A method for increasing maize yield

Abstract

The present invention discloses a method for increasing the yield of maize. The method is to overexpress an auxin influx carrier protein in maize, screen for transformed plants with significantly increased ear row number, grain weight per ear, or test weight, and increase the yield of maize. Overexpression of the auxin transport protein in the present invention can significantly increase the test weight and ear row number of maize kernels in inbred lines and hybrids, thereby significantly increasing the yield. Under the same genetic background, both the test weight and ear row number of kernels are positively correlated with the yield of maize. Selecting transgenic maize into which the auxin transport protein gene has been introduced using the test weight and ear row number can effectively obtain high-yield transgenic maize.

Description

(1) Technical Field

[0001] The present invention relates to a method for increasing the yield of corn. (2) Background Art

[0002] It is of great value to increase the yield potential of crops such as corn and rice. For a long time, through the genetic breeding of crops, the yields of crops such as corn and rice have been greatly increased. However, it has become increasingly difficult to further increase the yield using traditional breeding methods. Increasing the yield of crops through transgenic technology and gene editing technology may be an important technical method to further increase the yield potential of crops.

[0003] Examples of transgenic improvement of crop yields that have been reported so far include transgenic corn MON87403 expressing an Arabidopsis gene (USDA-APHIS Petition for the Determination of Nonregulated Status for Increased Ear Biomass MON 87403 Maize. United States Department of Agriculture, 2014. https: / / www.aphis.usda.gov / brs / aphisdocs / 14_21301p.pdf.); transgenic soybean HB4 expressing a Helianthus annuus gene (USDA-APHIS Pettition for determination of non-regulated status for the new plant variety HB4 soybean (IND-00410–5) intended for environmental release and food and feed use. United States Department of Agriculture, Animal and Plant Health Inspection Service: 2017. https: / / www.aphis.usda.gov / brs / aphisdocs / 17_22301p.pdf.); and transgenic corn DP202216 expressing the corn gene Zmm28 (Wu et.al., Proc Natl Acad Sci USA, 116:23850-23858). However, there is still a great need to further obtain new technologies for increasing the yields of crops such as corn.

[0004] Auxin is an important hormone that regulates plant growth and development. At the organ and whole-plant levels, auxin plays an important role from seedling to fruit maturity. The most obvious effect of auxin is to promote growth, but its promoting effect on the growth of stems, buds, and roots varies with concentration. Auxin importer carriers are a class of proteins that transport auxin from outside the cell into the cell. It was previously thought that auxin importer proteins play a regulatory role in plant root development and reproductive organ formation (Swarup, K., Benková, E., Swarup, R. et al. The auxin influx carrier LAX3 promotes lateral root emergence. Nat Cell Biol 10, 946–954 (2008).) (Swarup R, Friml J, Marchant A, et al. Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes & Development. 2001 Oct;15(20):2648-2653.).

[0005] Chinese Patent Application 201110376993.9 proposed a method for improving the root system, plant height, etc. of maize and sorghum by using auxin importer AUX1 / LAX family gene proteins. Although improving the root system and plant height of maize may increase maize yield, there is no direct positive correlation between root system, plant height and yield. In addition, 201110376993.9 did not provide examples of transgenic maize or sorghum with increased yield.

[0006] Our research unexpectedly found that overexpressing auxin transport proteins can significantly increase the test weight of maize kernels and the number of kernel rows per ear in maize inbred lines and hybrids, thereby significantly increasing yield, and these functions have not been discovered before. In particular, we found that under the same genetic background, the test weight of kernels and the number of kernel rows per ear are both positively correlated with maize yield. Selecting transgenic maize into which the auxin transport protein gene has been introduced using test weight and the number of kernel rows per ear can effectively obtain high-yield transgenic maize. (III) Summary of the Invention

[0007] The object of the present invention is to provide a method for increasing the yield of maize. By introducing an auxin influx carrier protein expression cassette and regulating the expression of the auxin influx carrier protein, the row number per ear, grain weight per ear, and test weight of maize kernels can be increased, thereby having the potential for yield increase.

[0008] The technical solution adopted by the present invention is as follows:

[0009] The present invention provides a method for increasing the yield of maize, which is to overexpress an auxin influx carrier protein in maize, screen for transformed plants with significantly increased row number per ear, grain weight per ear, or test weight, and increase the yield of maize.

[0010] Preferably, the auxin influx carrier protein includes, but is not limited to, auxin influx carrier proteins from plants such as maize, sorghum, rice, wheat, and Arabidopsis thaliana. The auxin influx carrier protein contains the polypeptide fragment of SEQ ID NO: 1, or contains a peptide segment having at least 75%, 78%, 80%, 85%, 90%, or 95% identity with the amino acid sequence of the polypeptide fragment of SEQ ID NO: 1. The identity of amino acids can be obtained by existing methods, such as Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 3364; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.). Those skilled in the art can clone the DNA sequence encoding the auxin influx carrier protein of the present invention from plants by searching databases or traditional gene cloning methods, or artificially synthesize the coding sequence of the auxin influx carrier protein according to the amino acid sequence.

[0011] Preferably, the auxin influx carrier protein of the present invention is a maize auxin influx carrier protein, including ZmAIC1 (also known as ZmAUX1), ZmATL1, ZmAIC2, ZmAIC3, or ZmAIC4, and their amino acid sequences are SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. A more preferred auxin influx carrier protein is maize ZmAUX1.

[0012] Preferably, overexpression of the auxin influx carrier protein of the present invention is achieved by introducing an expression cassette containing the auxin influx carrier protein-encoding gene into the maize genome. The expression cassette is functionally linked at the 5'-end with a promoter and at the 3'-end with a terminator to the auxin influx carrier protein-encoding gene. The auxin influx carrier protein overexpression cassette includes the ZmAUX1 expression cassette or the ZmATL1 expression cassette; the mRNA coding sequence of the ZmAUX1 gene in the ZmAUX1 expression cassette can be artificially synthesized according to the nucleic acid sequence of SEQ ID NO:7, then the natural promoter of the ZmAUX1 gene (1bp to 1775bp in SEQ ID NO:8) is added at the 5'-end, and the natural terminator of ZmAUX1 (5984bp to 6132bp in SEQ ID NO:8) is added at the 3'-end to obtain the ZmAUX1 expression cassette with a Hind III restriction site added at the 5'-end and a Kpn I site added at the 3'-end. The mRNA coding sequence of the ZmATL1 gene in the ZmATL1 expression cassette can be artificially synthesized according to the nucleic acid sequence of NM_001370703.1, then the natural promoter of the ZmATL1 gene (1bp to 1492bp in SEQ ID NO:12) is added at the 5'-end, and the natural terminator of ZmATL1 (7803bp to 8189bp in SEQ ID NO:12) is added at the 3'-end to obtain the ZmATL1 expression cassette with a Hind III restriction site added at the 5'-end and a Kpn I site added at the 3'-end.

[0013] Preferably, various different promoters can be used for overexpression of the auxin influx carrier protein. Commonly used constitutive promoters such as the maize ubiquitin promoter, the rice actin promoter, the CaMV35 promoter, etc. Organ- or tissue-specific promoters, such as spike-specific promoters, seed-specific promoters, etc. Usually, several promoters can be used simultaneously for one auxin influx carrier protein gene to find a suitable promoter that can obtain more excellent transgenic traits. A preferred promoter for obtaining high-yield transgenic maize in the present invention is the natural promoter of maize ZmAUX1, and its sequence is shown as 1-1775bp in SEQ ID NO:8.

[0014] Terminators are required for the normal expression of genes. The terminator of the present invention can be the natural terminator of the auxin influx carrier protein gene of maize or terminators from other sources, such as the NOS terminator, the CaMV35S terminator, the HSP terminator, etc. The preferred terminator of the present invention is the natural terminator of ZmAUX1 (5984-6132bp in SEQ ID NO:8).

[0015] To introduce the auxin input carrier protein of the present invention into the maize genome to obtain transgenic maize, a selectable marker gene expression cassette is usually also required to assist in the screening of transgenic plants. The selectable marker gene expression cassette can be tandemly arranged with the auxin input carrier protein expression cassette of the present invention in a single T-DNA construct, or can be screened by co-transformation in separate T-DNA constructs to obtain transgenic maize containing the overexpressed auxin input carrier protein of the present invention. The marker genes used in the present invention for screening transgenic maize with overexpressed auxin input carrier protein include, but are not limited to, the glyphosate resistance genes g10evo epsps, cp4 epsps, the glufosinate resistance genes pat, bar, the kanamycin resistance gene, the hygromycin resistance gene, etc. Preferably, the glyphosate screening marker expression cassette pCMP-g10evo epsps (SEQ ID NO:9).

[0016] Preferably, the method for increasing maize yield in the present invention is to insert the auxin input carrier protein overexpression cassette and the glyphosate screening marker expression cassette pCMP-g10evo epsps (SEQ ID NO:9) with a Kpn I site added at the 5' end and an Xho I site added at the 3' end into the pCAMBIA1300 vector backbone digested with (Hind III, Kpn I) by three-step digestion and ligation. The obtained construct is introduced into Agrobacterium tumefaciens LBA4404 by electroporation and then transformed into maize, and maize with increased yield is screened; the auxin input carrier protein overexpression cassette includes the ZmAUX1 expression cassette or the ZmATL1 expression cassette.

[0017] Preferably, the construct further includes replacing the promoter downstream of the auxin input carrier protein coding gene with the promoter p35S (SEQ ID NO:10) or inserting the plant virus enhancer 3xEnhancer (SEQ ID NO:11) at the Kpn I site.

