CN112457386A

CN112457386A - Protein EAD1 related to control of ear length and row grain number of corn as well as coding gene and application thereof

Info

Publication number: CN112457386A
Application number: CN202110066902.5A
Authority: CN
Inventors: 陈化榜; 裴元荣
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2021-03-09

Abstract

The invention discloses a protein EAD1 related to control of ear length and row grain number of corn, and a coding gene and application thereof. The protein provided by the invention is (a) or (b) as follows: (a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table; (b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1, is related to the length of the corn ear and the number of grains, and is derived from the sequence 1. The EAD1 gene can control the important yield traits such as ear length and row grain number in corn, namely, the homozygous mutation of the gene causes the ear length of corn to be shortened and the row grain number to be reduced. The invention provides a new gene resource for the research of the yield traits of plants, particularly corn, selects an inbred line with EAD1 excellent allelic genes as a donor for the genetic improvement of corn, and plays an important role in the application of the corn breeding field.

Description

Protein EAD1 related to control of ear length and row grain number of corn as well as coding gene and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a protein EAD1 related to control of ear length and row grain number of corn, and a coding gene and application thereof.

Background

Corn is the crop with the largest planting area in China, integrates grain, menses and feeding, and plays an important role in the grain safety and economic development in China. Corn yield is a primary goal in corn production and breeding and is a significant need for economic development. At present, the corn yield per unit area in China still has a great space for improving, but the analysis of key genetic loci of main corn yield factors is a bottleneck problem of corn genetic improvement. The rapid development of genome sequencing and assembly technology enables people to easily acquire a large amount of genetic information, but the genetic code is effectively read to establish the correlation between genotype and phenotype, so that the genetic improvement can be realized.

Ear length is a key factor of corn yield and is in positive correlation with the number of grains in a row. The genetic force of the spike length is high, but the spike length is often under the combined action of micro-effective polygenes, and the spike length is a typical quantitative character and has been a hotspot and difficulty of basic and application research for a long time. So far, based on linkage and association analysis of different populations, a total of 140 QTLs sites affecting spike length trait variation, 87 TAVs (trail-associated variation) were detected, and 47 QTLs sites affecting kernel number variation, 14 TAVs, were detected, forming different clusterings and distributed on multiple chromosomes (Li et al, 2018). However, in general, the detection of QTLs sites often lacks the cloning and in-depth mechanistic study of functional genes. The previous people utilize a plurality of groups to analyze the whole genome of the genetic structure of the maize ear length, isolate a candidate gene TPK1 of a key QTL locus, and predict that the gene can regulate the variation of the maize ear length through the change of the transcription level, but the function of the gene and the potential genetic regulation mechanism thereof, as well as the actual contribution rate to the ear length and the yield are further researched (Xiao et al, 2016). Jiahai Tao et al (Jia et al, 2020) cloned a major gene KNR6 controlling ear length and row size of maize, which encodes a serine threonine receptor protein kinase, and the study showed that the expression level of KNR6 was very significantly and positively correlated with ear length and row size, Zheng 58 and Chang 7-2 were genetically modified with excellent haplotypes, and it was found that the improved material was increased in both ear length and row size, and the yield of the hybrid was estimated to be increased by about 5.6%. These findings indicate that the superior haplotype of KNR6 can be used to increase panicle length and seed number of hybrids, thereby increasing hybrid yield.

On the one hand, due to lack of collection and creation of the maize ear length defect mutant, the maize ear length defect is often accompanied with the problems of poor phenotypic stability, difficult identification and the like; on the other hand, the mutants are often accompanied with development defects, so that the panicle size is smaller, the panicle number is reduced, and the direct breeding value is considered to be lacking. But it is undeniable that the ear length defect mutant is very important for disclosing the genetic regulation mechanism of the functional gene, the optimal allelic variation of the functional gene can be possibly cloned along with the development of the sequencing technology, the ear length and the row grain number of the corn can be effectively improved by coordinating the genetic action of the functional gene for production practice, and the blank of the related research based on the longitudinal improvement of the ear length and the row grain number of the corn is filled.

Disclosure of Invention

The invention aims to provide a protein EAD1 related to controlling the length of a corn ear and the number of grains in a row, and a coding gene and application thereof.

