Protein ZmDAP1 for regulating and controlling plant growth and development as well as coding gene and application of protein ZmDAP1

Abstract

The invention discloses a protein ZmDAP1 for regulating plant growth and development as well as a coding gene and application thereof, and belongs to the technical field of gene engineering. The amino acid sequence of the protein DAP1 is as follows: a. An amino acid sequence as shown in SEQ ID NO: 1; or b, an amino acid sequence which is obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1 and expresses a functional protein for regulating plant growth and development. The protein ZmDAP1 and the coding gene thereof disclosed by the invention are related to regulation and control of plant growth and development, and analysis of the function of the ZmDAP1 gene not only contributes to analysis of molecular mechanisms of corn ear development and plant type regulation and control, but also can provide a new gene resource for ensuring high and stable yield of corn.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a protein ZmDAP1 for regulating plant growth and development, a coding gene and an application thereof. Background Art

[0002] Maize (Zea mays L.) is one of the most important food crops and has made a significant contribution to meeting the huge demand for food for human beings. In the breeding of high-yield maize, the number of kernels per ear is one of the important breeding indicators. The number of kernels per ear depends on the normal development of the female ear and the male ear that can produce normal pollen grains. Whether the maize ear can develop normally directly affects the final yield level. In addition, the plant type of maize has an important influence on the size of the ear and the yield. The genes YIGE1 and YIGE2 that control the length of the maize ear affect the yield of maize by participating in the inflorescence development process. The maize glutaredoxin MSCA1 and its two homologous genes (ZmGRX2 and ZmGRX5) regulate ear development. The three homologous genes have strong functional redundancy. The triple mutant shows that the development and differentiation of spikelets are blocked and the plant type becomes smaller, which leads to a significant decrease in maize yield. At present, only a few ear length and plant type genes have been cloned in maize, so it is urgent to explore new genes that regulate ear development and plant type development. Summary of the invention

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

[0004] To achieve the above object, the technical solution adopted by the present invention is: to provide a protein ZmDAP1 for regulating plant growth and development, the amino acid sequence of the protein is:

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

[0006] b. The amino acid sequence as shown in SEQ ID NO: 1 is substituted, deleted and / or added with one or more amino acids, and expresses 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: the amino acid sequence of the tag in Table 1 is connected to the N-terminus or / and C-terminus of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the functional protein that expresses the regulation of plant growth and development is expressed.

[0008] Table 1 Amino acid sequences of tags

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

[0010] The present invention also discloses a gene ZmDAP1 encoding the above-mentioned protein for regulating plant growth and development, and the nucleotide sequence of this gene is as follows:

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

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

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

[0014] d. A nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 2 is substituted, deleted, and / or added with one or more nucleotides, and / or one or more base pair missense mutations are carried out, and encodes a functional protein for regulating plant growth and development.

[0015] The above-mentioned ZmDAP1 protein can be artificially synthesized, or its encoding gene can be synthesized first and then obtained through biological expression.

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

[0017] Further, the stringent conditions are hybridization and membrane washing 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 containing the above-mentioned recombinant vector.

[0020] The present invention also discloses the application of the gene ZmDAP1, the recombinant expression vector or the recombinant microorganism in regulating plant growth and development.

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

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

[0023] Further, by silencing or knocking out the ZmDAP1 gene, the plant height is reduced, and the number of branches of the male inflorescence, the length of the male inflorescence, the size of the ear, and the ear height are decreased.

[0024] Further, the plant is a monocotyledonous plant.

[0025] Further, the plant is maize.

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

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

[0028] By constructing a KO-ZmDAP1 knockout vector and transferring it into the maize inbred line B73-329 (hereinafter simply referred to as the wild type WT), compared with B73-329, the deletion of ZmDAP1 nucleotides in the transgenic T2 generation plants results in the change of ZmDAP1 amino acids, leading to the loss of ZmDAP1 protein function, thus causing phenomena such as the reduction of plant height and abnormal tassel development in the knockout transgenic plants. Other related agronomic traits, such as the number of branches of the tassel, tassel length, ear size, ear height, etc., all show different degrees of reduction. It shows that the gene ZmDAP1 and its encoded protein are related to maize ear development and plant type regulation, which not only helps to analyze the molecular mechanism of maize ear development and plant type regulation, but also provides new gene resources for ensuring high and stable maize yield. Description of the Drawings