[0018] The method of introducing a DNA fragment containing an auxin input protein gene expression cassette into a crop has been a mature technology. Generally, the Agrobacterium-mediated method can be used, and Agrobacterium-mediated maize transformation is a well-known technology to those skilled in the art. Other transformation methods, including but not limited to gene gun, protoplast transformation, etc., are also included in the method for obtaining transgenic maize with overexpressed auxin input carrier protein in the present invention.

[0019] Due to different factors such as the imported loci and copy numbers, the trait performances of different transformants into which the auxin input protein gene has been introduced are not the same. By comparing with conventional plants having the same genetic background, transformants with significantly increased test weight or ear row number or grain weight per ear are selected. Preferably, maize with an increase in ear row number by more than 2%, 3%, 5%, 8%, and / or an increase in grain weight per ear by more than 3%, 5%, 8%, 10%, and / or an increase in test weight by more than 5, 10, 13, 20 g / L.

[0020] The present invention also provides plants, plant cells, plant parts, seeds and grains in which a genetic modification is introduced at the auxin input carrier protein gene locus of the maize genome to increase the yield of maize, and the genomic auxin input carrier protein gene locus encodes the amino acid sequences of SEQ ID NO: 2-6. By genetically modifying these loci, the activity and / or level of the protein is increased, and maize with significantly increased ear row number and / or grain weight per ear and / or test weight can be obtained.

[0021] Preferably, the genetic modification includes inserting an exogenous enhancer into the genomic promoter region, or changing the nucleotide sequence of the locus promoter by gene editing. By editing the natural auxin input carrier protein gene locus of maize, gene-edited maize with increased test weight, ear rows, and grain weight is screened, and maize plants with increased yield are obtained.

[0022] The "genomic locus" generally refers to the position on the chromosome of a plant where a gene is found, such as a polynucleotide encoding a polypeptide. The "gene" includes nucleic acid fragments that express functional molecules, such as but not limited to specific protein coding sequences and regulatory elements, such as those before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence.

[0023] The "regulatory element" generally refers to a regulatory element involved in regulating the transcription of a nucleic acid molecule (such as a gene or a target gene). A regulatory element is a nucleic acid and can include a promoter, an enhancer, an intron, a 5'-untranslated region (5'-UTR, also known as a leader sequence), a 3'-UTR, or a combination thereof. Regulatory elements can act in "cis" or "trans", and usually act in "cis", that is, it activates the expression of a gene located on the same nucleic acid molecule (such as a chromosome) where the regulatory element is located.

[0024] The "enhancer" element is any nucleic acid molecule that can increase the transcription of a nucleic acid molecule when functionally linked to a promoter (regardless of its relative position).

[0025] The "cis element" generally refers to an element that affects or regulates the expression of an operably linked transcribable polynucleotide

[0026] Transcriptional regulatory elements, wherein the transcribable polynucleotide is present in the same DNA sequence. Cis-elements can function to bind transcription factors, which are trans-acting polypeptides that regulate transcription.

[0027] An "intron" is an intervening sequence in a gene that is transcribed into RNA but excised during the production of mature mRNA. The term is also used for the excised RNA sequence. An "exon" is a part of the sequence of a transcribed gene and is found in the mature messenger RNA derived from the gene, but is not necessarily part of the sequence encoding the final gene product. The 5′ untranslated region (5’UTR) (also known as the translational leader sequence or leader RNA) is the region of the mRNA that is directly upstream of the start codon. This region is involved in the regulation of the translation of the transcript by different mechanisms in viruses, prokaryotes, and eukaryotes. A "3′ non-coding sequence" refers to the DNA sequence located downstream of the coding sequence and includes the polyadenylation recognition sequence and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is typically characterized as affecting the addition of a polyadenylate tail to the 3′ end of the mRNA precursor.

[0028] "Genetic modification", "DNA modification", etc. refer to site-specific modifications that alter or change the nucleotide sequence at a specific genomic locus in a plant. The genetic modifications of the compositions and methods described herein can be any modification known in the art, such as, for example, insertion, deletion, single nucleotide polymorphism (SNP), and / or polynucleotide modification. Additionally, the targeted DNA modification at a genomic locus can be located at any position on the genomic locus, such as, for example, the coding region (e.g., exon) of the encoded polypeptide, the non-coding region (e.g., intron), regulatory elements, or untranslated region. A "targeted" genetic modification or "targeted" DNA modification refers to the direct manipulation of an organism's gene. The targeted modification can be introduced using any technique known in the art, such as, for example, plant breeding, genome editing, or single locus transformation.

[0029] The type and location of the DNA modification of the polynucleotide are not particularly limited as long as the DNA modification results in an increase in the expression and / or activity of the protein encoded by the corresponding polynucleotide.

[0030] Plants, plant cells, plant parts, seeds, and / or grains contain one or more nucleotide modifications in (a) the coding region; (b) the non-coding region; (c) the regulatory sequence; (d) the untranslated region, or (e) any combination of (a)-(d) of an endogenous polynucleotide encoding a polypeptide.

[0031] The DNA modification is the insertion of one or more nucleotides (preferably consecutive) into a genomic locus. For example, an expression regulatory element (FMV enhancer SEQ ID NO: 13) is inserted, which is operably linked to the target gene described herein. The targeted DNA modification can be the insertion of a promoter with high expression known in the art (such as the maize Ubi promoter, ZmAux1 promoter) into the 5' UTR of other auxin input carrier proteins, thereby controlling the expression of endogenous polypeptides through the inserted promoter. The DNA modification is a modification that optimizes the Kozak context to increase expression. The DNA modification is a polynucleotide modification or an SNP at a site that regulates the stability of the expressed protein.

[0032] As used herein, "increased", "increase", etc. refer to any detectable increase in the experimental group (e.g., a plant having the DNA modification described herein) compared to the control group (e.g., a wild-type plant without the DNA modification). Thus, increased protein expression includes any detectable increase in the total protein level in a sample and can be determined using conventional methods in the art, such as Western blotting and ELISA.

[0033] The genomic locus has more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) DNA modifications. For example, the translation region and regulatory elements of the genomic locus may each contain a targeted DNA modification. In certain embodiments, more than one genomic locus of a plant may contain a DNA modification.

[0034] DNA modification of genomic loci can be accomplished using any genomic modification technology known in the art or described herein. In certain embodiments, targeted DNA modification is performed by a genomic modification technology selected from the group consisting of: polynucleotide-guided endonucleases, CRISPR-Cas endonucleases, base editing deaminases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), engineered site-specific meganucleases, or Argonaute. Genomic modification can be facilitated by inducing double-strand breaks (DSBs) or single-strand breaks at defined positions in the genome near the desired alteration. Any available DSB inducer can be used to induce DSBs, including but not limited to, TALENs, meganucleases, zinc finger nucleases, Cas9-gRNA systems (based on bacterial CRISPR-Cas systems), the guided cpf1 endonuclease system, etc. In some embodiments, the introduction of DSBs can be combined with the introduction of a polynucleotide modification template. In additional embodiments, polynucleotide modifications can be introduced by prime editing methods (Zong, Y., Liu, Y., Xue, C. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat Biotechnol 40, 1394–1402 (2022).).

[0035] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that overexpression of the auxin transport protein in the present invention can significantly increase the test weight and ear row number of maize kernels in inbred lines and hybrids of maize, thereby significantly increasing the yield. Under the same genetic background, both the test weight and ear row number of kernels are positively correlated with the yield of maize. Transgenic maize into which the auxin transport protein gene has been introduced can be effectively obtained by selecting based on the test weight and ear row number to obtain high-yield transgenic maize. (IV) Specific Embodiments

[0036] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0037] The molecular biology and biochemistry methods used in the following embodiments of the present invention are all known techniques. They are described in detail in documents such as Current Protocols in Molecular Biology published by John Wiley and Sons, written by Ausubel, and Molecular Cloning: A Laboratory Manual, 3rd ED., published by Cold Spring Harbor Laboratory Press (2001), written by J. Sambrook et al.

[0038] Example 1. Obtaining the Coding Sequences of Auxin Influx Carrier Proteins in Different Species

[0039] According to the amino acid sequence of the maize auxin influx carrier protein (ZmAUX1 core region) shown in SEQ ID NO:1, a sequence search was performed in the NCBI Reference proteins database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).

[0040] SEQ ID NO 1(ZmAUX1 core region)

[0041] Whggsaydawfscasnqvaqvlltlpysfaqlgmlsgvlfqlfygllgswtaylisilyleyrtrrerekaadfrnhviqwfevldgllgrhwrnaglafnctfllfgsviqligcasniyyvndrldkrtwtyvfgaccattvfipsfhnyrvwsflglvmttytawymavaslvhgqvegvqhsgptrivlyftgatnilytfgghavtveimhamwrpqkfkaiyllatlyvltltlpsaaasywafgdellthsnalallprtpfrdaavvlmlihqfitfgfactplyfvwekliglhdcrslckraaarlpvvvpiwflaiifpffgpinsavgsllvsftvyiipalahmvtfrspqsrenaverpprfaggwtgayvinsfvvawvlvvgfgfggwasitnfvqqvntfglfakcyqcp.

[0042] The defined species are Zea mays, Oryza sativa, and Arabidopsis thaliana respectively. Auxin input carrier proteins with a sequence identity of at least 75% to SEQ ID NO:1 were searched and obtained, as shown in Table 1 below. Among them, numbers 1-5 correspond to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:3, and SEQ ID NO:6 respectively.