The protein provided by the invention is named as EAD1, is derived from corn (Zea mays) of Zea, and is (a) or (b) as follows:

(a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1, is related to control of the length and the grain number of the corn ear and is derived from the sequence 1.

Sequence 1 in the sequence table is an amino acid sequence of EAD1, and comprises 489 amino acid residues, wherein in the protein sequence, hydrophobic amino acids account for 72, hydrophilic amino acids account for 207, basic amino acids account for 52, acidic amino acids account for 46, the molecular weight of the protein is 52.03KD, and the isoelectric point is 5.85.

In order to facilitate the purification of the protein shown in the above (a), a tag shown in the following table may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid residue sequence of sequence 1 in the sequence listing.

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of (b) may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression. The gene encoding the protein of (b) above may be obtained by deleting one or several codons of amino acid residues and/or performing missense mutation of one or several base pairs from the coding region (positions 84-1554) in the DNA sequence shown in sequence 4 of the sequence listing.

Nucleic acid molecules encoding such proteins are also within the scope of the invention.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as an mRNA, hnRNA, or tRNA, and the like.

In one embodiment of the invention, the nucleic acid molecule is specifically a gene (named EAD1) encoding the protein, and the gene may be specifically a DNA molecule of any one of the following 1) to 6):

1) DNA molecule shown in sequence 2 in the sequence table;

2) DNA molecule shown in sequence 3 in the sequence table;

3) DNA molecule shown in sequence 4 in the sequence table;

4) DNA molecule shown in sequence 5 in the sequence table;

5) a DNA molecule which hybridizes with the DNA molecule defined in any one of 1) to 4) under strict conditions and codes a protein which is related to controlling the length and the number of grains per ear of corn and is derived from a sequence 1;

6) a DNA molecule which has more than 90 percent of identity with the DNA sequence defined in any one of 1) to 6) and codes a protein which is related to controlling the length of a corn ear and the number of grains in a row and is derived from the sequence 1.

Wherein, the sequence 2 is the sequence of the EAD1 gene in the wild maize HN321 genome; sequence 3 is the sequence of EAD1 gene in mutant EAD1 maize genome; the sequence 4 is a cDNA sequence of the EAD1 gene in the wild corn (wherein, the 84-1554 positions are coding region sequences); sequence 5 is the cDNA sequence of EAD1 gene in mutant EAD1 (wherein the 98 th-1564 th positions are coding region sequences).

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant microorganisms containing the above-described nucleic acid molecules are also within the scope of the present invention.

The recombinant vector can be a recombinant expression vector and can also be a recombinant cloning vector.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UBIN or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CaMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. Or directly screening the transformed plants in a stress environment without adding any selective marker gene.

The expression cassette consists of a promoter capable of driving expression of the gene, and a transcription termination sequence.

The transgenic cell line is a non-propagating material into which the gene is transferred.

The application of the protein or the nucleic acid molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant microorganism in any one of the following is also within the protection scope of the invention:

(a) plant breeding and/or seed production;

(b) regulating and controlling the length of the corn ear and the number of the grains in the row.

The invention also provides a method for cultivating the transgenic plant.

The method for cultivating the transgenic plant provided by the invention can be (A) or (B) as follows:

(A) a method of breeding transgenic plants with increased panicle length and increased grain number, comprising the steps of:

(a1) introducing an encoding gene of EAD1 protein into a receptor plant with the genotype of EAD1/EAD1 to obtain a transgenic plant expressing the encoding gene; the panicle length and the number of grains per row of the recipient plant are due to a loss or reduction in the ability of the recipient plant to express a functional said EAD1 protein;

(a2) obtaining a transgenic plant with increased panicle length and increased number of grains per row from the transgenic plant obtained in step (a 1); the coding gene can be introduced into the recipient plant through the recombinant expression vector p3300-EAD 1.

(B) A method of breeding transgenic plants with reduced panicle length and reduced number of seeds comprising the steps of:

(b1) inhibiting the expression of EAD1 protein in an EAD1/EAD1 receptor plant to obtain a transgenic plant;

(b2) obtaining transgenic plants having reduced panicle length and reduced number of grains per row from the transgenic plants obtained in step (b1), wherein the reduced panicle length and reduced number of grains per row are due to the loss of function of the EAD1 protein in the recipient plant.