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

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

[0031] Figure 3 is the comparison of the genotypes of the wild type WT and the knockout transgenic plants;

[0032] Figure 4 is the comparison of the plant types of the wild type WT and the knockout transgenic plants;

[0033] Figure 5 is the plant height statistics of the wild type WT and the knockout transgenic plants;

[0034] Figure 6 is the ear height statistics of the wild type WT and the knockout transgenic plants;

[0035] Figure 7 is the comparison of the tassels and ears of the wild type WT and the knockout transgenic plants; wherein, Figure 7 (a) is the comparison of the tassel phenotypes, Figure 7 (b) is the comparison of the ear phenotypes;

[0036] Figure 8 is the tassel length statistics of the wild type WT and the knockout transgenic plants;

[0037] Figure 9 is the tassel branch number statistics of the wild type WT and the knockout transgenic plants;

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

[0039] The following describes the detailed implementation of the present invention to facilitate those skilled in the art of this technology to understand the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. However, it should be clear that the present invention is not limited to the scope of the detailed implementation. For those ordinary skilled in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0040] An existing plant expression vector can be used to construct a recombinant expression vector containing the ZmDAP1 gene. When constructing a plant expression vector using the ZmDAP1 gene, any enhanced promoter or constitutive promoter can be added before its transcriptional start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiquitin promoter (pUbi), etc. They can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translational enhancers or transcriptional enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but they must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The plant expression vector carrying the protein-coding gene ZmDAP1 related to maize ear development and plant type regulation of the present invention can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroconductivity, Agrobacterium-mediated, gene gun, etc. The transformed plant host can be a monocotyledonous plant such as maize or a dicotyledonous plant such as Arabidopsis thaliana.

[0041] When identifying and screening transgenic plants or cells, in order to improve the screening efficiency, the used plant expression vector can be processed, such as adding an antibiotic marker with resistance (such as kanamycin) or an anti-chemical reagent marker gene (herbicide-resistant gene), a gene that expresses an enzyme or a luminescent compound that causes color change in plants (GUS gene, luciferase gene, etc.).

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

[0043] MSRPTGAWRLGMATCTIPAAPRAHVGRLPGIRQAARPALLLPSFSSSTLLCHRLPPILPLVLQLLRVRANHAMAASAGTVYEADAEAVVRRITPALDRARHKGQAGKIAVIGGCREYTGAPYFAAISALKVGADLSHVFCTKDAATVIKSYSPELIVHPILEESYSVRVDERASVSSKILTEVAKWMERFDCIVVGPGLGRDPFLLECVSNIMRHARQANIPTVVDGDGLFLVNNNLNLVEGNPLAILTPNVYEYKRLVQKVLNCDVDEESASEQLIALCQKIGDVTIMQKGKADVISDGKTVTQVSTFGSPRRCGGQGDILSGSVAVFASWARHFVLTNEEPTEKRVNPMTLGCIAASLLLRKAASHAFEKNKRSTVTSDIIEFLGKRSVCLHSVLNDLCSSSSF*(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 can be obtained from China Agricultural University. The primers and sequence information used are shown in Table 2.

[0047] Table 2 Primer Sequence Table

[0048] Primer Name Sequence 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 the maize ear development and plant architecture regulatory protein ZmDAP1.

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

[0051] Example 2 Obtaining and identification of ZmDAP1 transgenic maize.

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

[0053] The website CRISPR–P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) was used to design the knockout target sequence. Two knockout target sites were designed on the first exon of maize ZmDAP1. The sequence of the first target site (abbreviated as target site 1) is 5’-GCAGCACCGCGTGCGCATGT-3’, located at positions 174-193 of SEQ ID NO: 2; the sequence of the second target site (abbreviated as target site 2) 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 transgenic 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. The genomic fragment containing the target site was amplified and sequenced. The sequencing results were compared with the sequence of the transformation receptor material B73-329, so as to confirm whether it was a knockout transgenic plant and determine the knockout type. Two knockout transgenic plants were obtained, named KO-zmdap1-1 and KO-zmdap1-2 respectively.