[0043] SEQ ID NO 2 (ZmAUX1)

[0044] mhttpvskhrqhaqagkaldhrseglmaaggggggiadekapaaeafgghleaaemteaeeehsgvksrlsgllwhggsaydawfscasnqv

[0045] aqvlltlpysfaqlgmlsgvlfqlfygllgswtaylisilyleyrtrrerekaadfrnhviqwfevldgllgrhwrnaglafnctfllfgsviqligcasni

[0046] yyvndrldkrtwtyvfgaccattvfipsfhnyrvwsflglvmttytawymavaslvhgqvegvqhsgptrivlyftgatnilytfgghavtveimh

[0047] amwrpqkfkaiyllatlyvltltlpsaaasywafgdellthsnalallprtpfrdaavvlmlihqfitfgfactplyfvwekliglhdcrslckraaarlp

[0048] vvvpiwflaiifpffgpinsavgsllvsftvyiipalahmvtfrspqsrenaverpprfaggwtgayvinsfvvawvlvvgfgfggwasitnfvqqvntfglfakcyqcpphltaappaafmpppppmaaapsmppaatafnatglffpplpapapapspminfflrhhhrghhgrhgl*

[0049] SEQ ID NO 3 (ZmATL1)

[0050] MAREQLEESIVADGNGKEEEVGVMGIGAADGADDQHGGGKLSMKSLLWHGGSVWDAWFSCA

[0051] SNQVAQVLLTLPYSFSQLGMLSGVLLQIFYGFLGSWTAYLISVLYVEYRSRKEKEGVSFKNHVIQ

[0052] WFEVLDGLLGPYWKAAGLAFNCTFLLFGSVIQLIACASNIYYINDRLDKRTWTYIFGACCATTV

[0053] FIPSFHNYRIWSFLGLGMTTYTAWYLAIAALLNGQAEGVAHSGPTKLVLYFTGATNILYTFGGHA

[0054] VTVEIMHAMWKPAKFKYIYLLATLYVFTLTLPSSAAMYWAFGDELLTHSNAFSLLPKTRWRDAA

[0055] VILMLIHQFITFGFACTPLYFVWEKVIGMHDAKSIFKRALARLPIVVPIWFLAIIFPFFGPINSAVGA

[0056] LLVSFTVYIIPALAHVLTYRTASARMNAAEKPPFFLPSWTGMFVLNMFIVVWVLVVGFGLGGWASMVNFVRQIDTFGLFAKCYQCPKPPVPAAAQSPAPLPHH*

[0057] SEQ ID NO 4(ZmAIC-2)

[0058] MASEKVETIVAGNYMEMEHEPGGGGDHDQQPSGGAASSTSSSSRGGGKKKALSSLFWHGGSV

[0059] YDAWFSCASNQVAQVLLTLPYSFSQLGMASGVVFQLFYGLMGSWTAYLISILYVEYRTRKEREK

[0060] VDFRNHVIQWFEVLDGLLGKHWRNVGLFFNCTFLLFGSVIQLIACASNIYYINDKYDKRTWTYI

[0061] FGACCATTVFIPSFHNYRIWSFLGLLMTTYTAWYLTIAAIAHGQVEGVTHSGPSKMVLYFTGATN

[0062] ILYTFGGHAVTVEIMHAMWKPHKFKLIYLVATLYVLTLTLPSASAVYWAFGDMLLDHSNAFALL

[0063] PRSGFRDAAVIFMLIHQFITFGFACTPLYFVWEKLIGVHETGSVALRAAARLPIVAPIWFLAVVFPF

[0064] FGPINSTVGSLLVSFTVYIIPALAHMATFLPPAARENAVERPPRGLGGWAGMYAANFFVVAWVLVVGFGFGGWASTVNFVRQVNTFGLFTRCYQCPPRH*

[0065] SEQ ID NO 5(ZmAIC-3)

[0066] MSSEASSVVVADENGAAETVGVGRYVEMEKDQESSAAKSRLSGLLWHGGSAYDAWFSCASNQ

[0067] VAQVLLTLPYSFSQLGMLSGILFQLLYGLMGSWTAYLISVLYVEYRARKEREKADFRNHVIQWF

[0068] EVLDGLLGRHWRNVGLAFNCTFLLFGSVIQLIACASNIYYINDKLDKRTWTYIFGACCATTVFIP

[0069] SFHNYRIWSFLGLVMTTYTAWYLAVASLIHGQVDGVKHSGPTKMVLYFTGATNILYTFGGHAVT

[0070] VEIMHAMWRPQKFKAIYLMATLYVLTLTLPSAASVYWAFGDQLLTRSNALALLPRTAFRDAAV

[0071] VLMLAHQFITFGFACTPLYFVWEKLVGLHDCRSLCRRAAARLPVVVPIWFLAIIFPFFGPINSAVG

[0072] SLLVSFTVYIIPALAHMITFRSATARENAMEPPPRLLGRWTGAYMINAFVVAWVLVVGFGFGGW

[0073] ASMTNFVRQIDTFGLFTKCYQCPPPPPLPFPGGGLGNITMPFNGDGLPPTPAPSPAHYFFRHHRHHSHHRGL*

[0074] SEQ ID NO 6(ZmAIC-4)

[0075] MATGEQAEDAIVADVVGNGKGEEVRAMGDDAEQQRDGGKVSMKSLLWHGGSVWDAWFSCA

[0076] SNQVAQVLLTLPYSFSQLGMLSGVLLQVWYGLMGSWTAYLISVLYVEYRTRKEKEGVSFRNHV

[0077] IQWFEVLDGLLGPYWKAAGLAFNCTFLLFGTVIQLIACASNIYYINDRLDKRTWTYIFGACCATT

[0078] VFIPSYHNYRVWSFLGLGMTTYTAWYLTIAAAVHGQVPGVTHSGPSKLVPYFTGATNILYTFGG

[0079] HAITVEIMHAMWKPRKFKYIYLLATLYVFTLTLPSAAAMYWAFGDQLLTHSNAFSLLPRTPWRD

[0080] AAVVLMLVHQFITFGFACTPLYFVWEKAVGMHVTRSVFLRALVRLPIVVPVWFLAIIFPFFGPINS

[0081] AVGALLVSFTVYVIPALAHMLTYRSASARLNAAEKPPSFLPSWSGMFVLNAFVVAWMLVVGFGLGGWASVTNFIKQIDTFGLFAKCYQCPTKPHPGSPLPAPPHH*

[0082] Table 1. Auxin input carrier protein sequences of maize, rice and Arabidopsis

[0083]

[0084] Example 2: Obtaining constructs overexpressing the maize ZmAUX1 gene

[0085] Auxin influx carrier protein genes from different sources can be used for expression to screen for maize with increased ear row number, kernel weight per ear, and test weight in the present invention. The following is an example of obtaining T-DNA constructs overexpressing the maize auxin influx carrier protein ZmAUX1 (amino acid sequence shown in SEQ ID NO: 2) and ZmATL1 (amino acid sequence shown in SEQ ID NO: 3) genes.

[0086] 1. Construct of ZmAUX1

[0087] For convenient operation, the intron in the natural ZmAUX promoter-ZmAUX mRNA coding sequence (containing intron)-ZmAUX natural terminator shown in SEQ ID NO: 8 was removed. The specific operation is as follows:

[0088] The mRNA coding sequence of the ZmAUX1 gene can be artificially synthesized according to the nucleic acid sequence of SEQ ID NO: 7, and then the natural promoter of the ZmAUX1 gene (1 bp to 1775 bp in SEQ ID NO: 8) is added at the 5' end, and the natural terminator of ZmAUX1 (5984 bp to 6132 bp in SEQ ID NO: 8) is added at the 3' end to obtain a ZmAUX1 expression cassette with a Hind III restriction site added at the 5' end and a Kpn I site added at the 3' end.

[0089] The ZmAUX1 expression cassette and the glyphosate selection marker expression cassette pCMP-g10evo epsps (SEQ ID NO: 9) artificially synthesized with a Kpn I site added at the 5' end and an Xho I site added at the 3' end were inserted into the pCAMBIA1300 vector backbone digested with (HindIII, Kpn I) by three-step digestion and ligation. The obtained construct was denoted as AUCC.

[0090] Similarly, the pCMP from 1 bp to 364 bp in SEQ ID NO: 9 was replaced with p35S (SEQ ID NO: 10), and the obtained construct was denoted as AUCS.

[0091] An artificially synthesized plant virus enhancer 3xEnhancer (SEQ ID NO: 11) was inserted into the Kpn I site of AUCC, and the obtained construct was denoted as AUCE.