In step (b1) of the method, inhibiting expression of the encoded gene in the recipient plant is effected by: and (3) specifically shearing a genome DNA sequence encoding the EAD1 protein in the receptor plant by using CRISPR/Cas9 nuclease, so that the receptor plant loses the capability of expressing functional EAD1 protein.

Wherein the CRISPR/Cas9 nuclease performs on a genomic DNA sequence encoding an EAD1 protein in the recipient plantThe target fragment during specific shearing is a fragment which accords with the sequence arrangement rule of 5 '-NX-NGG-3' or 5 '-CCN-NX-3' in the genomic DNA sequence of the coding EAD1 protein in the receptor plant; n represents any one of A, G, C and T, 14 ≦ X ≦ 30, and X is an integer (e.g., X is 20), N_XRepresents X consecutive deoxyribonucleotides. More specifically, the target fragment is "5'-GAGTAGTGGTCTGGAATGGCGGG-3'" (i.e., position 113 and 136 of the sequence 2) in the genomic DNA sequence encoding the EAD1 protein in the recipient plant.

In the present invention, the plant may be either a monocotyledon or a dicotyledon. Wherein the monocotyledon is selected from Gramineae, specifically semen Maydis.

The invention adopts a map-based cloning strategy to combine the BC by using the maize short-ear mutant ead1 and the maize inbred line B73₁F₁The population maps the gene controlling the mutation trait to the corn five chromosome 199.62Mb to 199.97Mb, and uses the published B73 genome sequencing result as the reference physical distance of about 0.35Mb, and the reference physical distance comprises 9 genes. Wherein the sequence with the gene number Zm00001d017570 is different between the mutant and the wild type, the mutant has a deletion of 3bp and a substitution of 52 SNP, which results in the deletion of 1 amino acid and the change of 26 amino acids. The gene is complementarily expressed in the maize short ear mutant by using a transgenic technology, and the ear phenotype of the mutant can be recovered; the genes in the maize inbred line Zong31 are knocked out by using a transgenic technology, and the ear phenotype with the ear length and the grain number can be obtained. Therefore, the Zm00001d017570 gene is a target gene for controlling important yield traits of the corn ear length and the line grain number and is named as EAD 1.

The EAD1 gene can control the ear length and the seed number in corn, namely the homozygous mutation or deletion of the gene can shorten the ear length of corn, and the EAD1 gene which is normally expressed in the mutated or deleted gene can restore the ear length phenotype.

The invention provides a new gene resource for the research and application of important yield traits such as corn ear length, row grain number and the like.

Drawings

FIG. 1 is a graph comparing the phenotype of maize mutant ead1 with Wild Type (WT). Wherein, A, the plant shape and the female ear in the filling stage; b, fully mature female ears; c, counting the length of the female spike at the final stage of grouting; d, counting the number of the rows of the female ears at the final stage of grouting; and E, observing the development process of young ears of the wild type (left) and the mutant (right), wherein the scale bar is 1 cm.

FIG. 2 is a map of the ead1 gene. Wherein, A, gene localization; b, a candidate gene structure schematic diagram; c, the targeting sequence of the knockout vector and the type of mutation obtained; d, structural schematic diagram of the complementary vector.

FIG. 3 shows the functional verification of EAD 1. Wherein, A, the ear phenotype of homozygous knockout transgenic lines of the receptor plant Zong31 and EAD1 gene loci are compared; b, EAD1 mutant and acceptor plant Zong31 and EAD1 gene locus homozygous knockout transgenic plant are respectively crossed to obtain F₁Ear phenotype comparison of the generation plants; the ear phenotype of the complementary transgene negative (indicated by "-") and complementary transgene positive lines was compared against the C, ead1 mutant background (indicated by aa).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The wild maize inbred line HN321, mutant ead1 and conventional material B73 were collected and stored in the laboratory. The maize transgenic receptor Zong31 was provided by Beijing Bomeixing Oakology, Inc. Wherein the genotypes of HN321, B73 and Zong31 are EAD1/EAD1, and the mutant genotype is EAD1/EAD 1. The maize genome sequencing information is referenced to the MaizeGDB database linked as follows:http：//www.maizegdb.org/。

