CN111073896B - Gene for controlling corn grain filling, encoding product, primer, carrier and application - Google Patents

Abstract

The invention discloses a gene for controlling corn grain filling, a coding product, a primer, a carrier and application. A regulation mechanism of the artificial mutation corn grain filling mutant is analyzed through various means, a gene ZmGAMBB 1 for regulating and controlling corn grain filling and grain size is cloned, and the function of the gene is analyzed. The ZmGAMYB1 knockout can cause the expression quantity of downstream series genes to change, thereby causing the grain filling of the corn to be accelerated and the grain size to be increased. At the same time, the zmgamb 1 knockout caused increased resistance of maize seedlings to heavy arsenic. The invention can track and detect the ZmGAMYB1 gene knockout condition by using the specific detection primer of the gene knockout site. The knockout of the gene is associated with the grain filling and grain size properties of the corn and the resistance properties of the heavy metal arsenic in the seedling stage of the corn, can be used for improving the grain filling property, the grain size and the heavy metal resistance of the corn, and is applied to the breeding practice of improving the corn yield and the heavy metal resistance.

Description

Gene for controlling corn grain filling, encoding product, primer, carrier and application

Technical Field

The invention belongs to the technical field of corn genetic breeding, and particularly relates to a gene for controlling corn grain filling, a coding product, a primer, a vector and application.

Background

Corn (Zea mays L.) is one of the most important grain, feed and energy crops in the world, and plays an irreplaceable role in the aspects of guaranteeing the grain safety of the world, promoting economic development, relieving energy crisis and the like. The kernel is the most important component factor of corn biological yield, and the kernel development directly determines the kernel yield. Research shows that corn kernel development is a continuous biological process, relates to a series of complex physiological and biochemical metabolism, and can be divided into three main stages: (1) 0-15 days after pollination (0-15DAP), the embryo and endosperm cells are rapidly divided in the period, the cytological framework of the embryo and endosperm is mainly completed, and the potential accumulation space of the photosynthetic products is determined; (2) grains are quickly grouted 15-40 days (15-40DAP) after pollination, the speed of photosynthetic product accumulation is determined by the grouting speed during the period, and the time of photosynthetic product accumulation is determined by the effective grouting time; (3) and the grain dehydration and maturation period is 40-70 days (40-70DAP) after pollination, the period finishes the transportation of the nutrient substances converted from the gradually aged leaves and stems to the grains, and the grains are physiologically mature.

The corn kernel development process is controlled by a complex regulation network consisting of a plurality of regulation genes and functional genes. Through the interaction among various genes, the properties directly related to grain development, such as the cell framework, the grain filling rate, the effective grain filling time and the like of the corn grains are influenced, so that the grain yield difference among different lines of materials is caused. In the aspect of corn grain development research, in the prior art, a recombination inbred line group constructed by double parents of Nongda 108 is used for QTL analysis of two points in two years, and 6 genetic stable grain development related QTLs are identified; 33 genetically stable grain development non-conditional QTLs and 12 conditional QTLs were located using the "permanent F2" population. The rgf1 gene on the No. 2 chromosome of the corn is cloned by screening a grain development mutant, and the grain plumpness is reduced mainly by reducing starch accumulation; the study also showed that sugar uptake during the rapid cell division phase (especially 5-10DAP) determines the development of rgf1 genotype maize kernels. Through mutant screening and map-based cloning, the natural mutation of the maize Urb2 gene is revealed to significantly affect grain length and biological yield.

The corn grain filling related traits have important significance on the genetic improvement of grain size and the improvement of the corn yield per unit. However, the cloning of structural genes and non-coding genes related to corn grain filling is still relatively deficient at present, so that the development of excellent allelic variation of the grain filling related genes and the exploration of a regulation mechanism of the grain filling related genes are urgently needed, and further, theories and material support are provided for the breeding practice of corn yield improvement.

Disclosure of Invention

One of the purposes of the invention is to provide a gene ZmGAMYB1 for controlling corn grain filling, wherein the nucleotide sequence of the gene ZmGAMYB1 is shown in SEQ ID No. 1.

The second purpose of the invention is to provide an encoding product of the corn grain filling control gene ZmGAMYB1, wherein the amino acid sequence of the encoding product of the gene ZmGAYB 1 is shown in SEQ ID No. 2.

The third purpose of the invention is to provide a primer of the gene ZmGAMYB1 for controlling corn grain filling amplification, wherein the nucleotide sequence of the outer upstream primer is shown as SEQ ID NO.3, the nucleotide sequence of the outer downstream primer is shown as SEQ ID NO.4, the nucleotide sequence of the inner upstream primer is shown as SEQ ID NO.5, and the nucleotide sequence of the inner downstream primer is shown as SEQ ID NO. 6.

The fourth purpose of the invention is to provide a gene editing vector CRISPR/Cas 9-ZmGAMBB 1 for expressing the corn grain filling control gene ZmGAMBB 1, wherein the nucleotide sequence of the gene editing vector contains a PAM base sequence of a ZmGAMBB 1 gene, and the gene editing expression can be carried out on the gene.