[0092] SEQ ID NO 7 (ZmAUX1 mRNA)

[0093] gccacgtacagtacagccccagccccagaggccccgcggcccggcatgaaaagaaaggacgctgggtttctttatgccccagctgctggagtgctg

[0094] tctcccccccgggacatcgcctctccatatatacgacacccccgctcccccctgcaatgcacacaacaccagtaagcaagcaccgccagcacgcaca

[0095] agcaggcaaagctctcgaccatcgctctgagggattaatggcggcgggaggaggaggcggcggcatcgccgacgagaaggcccctgctgctgag

[0096] gcgttcggcgggcacctggaggcggcggagatgacggaggcggaggaggaacacagcggcgtcaagtcccggctgtcgggtctgctgtggcac

[0097] ggcgggtcggcgtacgacgcgtggttcagctgcgcgtcgaaccaggtggcgcaggtgctgctgacgctgccctactcgttcgcgcagctggggatg

[0098] ctgagcggcgtgctgttccagctcttctacggcctgctgggcagctggacggcgtacctgatcagcatcctgtacctggagtaccgcacccgtcggga

[0099] gcgcgagaaggccgcggacttccggaaccacgtgatccagtggttcgaggtgctggacgggctgctgggccggcactggcgcaacgccgggctg

[0100] gccttcaactgcaccttcctcctcttcggctccgtcatccagctcatcggctgcgccagcaacatctactacgtcaacgaccggctggacaagcggacg

[0101] tggacctacgtcttcggcgcctgctgcgccaccaccgtcttcatcccctccttccacaactaccgcgtctggtccttcctgggcctcgtcatgaccaccta

[0102] caccgcctggtacatggccgtcgcctcgctcgtccacggacaggtcgagggcgtccagcattcggggcccacaaggatcgtactctacttcaccggg

[0103] gcgaccaacatactctacacgttcgggggacacgcggtcacagtggagatcatgcacgcgatgtggcgtcctcagaagttcaaggccatctacctgct

[0104] ggcgacgctgtacgtgctgacgctgaccctcccttccgccgccgcctcctactgggcgttcggcgacgagctgctgacgcactccaacgcgctggcg

[0105] ctgctcccgcggacgcccttccgcgacgccgccgtggtgctcatgctcatccaccagttcatcaccttcggcttcgcctgcaccccgctctacttcgtct

[0106] gggagaagctcatcggcctccacgactgccgtagcctctgcaagcgcgccgccgccaggctccccgtcgtcgtccccatctggttcctcgccatcatc

[0107] ttccccttcttcgggcccatcaactccgccgtcggctcgctcctcgtcagcttcaccgtctatatcatcccggcgctcgcgcacatggtcaccttcaggtc

[0108] gccgcagtcgagggagaacgccgtggagcggccgccgaggttcgccggagggtggacaggcgcttacgtcatcaactccttcgtggtggcgtggg

[0109] tgctggtggtcggcttcggcttcggaggctgggccagcataactaacttcgtgcagcaggtcaacaccttcggcctcttcgccaagtgctaccagtgcc

[0110] cgccgcatctcactgctgccccgccggcggccttcatgccgccgccgccaccaatggccgcggcgccctccatgccaccggcggcgacggcgttc

[0111] aatgccaccggcctcttcttccctccgctgccagccccggctccggctccgtcgccgatgatcaacttcttcctccgccaccaccaccgtgggcaccac

[0112] ggccgccacggcctgtgacgtcgtcgtcgacgcgctgtgcattgcatgcatggtcgctagcgctgcgcctctgtcacgtacggccgattactccctgtc

[0113] gagtttgctccacagtagttttagctagttatatatcttggtcatcagcacgcagcaaaggtgcaagttaattagctactagcctgattagtaaaagttgtgtg

[0114] tcgtcaaatgccctgttgtacgttcatcagataaaagagatcgaggtacacgtggatgagagtctggacgtacgtacggtcataaggctaggtaactctg

[0115] cctacatgatcgtcaggatacgtttggagtcccttgtcctgtaccgtgtccttgtgtgtagctgctttcgagaaagaagctgagcagaaaaagaaaaaaaa

[0116] aagagaatggaatgttgttacgttttcctctcacgcagtcatatgccggcccgcgcgccttgtcgtagctctacgtgttctggttactgcttcgctgtttattactccggttatcaagtatttattgtctactagtttgtttttaaattaaaacgtaataaataaataaaagtataaatgag。

[0117] SEQ ID NO 8(ZmAUX1genomic)

[0118] acgatgggtagggcaatggtgacggttagggttttttcccctaattaaaaacagttttgttgttttttggatttttttggttcttaatttaccgagtgcttttttttgtc

[0119] gagtgcgcgaaaaaagtactcgacaaagaaccatttgccgataaataccgagtgtaatactcagcaaaggatttgtcgagtgcaaaaaggtctctgccg

[0120] agtatttgacacactcgacacagaaggccggtccagtagtgtatatgtggagtgtttacagaatgtcgtccgcagacagtggcttataagaggagtaaat

[0121] aatgatgacgacaaattctaactatggtcactaatatataccacgagggctctgtcccccacaccccctgcgtgcagggctacatgtgcttacgctcctgc

[0122] ccaagcctccctgcgatgggcctccgctccgctcgccaacacgacctccatatgcaaaatttagatcacaaactcaacaatctctaccttgagacaaatt

[0123] ccatcttgtagccataacatataatcaactttctgaataatagaagaaacaaactgctggtgccaacatagccactagggctaagcaattataccaattaag

[0124] ttcgagcaaagctcaaacttaaccccagaaacatgtttagtcatcatggaagctagattgaagtgtgtattgatcatgtatacccttaatttaccttccgtaac

[0125] tattttccggatataatgatacttaatatcaatgtgttttgtcctcgtttgaaacatttgatcctttgtaagataaataacactctgactgtcagaccaatgtaaata

[0126] atgatgacggcctaagcctcccgtcgatgggcctccacttctctcgacaacgtgagttccatatgcagaatttagattacaaactcaacaattgatatccaa

[0127] ttaagtcacactaggatactaacatatataaataaatctgggagccgaggtcagcaatagtgagttgctaaagacaagaggggatgggagacggcgat

[0128] gagaatatgggttgcgttggaggtgagcagaattttaggctggcgccggctacatttgataacaaaagaaaaaaaagaatggaaccgtgatctagcttg

[0129] tgtagaataatgcaataatgcaaggattatttgcacgtgccccacaacataattatatatacagagaaaagaaacatttttcaatataacatatattcttgtttc

[0130] aagttgtgttcttccatccaaaatatgtaaatggtatatgtgataaaaacatgcaagggacgatgtgaggagaagatccaagccatggaacacgcatcca

[0131] acggacaaacacgttcagttggatcttttagagacatcactcgagtgatgcacatactttcccggccccaaaagaaaaaaaaaggtgatgtgcatgcata

[0132] catgcattgcccccggcccacttagggcttgttcggttatttccaatccatacggattagaggggattgatacggattggaggggattttgacttactggag

[0133] attgaaaccccctcaatccccctcaatccatatggattggggtagaaccgaacaagcccttataatgtgtccaggcagaacactttcttcgtatgctctagc

[0134] aatgcacgctgctccgtccttgctttccacgggatttttcaccacgtcttttgatcacccgctcctttcgtttgctttcttgaacccaaataggatcgagtaata

[0135] ctatatattaatgataaaataaagagaggcgggtagcagccacgtacagtacagccccagccccagaggccccgcggcccggcatgaaaagaaag

[0136] gacgctgggtttctttatgccccagctgctggagtgctgtctcccccccgggacatcgcctctccatatatacgacacccccgctcccccctgcaatgca

[0137] cacaacaccagtaagcaagcaccgccagcacgcacaagcaggcaaagctctcgaccatcgctctgagggattaatggcggcgggaggaggaggc

[0138] ggcggcatcgccgacgagaaggcccctgctgctgaggcgttcggcgggcacctggaggcggcggagatgacggaggcggaggaggaacacag

[0139] cggcgtcaagtcccggctgtcgggtctgctgtggcacggcgggtcggcgtacgacgcgtggttcagctgcgcgtcgaaccaggtggcgcaggtgct

[0140] gctgacgctgccctactcgttcgcgcagctggggatgctgagcggcgtgctgttccagctcttctacggcctgctgggcagctggacggcgtacctga

[0141] tcagcatcctgtacctggagtaccgcacccgtcgggagcgcgagaaggccgcggacttccggaaccacgtgatccagtggttcgaggtgctggacg

[0142] ggctgctgggccggcactggcgcaacgccgggctggccttcaactgcaccttcctcctcttcggctccgtcatccagctcatcggctgcgccagcaac

[0143] atctactacgtcaacgaccggctggacaagcggacgtggacctacgtcttcggcgcctgctgcgccaccaccgtcttcatcccctccttccacaactac

[0144] cgcgtctggtccttcctgggcctcgtcatgaccacctacaccgcctggtacatggccgtcgcctcgctcgtccacggacaggtcgagggcgtccagca

[0145] ttcggggcccacaaggatcgtactctacttcaccggggcgaccaacatactctacacgttcgggggacacgcggtcacagtgtgagtatatctctgtcc

[0146] tgttagcctaattaatcgcatcattagtttatcagtctgtcccttgattactcctaatcggctagtagtagtagtagttaattaacagtaaaaaggcacagtagc

[0147] tcatatgcatgcgcgtgcgacgtgcgtgcgcgcacgctacacggctgcacgctctgccctcgcggctcggcacatgcatgctgcttgggaatgatgcg

[0148] ggccccatgtgtttttcttggggaccactgacatgcatatatatgggggggattaaataaatggccatgacggcgacaaagataaaagagagcagagc

[0149] agtgtcggtcttgctgccgctctcagttctaacttctcatcgccggcaagtgctagtgctaatgctactactaggtgcgtggtgtagtagcttaaaggacat

[0150] acagaccctcccacaacgcaacaaggacacgaccgttgcatgtactgtacgtataatacatgaggtacatgggtccccctgttcggctgttcacttccgt

[0151] ccatcggtcatatggagggagacccatgcacagcacagatgcatgcatgcatgcatgccaccgacacatgggtcccagcctgccacgaggcaccat

[0152] gactcgttgcttccgttgttgtgtgcgcttctttttgacgttgatacatacatatgtagggagatcatgcacgcgatgtggcgtcctcagaagttcaaggcca

[0153] tctacctgctggcgacgctgtacgtgctgacgctgaccctcccttccgccgccacctcctactgggcgttcggcgacgagctgctgacgcactccaac

[0154] gcgctggcgctgctcccgcggacgcccttccgcgacgccgccgtggtgctcatgctcatccaccagttcatcaccttcggcttcgcctgcaccccgct

[0155] ctacttcgtctgggagaagctcatcggcctccacgactgccgtagcctctgcaagcgcgccgccgccaggctccccgtcgtcgtccccatctggttcct