example 1 map-based cloning of the maize ear Length and seed number Gene EAD1

Phenotype of maize mutant ead1

Maize mutant ead1 was not abnormal in plant overall morphology (a in fig. 1) compared to normal plants, but the mutant had significantly reduced ear length and kernel number (B in fig. 1), with an average reduction in ear length of 55.2% (C in fig. 1) and an average reduction in kernel number of 57% (D in fig. 1). By observing the development process of the ears of the wild type and the mutant (E in figure 1, the scale is 1cm), it is found that when the ears grow and develop to about 2cm, ead1 phenomena of hydration, browning and growth retardation occur at the tops of the ears, and the middle and lower parts of the ears can still grow and develop normally. At this stage, the apical inflorescence meristem of the ear of wild type HN321 has not been differentiated and still has differentiation ability, but the tip of the mutant is abnormal before the differentiation is completed, so that it is considered that the ear length of the mutant is shortened and the number of seeds is reduced due to the abnormal growth at the terminal stage of the differentiation and development of the tip of the young ear of the mutant.

Second, construction of genetic mapping population

We cross the short spike mutant ead1 with maize inbred line B73 to form F₁，F₁The development of the generation female ears is normal, and the ear length shortening phenotype is not generated, which indicates that ead1 is recessive gene mutation. Backcross F with ead1₁Construction of segregating population BC₁F₁And performing phenotype identification on 862 single plants in the segregating population, and analyzing that the phenotype segregation ratio of normal development of the long panicle and abnormal development of the top of the short panicle by using a fitness chi-square test conforms to the Mendel segregation law (Table 1) of 1: 1, so that ead1 shows that the phenotype of the shortened panicle length and the abnormal development of the top is controlled by a single gene. We will BC₁F₁The population is expanded and used as a genetically mapped population for map-based cloning of the gene of interest.

TABLE 1.BC₁F₁Fitness chi-square test of isolated populations

Note: chi shape² _0.05(1)＝3.84

Map-based cloning of EAD1 Gene

First, genomic DNA of mutants ead1 and B73, which are two parents for constructing a genetically mapped population, were used as templates, and primers with polymorphisms between mutants ead1 and B73 were screened using corn genome-wide primers. Then theFrom BC₁F₁And selecting 20 plants of the short ear tip dysplasia single plant and the long ear normal single plant with extreme phenotypes from the population, and verifying whether the polymorphic primers are linked with the ear length traits. Screening out linkage primers for determining genotypes of 200 individuals in a population, screening out exchange individuals with the genotypes not meeting the phenotypes by combining the phenotypes of the ear length and the normal or non-normal apical development, and determining a positioning interval according to a reduction trend according to the difference of the number of the screened exchange individuals of different primers, thereby positioning the EAD1 gene between primer markers IDP6939(187.8Mb) and IDP6872(207Mb) of the maize chromosome five. Development of polymorphic molecular markers between IDP6939 and IDP6872 and use for detection of BC₁F₁All individuals of the population (3,491 strain), eventually mapped the EAD1 gene between markers 199-4(199.62Mb) and 200-3(199.97Mb), were physically separated by approximately 0.35Mb, with reference to published maize inbred B73 genome sequencing results (FIG. 2A). Wherein, the sequences of the molecular marker primers for gene mapping are shown in Table 2.

TABLE 2 molecular marker primer sequences for gene mapping

Cloning of EAD1 Gene

Referring to the maize genome sequencing information, the 0.35Mb range of the localization interval contains 9 genes, which are Zm00001d017561, Zm00001d017562, Zm00001d017563, Zm00001d017564, Zm00001d017566, Zm00001d017567, Zm00001d017568, Zm00001d017570, and Zm00001d017571, respectively. The 9 genes are amplified by taking genome DNA of a wild type and genome DNA of a mutant as templates, and sequence difference is compared, so that only the gene Zm00001d017570 has DNA sequence difference between the wild type and the mutant, and the mutant has 3bp base deletion and 52 single base substitution in the sequence, so that 1 amino acid deletion and 26 amino acid changes are caused. Thus, it is speculated that Zm00001d017570 may be a candidate gene for EAD1, and fig. 2B shows the gene structure of Zm00001d017570, consisting of 3 exons (grey boxes) and 2 introns (black line segments).

