation knockout transgenic plants exhibited alterations in ear development and plant type, mainly manifested as reduced plant height, reduced ear height, smaller tassels, fewer tassel branches, varying degrees of degeneration of tassel spikelets, and smaller ears. Figure 4 and Figure 7 ).

[0059] from Figure 5 , Figure 6 , Figures 8-10 It can be seen that the plant height, ear height, and tassel length of the knockout transgenic plants are significantly lower than those of the wild type, and the number of tassel branches and ear length of the knockout transgenic plants are significantly less than those of the wild type.

Claims

1. A protein ZmDAP1 or its coding gene for regulating the growth and development of corn, wherein the protein ZmDAP1 or its coding gene is applied to regulate the growth and development of corn. By silencing or knocking out the coding gene, the plant height of the corn is reduced, the branch number of the tassel, the tassel length, the ear size and the ear position height are reduced; the amino acid sequence of the protein ZmDAP1 is: the amino acid sequence shown as SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The coding gene is ZmDAP1 a gene whose nucleotide sequence is: a. the nucleotide sequence shown as SEQ ID NO:2; or b. the nucleotide sequence shown as SEQ ID NO:2 from the 5' end 120-1340.

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