[0156] cgccatcatcttccccttcttcgggcccatcaactccgccgtcggctcgctcctcgtcagcttcaccgtctatatcatcccggcgctcgcgcacatggtca

[0157] ccttcaggtcgccgcagtcgagggaggtacgtaattgccttcgctagctcattcatactactccgtaacacagcatgtacagcagcactgtgcagctttg

[0158] gttgttgtccatatacatacatgagtaactgtcttgatcgaccatccacttgacgagacagtttcttggatgtgcatgactacactggggtccatgcacgca

[0159] aagagatagctagctagctcgccattgtcgcacgcatgcagtcgaccaaactgcttgcagtttgctcagattctcgctactacaaaacacgcacggcgc

[0160] accctgtccatcacctgctacatctgctagccgctgcatgcgcgacccaatcgatgagctagctgctagcctgcatggcgaggcgcgtcggtggtggc

[0161] catgcatgacctggacaacacactgattggagtccatgcaaacctgtagcgagatctcgtaacatccatcgtccacagatcgacctagctagctgtacat

[0162] gcatgcttacaaataagccacggatcggatgggtaccataccattaccataccttatatatatacacacatgcatgcattttctagtaccgccggcgcaatc

[0163] agcgtcgtgtcaccgaacgaaggcaaagacaaaactgataatccctttttgttattatcggcacccaagtagtggtgataataatagaaccactgtactgg

[0164] aaagccgcgcacaagggcgcagagacgagcatgcctttgtaccttgtggtgtccgggagctcttggcatgcatggatggagtggagggaggagcgg

[0165] agggcatacgactacaccatgcagtgatgcggcccacaaccatgtctgagcggggggagaaaaaaaaaagcaagcagttccaggaacactcttgag

[0166] agagcctcagctctcacatgcagtcacatccattgcactagacaagcccactttgtcatcagtaaggatagatcattatatatattctttctccccttcctgaa

[0167] agagaaaagacgatatatatttcattttccccattgtatatatatatatttatttcgcttggtggtagattgcatatatattaacaggggactaacatacaatgata

[0168] tgcatgtaactaactgcgcagaacgccgtggagcggccgccgaggttcgccggagggtggacaggcgcttacgtcatcaactccttcgtggtggcgt

[0169] gggtgctggtggtcggcttcggcttcggaggctgggccagcataactaacttcgtgcagcaggtcaacaccttcggcctcttcgccaagtgctaccagt

[0170] gcccgccgcatctcactgctgccccgccggcggccttcatgccgccgccgccaccaatggccgcggcgccctccatgccaccggcggcgacggc

[0171] gttcaatgccaccggcctcttcttccctccgctgccagccccggctccggctccgtcgccgatgatcaacttcttcctccgccaccaccaccgtgggca

[0172] ccacggccgccacggcctgtgacgtcgtcgtcgacgcgctgtgcattgcatgcatggtcgctagcgctgcgcctctgtcacgtacggccgattactcc

[0173] ctgtcgagtttgctccacagtagttttagctagttatatatcttggtcatcagcacgcagcaaaggtgcaagttaattagctactagcctgattagtaaaagtt

[0174] gtgtgtcgtcaaatgccctgttgtacgttcatcagataaaagagatcgaggtacacgtggatgagagtctggacgtacgtacggtcataaggctaggtaa

[0175] ctctgcctacatgatcgtcaggatacgtttggagtcccttgtcctgtaccgtgtccttgtgtgtagctgctttcgagaaagaagctgagcagaaaaagaaa

[0176] aaaaaagagaatggaatgttgttacgttttcctctcacgcagtcatatgccggcccgcgcgccttgtcgtagctctacgtgttctggttactgcttcgctgttt

[0177] attactccggttatcaagtatttattgtctactagtttgtttttaaattaaaacgtaataaataaataaaagtataaatgagctttgttgaccacgctgtcgttaac

[0178] gacacgcaaacaggatgagaacctcatagtatgggtgtgtttggttgcctgcatctgcgcgtgcttgcatcgtgggatgcgggtagctactgtttgattgctgtgagccaggctatatgcgtgc。

[0179] SEQ ID NO 9 KpnI-pCMP-G10-XhoI

[0180] ggtacctacgtaactagtctggcagacaaagtggcagacatactgtcccacaaatgaagatggaatctgtaaaagaaaacgcgtgaaataatgcgtctg

[0181] acaaaggttaggtcggctgcctttaatcaataccaaagtggtccctaccacgatggaaaaactgtgcagtcggtttggctttttctgacgaacaaataaga

[0182] ttcgtggccgacaggtgggggtccaccatgtgaaggcatcttcagactccaataatggagcaatgacgtaagggcttacgaaataagtaagggtagttt

[0183] gggaaatgtccactcacccgtcagtctataaatacttagcccctccctcattgttaagggagcaaaacaatggcggcgaccatggcgtccaacgctgcg

[0184] gctgcggctgcggtgtccctggaccaggccgtggctgcgtcggcagcgttctcgtcgcggaagcagctgcggctgcctgccgcagcgcgcggagg

[0185] gatgcgggtgcgggtgcgggcgcggggtcggcgggaggcggtggtggtggcgtccgcgtcgtcgtcgtcggtggcagcgccggcggcgaagg

[0186] ctgaggacgccctgcccgccaccttcgacgtgatcgtgcatccagctcgcgaactccgcggcgagcttcgcgctcagccatccaagaactacaccac

[0187] tcgctacctcctcgccgctgccctcgctgagggcgagacccgcgtggtgggcgtggctacctctgaggacgccgaggccatgctccgctgcctccg

[0188] cgactggggcgctggcgtggagcttgtgggcgatgacgccgtgatccgcggtttcggcgctcgcccacaggccggtgtgaccctcaacccaggcaa

[0189] cgctggcatggtggcccgctccctcctcggcgtggccgctctcacctctggcaccactttcgtgaccgactacccggactccctcggcaagcgccctc

[0190] agggcgacctccttgaggccctcgaacgcctcggtgcctgggtgtcctccaacgacggtcgcctcccgatctccgtgtccggcccagtgcgcggtgg

[0191] caccgtggaggtgtccgccgagcgctcctcccagtacgcctccgccctcatgttcctcggccctctcctcccggacggactcgaactccgcctcaccg

[0192] gcgacatcaagtcccacgctccgctccgccagacactcgacaccctctctgacttcggcgtgcgcgccactgcctccgacgacctccgccgcatctcc

[0193] atcccgggtggccagaagtaccgcccaggccgcgtgctcgtgccgggcgactacccgggctccgctgccatcctcaccgccgctgccctcctccca

[0194] ggcgaggtgcgcctctctaacctccgcgagcacgacctccagggcgagaaggaggccgtgaacgtgctccgcgagatgggcgctgacatcgtgcg

[0195] cgagggcgataccctcaccgtgcgcggtggccgccctctccacgccgtgactcgcgacggcgattccttcaccgacgccgtgcaagccctcaccgc

[0196] cgctgctgccttcgccgagggcgacaccacctgggagaacgtggccactctccgcctcaaggagtgcgaccgcatctctgacacccgcgctgagctt

[0197] gagcgcctcggcctccgcgcacgcgagaccgccgactctctctccgtgactggctctgctcacctcgctggtggcatcaccgccgacggccacggc

[0198] gaccaccgcatgatcatgctcctcaccctcctcggcctccgcgcagacgctccactccgcatcaccggcgcacaccacatccgcaagtcctaccctcagttcttcgctcacctcgaagccctcggcgctcgcttcgagtacgctgaggccaccgcctaactcgag。

[0199] SEQ ID NO 10(p35S)

[0200] ctagagcagcttgccaacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccaaagggctattgagacttttc

[0201] aacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcaaaaggacagtagaaaaggaaggtggcacctacaaat

[0202] gccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaa

[0203] agaagacgttccaaccacgtcttcaaagcaagtggattgatgtgaacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctc

[0204] agaagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcaaaaggaca

[0205] gtagaaaaggaaggtggcacctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacc

[0206] cccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg

[0207] cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctgaaatcaccagtctctctctacaaatctatctct。

[0208] SEQ ID NO 11(3xEnhancer)

[0209] catcgagcagctggcttgtggggaccagacaaaaaaggaatggtgcagaattgttaggcgcacctaccaaaagcatctttgcctttattgcaaagataa

[0210] agcagattcctctagtacaagtggggaacaaaataacgtggaaaagagctgtcctgacagcccactcactaatgcgtatgacgaacgcagtgacgac

[0211] cacaaaactcgagcaacgagatcatgagccaatcaaagaggagtgatgtagacctaaagcaataatggagccatgacgtaagggcttacgcccatac

[0212] gaaataattaaaggctgatgtgacctgtcggtctctcagaacctttactttttatgtttggcgtgtatttttaaatttccacggcaatgacgatgtgaccgtcga

[0213] cccactaaaacattgctttgtcaaaagctaaaaaagatgatgcccgacagccacttgtgtgaagcatgagaagccggtccctccactaagaaaattagtgaagcatcttccagtggtccctccactcacagctcaatcagtgagcaacaggacgaaggaaatgacgtaagccatgacgtctaatccca。

[0214] 2. Construct of ZmATL1

[0215] Similarly, the intron in the natural ZmATL1 promoter - Zm ATL1 mRNA coding sequence (containing intron) - ZmATL1 natural terminator shown in SEQ ID NO:12 was removed. The specific operation is as follows: The mRNA coding sequence of the ZmATL1 gene can be artificially synthesized according to the nucleic acid sequence of NM_001370703.1, and then the natural promoter of the ZmATL1 gene (1bp to 1492bp of SEQID NO 12) is added at the 5' end, and the natural terminator of ZmATL1 (7803bp to 8189bp of SEQ ID NO:12) is added at the 3' end to obtain the ZmATL1 expression cassette with a Hind III restriction site added at the 5' end and a Kpn I site added at the 3' end.