Carrying out PCR amplification on the wild type inbred line material HN321 genome DNA serving as a template by using a primer pair F1/R1, wherein the sequence of the obtained PCR product is a sequence 2 in a sequence table, and the sequence 2 is the sequence of the EAD1 gene in the wild type corn genome; and (3) performing PCR amplification by using the genome DNA of the mutant EAD1 material as a template and also using a primer pair F1/R1, wherein the sequence of the obtained PCR product is the sequence 3 in the sequence table, and the sequence 3 is the sequence of the EAD1 gene in the mutant maize genome. Extracting total RNA of a wild type inbred line material, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification on a primer pair F1/R1, wherein the sequence of an obtained PCR product is a sequence 4 in a sequence table, the sequence 4 is a cDNA sequence of an EAD1 gene in wild type corn, and the 85-1554 th site is a coding region sequence; extracting total RNA of the mutant EAD1 material, performing reverse transcription to obtain cDNA, performing PCR amplification on F1/R1 by using a primer pair, wherein the sequence of the obtained PCR product is a sequence 5 in a sequence table, the sequence 5 is a cDNA sequence of the EAD1 gene in the mutant EAD1, and the 98 th-1564 th position is a coding region sequence. Both the sequence 2 and the sequence 4 encode EAD1 protein shown in the sequence 1 in the sequence table.

F1：5’-CGCTGCAGGGTCACATTCTGTTGCC-3’；

R1：5’-TGCGATCATCGGATTTCGGA-3’。

Example 2 functional verification (Gene knockout) of Gene controlling ear Length and seed number of corn EAD1

Genetic transformation experiment of EAD1 gene knockout vector

The inventor designs an experiment to edit EAD1 gene of a maize inbred line Zong 31. Specifically, a CRISPR-Cas9(Clustered regular short palindromic repeats associated 9) gene editing system is adopted to carry out site-directed editing on the genome sequence of EAD1 gene in a corn acceptor plant Zong 31. The CRISPR-Cas9 technology can cut DNA at a specific site on a genome, and by utilizing the characteristic that the repair of an organism to a DNA chain cannot guarantee 100% correctness every time, the reconnected DNA chain is different from the DNA chain which is not cut in sequence, so that the gene sequence is changed, and the encoded protein is changed.

Specifically, in the experiment, according to the characteristics of a CRISPR-Cas9 system, a specific sequence on a first exon of an EAD1 gene is selected as a sgRNA (single guide RNA) target sequence (a target position shown by a red segment B in a figure 2 and a target sequence 5'-GAGTAGTGGTCTGGAATGGCGGG-3' shown in a figure C, namely the 113 th and 136 th positions of a sequence 2), and the target sequence is connected to a pBUN411 vector (herbicide resistance) to construct an EAD1 gene knockout vector, and an agrobacterium-mediated method is adopted to transform a corn receptor plant Zong 31. Corn genetic transformation experiment genetic transformation of the recombinant pBUN411-EAD1 vector into a corn Zong31 inbred line is completed by Tianjin Jinopo Biotech Co., Ltd, and the specific transformation method is a conventional agrobacterium-mediated genetic transformation method of corn immature embryos.

Functional verification of knockout transgenic offspring

Extracting genome DNA from a single plant in a gene knockout transgenic progeny population, taking the genome DNA as a template, carrying out PCR amplification on F1/R1 by adopting the primer pair, sequencing a product, and screening an editing site as a homozygous mutant plant. The ear phenotype of the homozygous mutant strain was observed, and when the phenotype of mutant ead1, i.e., shortening of ear length, reduction of number of seeds, abnormal development of ear tip, etc., appeared, the gene was the target gene.