[0056] Figure 3 The genotypes of B73-329, KO-zmdap1-1 and KO-zmdap1-2 are as follows. The genotype detection method is as follows: The genomic sequence of 325 bp containing target site 1 was amplified using the primer pair YM-1F and YM-1R, and at the same time, the genomic sequence of 507 bp containing target site 2 was amplified using the primer pair YM-2F and YM-2R. The amplified results were sent for sequencing and compared with the sequence of B73-329. According to the comparison results, in the knockout transgenic plant KO-zmdap1-1, the nucleotide sequence "T" at the 191st position corresponding to SEQ ID NO: 2 in the ZmDAP1 gene was deleted, and a nucleotide sequence "G" was inserted between the 371st and 372nd positions of SEQ ID NO: 2. In the knockout transgenic plant KO-zmdap1-2, the nucleotide sequence "T" at the 191st position corresponding to SEQ ID NO: 2 in the ZmDAP1 gene was deleted, and the nucleotide sequence "5'-GGACGCG-3'" between the 368th and 374th positions of SEQ ID NO: 2 was replaced by "5'-CGTCGTGCGCCGGATCACGCCGGCACTCGACCGCCGG GACGGTGTAC-3'". The changes in the above nucleotide sequences led to the change of the ZmDAP1 amino acid sequence, resulting in the loss of the function of the ZmDAP1 protein and affecting the plant phenotype.

[0057] III. Phenotypic Identification of Transgenic Maize

[0058] The genomic sequence containing the target site was amplified with specific primers and sequenced. The sequencing results were compared with the transformation receptor material B73-329 to determine the mutation type of the knockout transgenic plant. The KO-ZmDAP1 knockout vector was introduced into B73-329, and the change in the ZmDAP1 nucleotide sequence led to the loss of the function of the ZmDAP1 protein. Phenotypic observation showed that the ear development and plant type of the T2 generation knockout transgenic plants were both changed, mainly manifested as reduced plant height, reduced ear height, smaller tassel, fewer tassel branches, different degrees of degeneration of tassel spikelets, smaller ear, etc. ( Figure 4 andFigure 7 )。

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

Claims

1. A protein ZmDAP1 that regulates plant growth and development, characterized in that: The amino acid sequence of this protein is: a. the amino acid sequence as shown in SEQ ID NO: 1; or b. The amino acid sequence as shown in SEQ ID NO: 1 is substituted, deleted and / or added with one or more amino acids, and expresses an amino acid sequence of a functional protein that regulates plant growth and development.

2. A gene ZmDAP1 encoding the protein for regulating plant growth and development according to claim 1, characterized in that: The nucleotide sequence of the gene is: a. a nucleotide sequence as shown in SEQ ID NO: 2; or b. a nucleotide sequence of positions 120-1340 from the 5' end as shown in SEQ ID NO: 2; or c. a nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and encodes a nucleotide sequence that is a functional protein that regulates plant growth and development; or d. The nucleotide sequence as shown in SEQ ID NO: 2 is substituted, deleted and / or added with one or more nucleotides, and / or undergoes missense mutation of one or more base pairs, and encodes a nucleotide sequence of a functional protein that regulates plant growth and development.

3. A recombinant expression vector, characterized in that: Comprising the gene according to claim 2.

4. A recombinant microorganism, characterized in that Comprising the recombinant vector according to claim 3.

5. Use of the gene ZmDAP1 according to claim 2, the recombinant expression vector according to claim 3 or the recombinant microorganism according to claim 4 in regulating plant growth and development.

6. The use according to claim 5, characterized in that: By silencing or knocking out the ZmDAP1 gene, plant growth and development can be regulated.

7. The use according to claim 6, characterized in that: By silencing or knocking out the ZmDAP1 gene, the plant height was reduced, and the number of stalks, tassel length, ear size and ear height of the tassel were reduced.

8. The use according to claim 5, characterized in that: The plant is a monocotyledonous plant.

9. The use according to claim 8, characterized in that: The plant is corn.

10. A preparation for regulating plant growth and development, characterized in that: Included are agents that inhibit the expression of the ZmDAP1 gene.

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