[0216] The ZmATL1 expression cassette and the glyphosate selection marker expression cassette pCMP-g10evo epsps (SEQ ID NO:9) with a synthetic Kpn I site added at the 5' end and an Xho I site added at the 3' end were inserted into the pCAMBIA1300 vector backbone digested with (HindIII, Kpn I) by three-step digestion and ligation. The resulting construct was designated as ATC.

[0217] Similarly, the pCMP at 1bp - 364bp in SEQ ID NO:9 was replaced with p35S (SEQ ID NO:10), and the resulting construct was designated as ATS.

[0218] The synthetic plant virus enhancer 3xEnhancer (SEQ ID NO:11) was inserted into the Kpn I site of ATC, and the resulting construct was designated as ATE.

[0219] SEQ ID NO 12 (ATL1 genomic)

[0220] ctggctggggcttcgcccactaacggaagcccagggcttccattacctcttccctatagatatagtatttagagccttatatatatatatatatataacatata

[0221] cattattgtatggatccagtaaaaggataaaacaaattttagtttaagacggagagagtatatattttcatatgcctatttaaactaaggactattgataccata

[0222] tttttataaaatgagcaagatctaaaatagttttgttggttacttattttcaattgttgtgaataaaaaaagacaacataaatattaaacattaagaaccttcatcc

[0223] atcgaagcattatcttcttttagaaataatggattttgaacgaaggttatgaagggcatgtcttcatttttcatacaaataatattacaatgtaacattatgacaa

[0224] gcacttatttcatattgcatgtcagaataatgtcaagatctgcattatatgttaataaagtgatacaagccgcatattacgcttgtaccttcggcttgctcggaa

[0225] gagagaatgcaagtacaaccttccaataaaaagcgtgaacagtacggtgtactattcatctatttatagacatggacgcaaaccaaacaaaagtacaatt

[0226] ataaccctgatatctatacattgctctaatacaaatatcaaggataaacaggtcttttcattgctggcttgagttcatgtatgaccttcgtgatcaccttatggc

[0227] gaacctttaattcttgccgaagcttcactgtattttctgcgtgtctgtcacaacttcggcacttagttcctttaacgtatctttgtcagaatgaattacaaacgatt

[0228] tcttcatcctgagaaccttcgacagagaaggagacccccaacaaatttgacttacaccagatctaaggtaattcgtgttcgctacggctttaatccggcttt

[0229] ggttgcggcttctaccgtattttatatctgtagattgcagctgtagcagtccaacagttacaacagctgcggctgcagggcgcaaaagaaatattcacaac

[0230] ataatatcgaccatcatgcagagtgctttgacaatatatggctctctttggaaacaaagtttttaaaaaccacaatatttgaaataatacaacataatttagtca

[0231] agacaataccacagtgtatattgcagttttaaaaactaggtctagatctaagtttagtataccttaaaataactatagtttttgcaatacttaatttttgaaaacag

[0232] atattttatctataagaccagtccacaaataaagaatgagcatttgtctgataatttatctatctttctatctataagaaagaagtaccagagaggagatataaa

[0233] ctaatagaaaacaaaacaaaaaaaagcggaagaacgaagtgtgaaaaggccgagatagaaggctcgcctcggcagtctgcaaggcgcttgttataa

[0234] agccgcaagtggtaccatttctcactcgccctctctccgctcgctcgctctctctctcaccgaatcacacacacaggcaggccagtgaggcaggggca

[0235] cgaaggcgcggctccctctctcaagcccctcgccattactcgctcataaaggaagaacagagcgagcgggtagcgaggaagaagctgggccgcgg

[0236] ggcaagaggagagaggtccccagccgtaggtctgctgtgcaactccggctcgccgcctgccaaccgcgcggcgcggccaccacgctaggcccct

[0237] cgccctcctcgtaggtgaacagagcctgcagtcgatacaacagtaaattccgcggcattccattcgctttccctctcgaaacgcaccctgcatttactgca

[0238] cgtcgctcgccctccggccatccgcgatcttcaaatcttccactgtaaaaaggttcgtgctgttgttttttttttacttttttcgtgcgttcgcgttcgacgcctc

[0239] gctcggctggttccttccttccttccgctgtgttcccttctgtttctgtacgggcggtgtatatatttttgttttatatcgttgtttggatcttgatctggccggctga

[0240] tgaatacgtgcttctctctccgacgaacgacgcagcgggcggagacgggaaggccgtgcgctcaaggatggcgcgcgagcagctggaggagtcc

[0241] atcgtggccgacggcaacggcaaggaggaagaggtcggggtcatgggcatcggcgccgcggacggcgccgacgaccagcacggcggcggca

[0242] agctcagcatgaagagcctgctgtggcacggcggctccgtctgggacgcgtggttcagctgcgcctccaaccaggttggcctcacgcacctcacgtc

[0243] ctcacctcactcatggcggagccgctccgcgcctccgccttgagttcgttcatgcagatggacagcggagcgccccgactgactgccactgaatttgc

[0244] gtcttggtcctggtcccgcaggtggcgcaggtgctcctgacgctgccgtactccttctcccagctggggatgctgtccggcgtgctgctgcagatcttct

[0245] acggcttccttggcagctggaccgcgtacctcatcagcgtcctctacgtcgagtaccgctcccgcaaggagaaggagggcgtcagcttcaagaacca

[0246] cgtcatccaggtatggctctcgtcgtcgtcgtcgtcgtccgtccccgctccactttctcctccccgccataccatgtgtaatactccaactgactacagtttc

[0247] gtccctttccatccgggctccggcttttgccacgaaaaccgatcctttttttgggggcgctgggttccgtacttccgttcggtcgttatcgatccgccgtgtc

[0248] tgcgaaaattgcggccttttgtgtaaacgcgagaggggtatcgatcccccggcctcggccactgatatgcatgcttttctgttgtgcttcgtgcttgccattt

[0249] ggccaaacgaaaagcgagcgacggggcatcgacccgtggtgatcctaacttatctcccgcgagatcggaaggaaccgggatttaagcgtgatgggt

[0250] tttgattaagcagtcgtgtatatatacttaataataataaaacaaaaaaaaacaaaagattactagtagtagaaagaagataggaagttggtggtgatatgg

[0251] aggagtaataatcttgtgaatgtgatgttgaccagtggttcgaggtgctggacgggctgctgggcccctactggaaggcggcgggtctggccttcaact

[0252] gcacgttcctgctgttcggctccgtcatccagctgatcgcctgcgcgaggtgggtcttcgcttccatcgtgctctcctgctactctgcacataggtagctgc

[0253] tgtggcaaggggagctgaaccgtggtgacatctctctctctctctctctctctctctccgtgcagcaacatctactacatcaacgaccggctggacaag

[0254] cggacatggacgtacatcttcggcgcctgctgcgccaccacggtgttcatcccgtccttccacaactaccggatctggtccttcctgggcctcggcatga

[0255] ccacctacaccgcgtggtacctcgccatcgccgcgctcctcaacggccaggtgagccccccccacagccaccgccaccggatccgatcgctattgtc

[0256] taccactgattcaccgtccccgtcgtcctcgccgacgcacatgcaggccgaaggcgtggcgcactccggccccaccaagctcgtgctctacttcaccg

[0257] gcgccaccaacatcctctacaccttcggcggccacgccgtcacagtgtaagcaaagccttctctttcccacaaccaactcaacaaattactgactactag

[0258] taagtagtacgcacgtgctaccacttctactgcagctttagcaggacgttagttaactagtactaatcctaccaagttccttgtttttgttgttaaataaatactc

[0259] tgcaattagcgctgctgttaagttcaccctggagcagagctgcagaagcaatggccccccctccccgcctccattaatggtgttcgggtaccagtgtacc

[0260] acaccagtagctgcaggaccacactgcaacagtgtagtgtagttaatcctaactacattcatgattgtggctttgcagctactagcttattagcttagcttgc

[0261] tccacgacgagcagatcccccatgaacagtcagggccacctatatatatacacacacataaaaaaggtaactgtaggcaggctgcgcttgtgcagttgt

[0262] gcggggtgggaaccagctgcggcctgcaggcatccggcgataaaaaaaattcagccgctaaagcccggtgcgctcatgtgcatttgagtgatgcatc

[0263] ttccaaaaggcccctcccctaccagtagaagatatagcctacgaggggcagagagaaaagggatgcagtgttggctagccactttgcattatagaata

[0264] ggcggctactacctcccaggatgatccctttattcctactagtcgtgttcatcgcttgcccgagctttcatctattctattgcctggattattgactggattttatt

[0265] gacatggctgcggcggcatgattaagaggccacgcgagcggggaataattagcaacagcgctgctcagctcgagtgcgcccggccgggtctttaag

[0266] cggcctcggagatctggggattttctatcccaagccagcatgtgggtgctcggttccctagctgcgcttgttctagtgcaggagggggtggggtgggaa

[0267] gggaacgggaaaagagggcgcgcttcattgaattcagccagcggccagcgcggagctctcacgtcatggcctgcctgcaggcgcaattcattccctt

[0268] gttgccctcatgcaaggggcgaaatgatgatgatgataagctttgcgaccatccatggattgggacacattttggtgtttggagctgtaggctgtagccag

[0269] ggaaaaaccttgcttggtggagcagtggcgacacacatgcccactttgagagcaaatgcatcctctgagagagagagagagagagagagagagag

[0270] agagagagagagagagagagagagagagagagagagagagagagagagagaggtggtggtgagtgtctctgtccatcttgactcacagtgatcca