The sequencing results were analyzed and it was found that compared to the wild type, the transgenic plants produced a single base insertion in the first exon of the EAD1 gene, resulting in an amino acid frame shift and premature termination of translation (C in fig. 2). Observation T₂Ear phenotype of generation homozygous mutant lines ko1 and ko2 of 15 individuals each showed a phenotype of shortened ear length, reduced row grain number and retrogression of ear tip, similar to mutant ead1, compared with conventional recipient plant Zong31 (FIG. 3A).

Also to verify that the ear phenotype of the knockout line was indeed due to the editing mutation of the EAD1 gene, rather than to an unknown off-target effect, we crossed conventional recipient plants Zong31 and homozygous knockout mutants ko1 and ko2, respectively, with mutant EAD1 to construct F₁And performing allelic validation. Observe each combination F₁Each 22 strains, the results of which are shown in FIG. 3B, are shown only when the EAD1 gene is homozygously mutatedThe ear phenotype with shortened ear length and reduced row grain number is shown, while the short ear phenotype of the knockout mutant cannot be recovered by the mutant EAD1, which indicates that EAD1 is the target gene for controlling the reduction of ear length and row grain number of corn in the mutant.

Example 3 functional verification (complementary function) of the Gene controlling ear Length and seed number of corn EAD1

First, construction of EAD1 complementary vector and genetic transformation experiment

The invention utilizes a transgenic complementation experiment to prove that EAD1 has the function of regulating and controlling the phenotype of the corn ear length and the kernel number. Using genome DNA of the wild maize inbred line material as a template, amplifying a promoter sequence of EAD1 (a sequence 6 in a sequence table is a promoter sequence of EAD1) by using a primer pair F2 and R2, carrying out double digestion on a p3300-eGFP-nos vector existing in a laboratory by using EcoR I and BamH I, detecting and recovering a DNA product by using agarose gel electrophoresis, and connecting the EAD1 promoter sequence with a linear vector after digestion by using a homologous recombination method to construct an intermediate vector p3300-Native promoter: : eGFP-nos vector. The cDNA of the root system of the wild type maize inbred line material is taken as a template, a primer pair F3 and R3 is used for amplifying the coding region sequence of an EAD1 deletion terminator (84-1554 th site of a sequence 4 in a sequence table), an intermediate vector is subjected to double enzyme digestion by BamH I and Nco I, and a homologous recombination method is utilized to construct a complementary vector C-constraint (p3300-Native promoter:: EAD1-eGFP-nos, figure 2D), namely the recombinant expression vector p3300-EAD1 (the promoter and the coding region sequence of the wild type EAD1 are fused). The corn recipient plant Zong31 was transformed using an Agrobacterium-mediated method. In the corn genetic transformation experiment, the genetic transformation of a recombinant p3300-EAD1 vector to a corn Zong31 inbred line is completed by Beijing Bomeixing Olympic technology, Inc., and the specific transformation method is a conventional agrobacterium-mediated genetic transformation method of corn immature embryos.

F2：5’-CTATGACATGATTACGAATTCGGTCCAAGTTTGCATGTCTA-3’；

R2：5’-GCCCTTGCTCACCATGGATCCGCTGACCGGTACAAGAGCGT-3’；

F3：5’-CGCTCTTGTACCGGTCAGCATGGACGCCGCCGCGAGGG-3’；

R3：5’-TCGCCCTTGCTCACCATGGATCCAGCCTTGCTTGTCTGGTTT-3’。

Second, functional verification of complementary transgenic offspring

To verify the function of the EAD1 gene, a complementary transgenic positive individual was crossed with mutant EAD1 to construct F₂Separating the population, identifying the genotype, finding out positive individuals containing transgenes under the background of the mutant and negative individuals without transgenes under the background of the mutant, and carrying out identification and analysis on the ear phenotype. The identification primer of the homozygous mutant is F4/R4, and if the obtained product is 175bp single band, the genomic DNA can be determined to be from the mutant ead 1. The transgene positive identification primer is F5/R5, if the target band with the size of 400bp is obtained through amplification, the transgenic positive plant is obtained, and if no target product exists, the transgenic negative plant is obtained.

F4：5’-GTCGTTGTCTTCGAATACACTGT-3’；

R4：5’-CCACTCGGTTGAAACACTTGT-3’；

F5：5’-CGTGAAGCTGGGAGAGACGT-3’；

R5：5’-CTTCAGGGTCAGCTTGCCGTA-3’；

The present invention detects two F combinations of two complementary transformation events (C1, C2)₂Separating the population, and finding that the positive individual plant containing the transgene shows a phenotype of long spike and large number of grains in the line under the background of the mutant (indicated by aa), and the spike mutation character is recovered; however, negative individuals containing no transgene in the mutant background still showed the phenotype of short ears and small number of seeds, and the ear mutation character could not be recovered (FIG. 3). It follows that the transformation of the EAD1 mutant with normally expressed EAD1 gene can restore the short spike phenotype of EAD1 homozygous mutation.

The results prove that the corn EAD1 gene is a functional gene for controlling important agronomic traits of corn ear length and grain number.

Combining the results of the above examples, it can be seen that: through map-based cloning and transgenic functional verification, the cloned EAD1 gene is a gene related to important agronomic traits for controlling the ear length and the row grain number of corn, and the ear length of corn can be shortened and the row grain number can be obviously reduced after mutation of protein encoded by the gene. The invention provides a new gene resource for the research of the yield traits of plants, particularly corn, selects an inbred line with EAD1 excellent allelic genes as a donor for the genetic improvement of corn, and plays an important role in the application of the corn breeding field.

Claims

1. A protein which is (a) or (b) below:

(b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1, is related to the length of the corn ear and the number of grains, and is derived from the sequence 1.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a gene encoding the protein of claim 1, and the gene is a DNA molecule selected from the group consisting of 1) to 6) below:

1) DNA molecule shown in sequence 2 in the sequence table;

2) DNA molecule shown in sequence 3 in the sequence table;

3) DNA molecule shown in sequence 4 in the sequence table;

4) DNA molecule shown in sequence 5 in the sequence table;

6) a DNA molecule which has more than 90 percent of identity with the DNA sequence defined in any one of 1) to 5) and codes a protein which is related to controlling the length of a corn ear and the number of grains in a row and is derived from the sequence 1.

4. A recombinant vector, expression cassette, transgenic cell line or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.

5. The recombinant vector according to claim 4, wherein: the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

6. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or 3 or the recombinant vector, expression cassette, transgenic cell line or recombinant microorganism of claim 4 or 5 in any one of:

(a) plant breeding and/or seed production;

7. The method for cultivating the transgenic plant comprises the following steps:

the method for cultivating the transgenic plant provided by the invention can be A or B as follows:

A. a method of breeding transgenic plants with increased panicle length and increased grain number, comprising the steps of:

(a1) introducing a coding gene of the protein shown in the sequence 1 into a receptor plant to obtain a transgenic plant expressing the coding gene; the trait of spike length and row grain number of the receptor plant is caused by the loss or reduction of the capability of the receptor plant to express a functional protein shown in sequence 1;

(a2) obtaining a transgenic plant with increased panicle length and row grain number traits from the transgenic plant obtained in step (a 1); the encoding gene can be introduced into the recipient plant by the above recombinant expression vector;

B. a method of breeding transgenic plants with reduced panicle length and reduced number of seeds comprising the steps of:

(b1) inhibiting the expression of the protein in the genotype receptor plant to obtain a transgenic plant;

(b2) obtaining transgenic plants having reduced panicle length and reduced number of grains per row from the transgenic plants obtained in step (b1), the reduced panicle length and reduced number of grains per row of the resulting transgenic plants being caused by the loss or reduction of function of the protein expressed by the recipient plant.

8. The method of claim 7, wherein: in the step (a1), the encoding gene is introduced into the recipient plant by the recombinant expression vector of claim 5; in step (a3), inhibiting the expression of said protein in said recipient plant is effected by: and (3) specifically shearing the genome DNA sequence of the encoding gene in the receptor plant by using CRISPR/Cas9 nuclease, so that the receptor plant loses the capability of expressing functional protein.

9. Use or method according to claim 7 or 8, characterized in that: the plant is a monocotyledon or a dicotyledon.

10. Use or method according to any of claims 7-9, wherein: the monocotyledon is a gramineous plant;

the Gramineae plant is corn.