[0271] gagcttattatgccagttaggctgttactgctctggagactatgcgagtgatgtgacgacgaccatttttagagtactagtgtatactagctagctcttattaa

[0272] catgcatcacgaggtcttcctatgctggagctgagttgtgttggcagcagcagcaggtgattagtttattactggtaacggccttgtttggccgcgtttggc

[0273] ggaggtgctccatcatttgactggaggctcgtgcatgtgttggcgagattcctcctgccgacgacgagatctttgccctgctgtgccggtgcgtgagtct

[0274] gactgggagaaccacttggccgcgccggattttcctttttgccgtacgtcttcacgtatcatttctcgtcaactactaaagcctcgggcctgggcatatagt

[0275] gttgcctcgcatgtgcagggccgttttgtccgttaatttgtactagaacatatatatacactgatgatgaacagctttactgaatgcaggtagccgaaagcta

[0276] gagaacaagctaacaataataccacgtttgtgttcttggtttgtttggcagcgagatcatgcacgcgatgtggaagcccgccaagttcaaatacatctacc

[0277] tgctggcgacgctgtacgtgttcacgctgacgctgccgtcgtcggcggccatgtactgggcgttcggcgacgagctgctgacccactcgaacgccttc

[0278] tcgctgctgcccaagacccggtggcgcgacgcggcggtgatcctgatgctgatccaccagttcatcaccttcggcttcgcgtgcacgccgctctacttc

[0279] gtgtgggagaaggtgatcgggatgcacgacgccaagagcatcttcaagcgcgcgctggcgaggctgcccatcgtcgtgcccatctggttcctcgcca

[0280] tcatcttccccttcttcgggcccatcaactccgccgtcggcgcgctgctcgtcagcttcaccgtctacatcatcccggccctggcgcacgtcctcacctac

[0281] cgcacggcgtccgcgcgcatggtacgtatacatatgcatgcgtgcatttgcattgcattgcacccccgtcgtttagcaaacaataacaacggcgtcctgt

[0282] ggcctggccggccgtcgcgttgcctaatgcctactggttcgggtacgagtaagtgacccgatccgttcatccccgttgcctagctagagtgcagtagta

[0283] ccgccaccagtcgcagcagctgtcacgaacagtggcaggactgcaacacgaaatagacagtggagatccgtgcacacgcagcagccagttcacgtt

[0284] agatgcaaagcaccggtcccccacttgggcaaatcccccaatcattgaatggagacaacagccacattcaattcgaagccggccccgcccccgcgg

[0285] cgcccgacaacgatatgggacgacggatcggggatcggaacaccgcatcacccccacgtcatcatccgcgtctctgcccgtcgcctcgccgaatgc

[0286] cggtgcccatggcagatgtaggcttcactgtgccggttccgacagctggcgggggcggtttcggagcgacttttttgcgagtacggcacggcggcgat

[0287] ggcaacgccggcgtgggtcaaggaaccgcttgcctgtgacggaagatctgacgtagcgccgatgcggtgctgacgttgggtgtcgcgctgtgacgg

[0288] aagaccctcctggcgaggcgcgacgcgacggtggtcgcgacgtcgcggcactggcgcgcgtctggacgcactcggccggccaatcgcgctccca

[0289] ggtcccagggcccggcatcgtgtggcggtgtggctggaacgcgagagatgatggctcctctgtcctccgtagacaacgggatggcgatggagatgt

[0290] gagatctcggtgtgcgctggagggttcctttcctcgccctccgttttccccagctttttatttccgaaaggaattcgagcagtgaccctgtgaaaatgcctc

[0291] gaaaaggatagggctaggaacaagctaaggcggggcacgccgcgatgcttatcttttattcatgtcagtgcttctggcgatcgatttcattctgacgactg

[0292] ctgctctgtgtttccctgcaaaaaaaaaattatttgtttttgcaacagaacgccgcggagaagccgcccttcttcctgccgagctggacggggatgttcgt

[0293] cctcaacatgttcatcgtggtgtgggtgctggtggtcggcttcgggctgggcggctgggccagcatggtcaacttcgtgaggcagatcgacacgttcg

[0294] ggctgttcgccaagtgctaccagtgcccgaagccgccggtcccggcggccgcgcagtcaccggcgccgctgccgcaccactagggcgcgcaggc

[0295] cccgtgacggcatggagctagcgtcgctgcttgcattgcagccggtgttaattgctactaggttttggtcgccttgtaatatacaagcctcctctagggtcg

[0296] tagaactcgatcacagcagagtcacgcaaaggcggctcgtgccgtgccgtgccgccgccgcgttgctttcccgcccgccctcctctgcctgatcggtc

[0297] cgtcggcattggtgtcgttttgttttcgtttggcttgccaccggtattaaaaagttgagtgatggtcatggcttcttaatccatatgatgatatgacatacgtatc

[0298] ctatataggttcattagttatttttccttgttggttcgtggaggtcggtccatgttgtgtagcgtgtgggtgactgtaatggactgcgctggaatgctagctaaa

[0299] ggcagaaaacaaaaaaccacagtgcccttttgtttgttattgccctctgctcattagtttctcgctggaatttttattatgggcttcttcgaaaggaccacaaa

[0300] gcgatcatccaagttccaagggagagaaggaaagcgagcagcaaaggaagaggcgctggctggctgatggatcgatgctgcggccgcggcgtgg

[0301] gtcggcgcggaccataattgcacgcactaccagggcctttctgaggctggattgcaacggaatgggggtcgtaaaagccat。

[0302] The binary vectors containing constructs AUCC, AUCS, AUCE, ATC, ATS, and ATE were respectively introduced into Agrobacterium tumefaciens LBA4404 by electroporation for subsequent transformation of maize.

[0303] Due to the differences in the ZmAUX1 and ZmATL1 genes, as well as the regulatory elements downstream of the 3' end of the expression cassette, introducing different constructs into maize is expected to obtain transgenic maize with overexpression of the ZmAUX1 and ZmATL1 genes to varying degrees in different tissues, so as to screen for target traits, namely transgenic maize with increased ear rows, ear grain weight, test weight, or yield.

[0304] Example 3. Transformation of transgenic lines

[0305] Agrobacterium-mediated genetic transformation of maize was carried out. Specifically, it was carried out according to the method and culture medium formula reported by Frame et al. (Plant Physiol, 2002, 129: 13 - 22). Glyphosate was used as the screening reagent, and the transformation steps were as follows: Maize inbred line Shen 3336 was planted. Maize ears 8 - 10 days after pollination were taken, and immature embryos with a size of 1.0 - 1.5 mm were collected. Agrobacterium containing the transformation vector was mixed in the infection medium, and the OD660 of the bacterial suspension was adjusted to 0.5 - 0.6. The collected immature embryos were placed in the Agrobacterium-containing infection solution and left standing at room temperature for 5 min. The embryos were then poured onto the co-culture medium, and the liquid was blotted dry. The embryos were placed with the flat side down on the co-culture medium and cultured at 22 °C for 3 - 5 days. The cultured immature embryos were transferred to the callus induction medium containing the final concentration of 200 mg / L ticarcillin antibiotic (GlaxoSmithKline, USA) and cultured in the dark at 28 °C for 10 - 14 days to kill Agrobacterium. All the calli after induction culture were transferred to the screening medium containing the final concentration of 2 mM glyphosate and cultured in the dark at 28 °C for 2 - 3 weeks. After induction culture, all the calli were transferred to fresh screening medium containing 2 mM glyphosate and cultured in the dark at 28 °C for 2 - 3 weeks. The surviving embryogenic tissues were transferred to the regeneration medium and cultured in the dark at 28 °C for 10 - 14 days and then transferred to fresh regeneration medium and cultured under light at 26 °C for 10 - 14 days. Well-developed plants were selected and transferred to the rooting medium and cultured under light at 26 °C until the roots were fully developed. The regenerated seedlings after rooting were transplanted into the greenhouse for growth and seed multiplication. A total of 140, 120, 55, 72, 59, and 61 independent transgenic single plants were produced in this example. Plants with single-copy insertion of T-DNA were screened by taqman probe quantitative PCR. The T0 generation plants were self-pollinated to harvest seeds, and the ear row numbers were recorded. The results are shown in Table 2.

[0306] Table 2. Changes in ear rows of transformants with different constructs

[0307]

[0308] Example 4, Agronomic Trait Determination and Selection of Transgenic Lines

[0309] Select two transformants with increased ear rows for each construct in Example 3. The transgenic maize and the control maize (maize inbred line Shen 3336) were designed according to a randomized block design with 3 replicates. The row spacing for experimental planting was 0.5 m and the density was 4,500 plants per mu. The ear row number, ear grain weight, and test weight of each treatment were measured. The results are shown in Table 3. The results indicate that the ear row number, ear grain weight, and test weight of these transformants were significantly increased compared to the non-transgenic control maize.

[0310] Table 3. Screening and Analysis of Key Traits of Different Transformants Overexpressing Auxin Input Carrier Protein

[0311]

[0312]

[0313] Example 5, Yield Determination of Transgenic Hybrid Maize

[0314] Select two transformants with increased ear rows for each construct in Example 3 as male parents, and hybrid seeds (transgenic Shen 3336xD5433) were prepared by crossing with the conventional female maize inbred line D5433. Using maize inbred line Shen 3336 as the male parent, hybrid seeds (Shen 3336xD5433) prepared by crossing with the conventional female maize inbred line D5433 were used as the control. The transgenic maize hybrids and the control maize hybrids (Shen 3336xD5433) were designed according to a randomized block design with 3 replicates. The row spacing for each experiment was 0.5 m and the density was 4,500 plants per mu. The yield of each treatment was measured. The results are shown in Table 4. The results indicate that the yield of these maize plants overexpressing the maize auxin input carrier protein and with increased ear rows, ear grains, and grain weight also increased.

[0315] Table 4. Yield Determination Results of Transgenic Maize Overexpressing Auxin Input Carrier Protein

[0316]

[0317] Example 6, Obtaining Maize with Increased Yield by Editing Leader Sequences

[0318] Through methods such as prime editing, e.g., Zong, Y., Liu, Y., Xue, C. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat Biotechnol 40, 1394–1402 (2022), and Wang, J., He, Z., Wang, G. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat Methods 19, 331–340 (2022), exogenous enhancers can be inserted into the genome to enhance the expression of genes near the insertion sites.

[0319] As an embodiment of the present invention, three pairs of PE gRNAs were designed (Table 5). Using the PE gRNAs shown in SEQ ID NO:14 and SEQ ID NO:15, the FMV enhancer (SEQ ID NO:13) was inserted into the promoter region of the ZmAUX1 gene between -860 bp and -616 bp from the translation start site (corresponding to 1068 bp to 1311 bp in SEQ ID NO:8), and the construct TEGE-1 was obtained.

[0320] Using the PE gRNAs shown in SEQ ID NO:16 and SEQ ID NO:17, the FMV enhancer (SEQ ID NO:13) was inserted into the promoter region of the ZmAUX1 gene between -1207 bp and -999 bp from the translation start site (corresponding to 721 bp to 928 bp in SEQ ID NO:8), and the construct TEGE-2 was obtained.

[0321] Using the PE gRNAs shown in SEQ ID NO:18 and SEQ ID NO:19, the FMV enhancer (SEQ ID NO:13) was inserted into the promoter region of the ZmAUX1 gene between -1597 bp and -1411 bp from the translation start site (corresponding to 331 bp to 516 bp in SEQ ID NO:8), and the construct TEGE-2 was obtained.

[0322] The above constructs were transformed into maize to obtain gene-edited maize with the FMV enhancer inserted into the promoter region, resulting in an up-regulated expression level of ZmAUX1. The obtained gene-edited maize showed an increase in the number of kernel rows, kernel weight per ear, test weight, and yield compared to the control maize.

[0323] Table 5. pegRNAs that insert enhancers into the promoter region to enhance the expression of the ZmAUX1 gene

[0324]

[0325] SEQ ID NO 13 (FMV enhancer)

[0326] catcgagcagctggcttgtggggaccagacaaaaaaggaatggtgcagaattgttaggcgcacctaccaaaagcatctttgcctttattgcaaa gataaagcagattcctctagtacaagtggggaacaaaataacgtggaaaagagctgtcctgacagcccactcactaatgcgtatgacgaacgcagtga cgaccacaaaa。

[0327] SEQ ID NO 14 (AUXTET1)

[0328] gcagaattttaggctggcgcGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCacttgtactagaggaatctgctttatctttgcaataaaggcaaagatgcttttggtaggtgcgcctaacaattctgcaccattccttttttgtctggtccccacaagccagctgctcgatgccagcctaaaTCATCTCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTTTTT。

[0329] SEQ ID NO 15 (AUXTET2)

[0330] tggatgcgtgttccatggctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCttgcctttattgcaaagataaagcagattcctctagtacaagtggggaacaaaataacgtggaaaagagctgtcctgacagcccactcactaatgcgtatgacgaacgcagtgacgaccacaaaacatggaacacTCATCTCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTTTTT。

[0331] SEQ ID NO 16(AUXTET3)

[0332] taccttccgtaactattttcGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCacttgtactagaggaatctgctttatctttgcaataaaggcaaagatgcttttggtaggtgcgcctaacaattctgcaccattccttttttgtctggtccccacaagccagctgctcgatgaatagttacggaTCATCTCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTTTTT。

[0333] SEQ ID NO 17(AUXTET4)

[0334] gtatcctagtgtgacttaatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCttgcctttattgcaaagataaagcagattcctctagtacaagtggggaacaaaataacgtggaaaagagctgtcctgacagcccactcactaatgcgtatgacgaacgcagtgacgaccacaaaaaagtcacactagTCATCTCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTTTTT。

[0335] SEQ ID NO 18 (AUXTET5)

[0336] atgacgacaaattctaactaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCacttgtactagaggaatctgctttatctttgcaataaaggcaaagatgcttttggtaggtgcgcctaacaattctgcaccattccttttttgtctggtccccacaagccagctgctcgatgttagaatttgtcgTCATCTCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTTTTT。

[0337] SEQ ID NO 19 (AUXTET6)

[0338] tatatgttatggctacaagaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCttgcctttattgcaaagataaagcagattcctctagtacaagtggggaacaaaataacgtggaaaagagctgtcctgacagcccactcactaatgcgtatgacgaacgcagtgacgaccacaaaatgtagccataacTCATCTCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTTTTT。

[0339] Example 7. Obtaining maize with increased yield through promoter gene editing.

[0340] Generally, changes in the nucleotide sequence of the promoter region can lead to the enhancement or weakening of the promoter. By using gene editing methods to change the nucleotide sequence of the promoter, multiple gRNAs are designed in the promoter region, and the gRNAs are combined pairwise and introduced into cells together with the Cas9 gene to obtain plants with mutated promoters (the main type is deletion mutation). The specific operations are as follows:

[0341] Seven sgRNAs were designed in the promoter region of the ZmAUX1 genome. These sgRNAs were combined pairwise, and it was expected to obtain gene-edited maize with multiple types of ZmAUX1 promoter mutations. By examining the number of kernel rows per ear, kernel weight per ear, test weight, gene-edited maize with increased number of kernel rows per ear, and / or increased kernel weight per ear, and / or increased test weight, and / or increased yield was screened.

[0342] Table 6. sgRNAs for editing the promoter region of the ZmAUX1 gene

[0343]

Claims

1. A method for increasing maize yield, characterized in that, The method is to overexpress an auxin influx carrier protein in maize to obtain transgenic plants with significantly increased ear row number, kernel weight per ear and test weight, thereby increasing the maize yield; the amino acid sequence of the auxin influx carrier protein is one of SEQ ID NO: 2 and SEQ ID NO:

3.

2. The method for increasing maize yield according to claim 1, wherein The overexpression of the auxin influx carrier protein is achieved by transferring an auxin influx carrier protein overexpression cassette into the maize genome. The auxin influx carrier protein overexpression cassette is formed by functionally connecting a promoter to the 5'-end and a terminator to the 3'-end of the auxin influx carrier protein coding gene.

3. The method for increasing maize yield according to claim 2, characterized in that, The auxin influx carrier protein overexpression cassette includes a ZmAUX1 expression cassette or a ZmATL1 expression cassette. The mRNA coding sequence of the ZmAUX1 gene in the ZmAUX1 expression cassette is artificially synthesized according to the nucleic acid sequence of SEQ ID NO: 7, and then a natural promoter of the ZmAUX1 gene shown by 1 bp to 1775 bp in SEQ ID NO: 8 is added to the 5'-end, and a natural terminator of ZmAUX1 shown by 5984 bp to 6132 bp in SEQ ID NO: 8 is added to the 3'-end, resulting in a ZmAUX1 expression cassette with a Hind III restriction site added to the 5'-end and a Kpn I restriction site added to the 3'-end; the mRNA coding sequence of the ZmATL1 gene in the ZmATL1 expression cassette is artificially synthesized according to the nucleic acid sequence of NM_001370703.1, and then a natural promoter of the ZmATL1 gene shown by 1 bp to 1492 bp in SEQ ID NO: 12 is added to the 5'-end, and a natural terminator of ZmATL1 shown by 7803 bp to 8189 bp in SEQ ID NO: 12 is added to the 3'-end, resulting in a ZmATL1 expression cassette with a HindIII restriction site added to the 5'-end and a Kpn I restriction site added to the 3'-end.

4. The method for increasing maize yield according to claim 2, wherein, The method is to co-transfer the auxin influx carrier protein overexpression cassette and a selectable marker gene expression cassette into the maize genome. The selectable marker gene expression cassette includes a glyphosate selection marker expression cassette pCMP-g10evo epsps.

5. The method for increasing maize yield according to claim 4, characterized in that, The nucleotide sequence of the glyphosate selection marker expression cassette pCMP-g10evo epsps is as shown in SEQ ID NO:

9.

6. The method for increasing maize yield according to claim 3, characterized in that, The method is to insert the auxin influx carrier protein overexpression cassette and the glyphosate selection marker expression cassette pCMP-g10evo epsps with a Kpn I restriction site added to the 5'-end and an Xho I restriction site added to the 3'-end into the pCAMBIA1300 vector backbone by triple digestion and ligation. The obtained construct is introduced into Agrobacterium tumefaciens LBA4404 by electroporation and then used to transform maize, and maize with increased yield is screened.

7. The method for increasing maize yield according to claim 6, characterized in that, The construct replaces the promoter downstream of the auxin influx carrier protein-encoding gene with the promoter p35S or inserts the plant virus enhancer 3xEnhancer at the Kpn I restriction site. The nucleotide sequence of the promoter p35S is SEQ ID NO: 10; the nucleotide sequence of the plant virus enhancer 3xEnhancer is SEQ ID NO:

11.

8. The method for increasing maize yield according to claim 1, characterized in that, The method introduces genetic modifications at the auxin influx carrier protein gene locus of the maize genome to increase maize yield; the genomic locus encodes the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; the genetic modifications include inserting an exogenous enhancer into the promoter region of the genomic locus or altering the nucleotide sequence of the locus promoter by gene editing.

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