The fifth purpose of the invention is to provide a positive clone for controlling corn grain filling gene ZmGAMBB 1 gene editing by using the gene editing vector amplification.

The sixth purpose of the invention is to provide a primer for tracking and detecting the positive clone, which is characterized in that the nucleotide sequence of the upstream primer is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8.

The seventh purpose of the invention is to provide the application of the corn grain grouting control gene ZmGAMBB 1 in the regulation and control of the size of corn grains.

The eighth purpose of the invention is to provide application of the corn grain grouting control gene ZmGAMBB 1 in regulation and control of heavy metal arsenic resistance of plants.

The ninth purpose of the invention is to provide the application of the corn grain filling gene ZmGAMBB 1 in expression regulation and control of downstream genes containing TAACTTCCTA die bodies in a promoter region in corn.

The tenth purpose of the invention is to provide the application of the corn grain grouting control gene ZmGAMBB 1 in corn genetic breeding.

Compared with the prior art, the invention has the beneficial technical effects that:

1. the invention discovers the gene ZmGAMBB 1 for regulating and controlling the corn grain filling, clones the cDNA sequence of the gene and analyzes the amino acid sequence of the coding product of the gene, and lays a foundation for the application practice of the gene in corn breeding.

2. The ZmGAMBB 1 can cause the corn grains of gene editing strains to be enlarged.

3. The ZmGAMYB1 can affect the expression quantity change of downstream genes of TAACTTCCTA motif contained in a promoter.

4. The ZmGAMYB1 can lead the resistance of gene editing strains to heavy metal arsenic stress to be enhanced, and helps breeding practice for improving the heavy metal resistance of corn.

5. The primer capable of tracking and detecting the ZmGAMYB1 gene editing site developed by the invention can be used for improving the size and filling of corn grains and provides help for breeding practice for improving the corn yield.

Drawings

FIG. 1 shows the positive cloning condition of CRISPR/Cas9-GAmyb gene editing positive event detected in different transgenic plants.

FIG. 2 shows CRISPR/Cas9-GAmyb target site editing.

FIG. 3CRISPR/Cas9-GAmyb transgenic maize compared with wild type in grain width, grain length, and hundred grain weight. A. B respectively shows that the CRISPR/Cas9-GAmyb transgenic corn is compared with the wild type in grain width and grain length; C. d, E shows the analysis of variance between CRISPR/Cas9-GAmyb transgenic corn and wild type in grain width, grain length and hundred grain weight; and indicates that the difference reached a significant level of p-0.05 and p-0.01, respectively.

Figure 4 is a phylogenetic tree of gamb 1 and homologues.

FIG. 5 shows the construction of GAMYB transcription factor DAP library.

FIG. 6 shows motif binding to GAYB transcription factor specificities.

Fig. 7 is a GO annotation of downstream regulatory genes of the GAMYB transcription factor.

Figure 8 is a comparison of the phenotype of CRISPR/Cas9-GAMyb transgenic maize treated with and without arsenic.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise specified, and the materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Corn grain filling gene ZmGAMBB 1 for controlling corn grain filling and grain size discovery and confirmation

Construction of ZmGAMYB1 gene CRISPR/Cas9 knockout vector

(1) gRNA primer design

When designing a primer, BsaI restriction enzyme recognition sites are inserted, Cas9 recognition PAM sites are searched in a gene single copy sequence, 19nt before NGG sites in the single copy sequence are searched, and the primer sequences are used as primer sequences to be handed over to Wuhan Olympic department Dingsheng Biotech company to complete primer synthesis.

(2) Four primer amplification

4-primer PCR amplification was performed using the pCBC-MT1T2 plasmid as a template. Zma-GAMMA 1-MT1T 2-F/Zma-GAMMA 1-MT1T2-R is 10 mM; Zma-GAMMA 2-MT1T 2-F0/Zma-GAMMA 2-MT1T2-R0, and the primer sequences are referred to Table 1. A50. mu.L system was amplified with 2 XEs Taq Master Mix, the reaction sequence is shown in Table 2, the reaction time at 72 ℃ was extended to 1min, and then the PCR product was purified and recovered.

TABLE 1 primer sequence information

TABLE 2 reaction procedure

(3) And (3) purifying and recovering the PCR product, and comprising the following steps:

a, adding 450 mu l of CP into a 1.5ml centrifuge tube, and uniformly mixing with the PCR product;

b, putting the adsorption column into a 2ml collecting pipe, adding the mixed solution into the adsorption column, centrifuging at 12000rpm for 1 minute, pouring the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;

c, adding 600ul of rinsing liquid WB into the adsorption column, centrifuging at 12000rpm for 1min, and pouring off waste liquid;

d, repeating the step d;

e, putting the adsorption column back into the collection tube, and centrifuging at 12000rpm for 2 min;

f, placing the adsorption column in a new 1.5ml centrifugal tube, and standing at room temperature for 10min for airing;

g after drying, adding 30ul of sterilized water preheated at 65 ℃ into the adsorption column, standing for 2min, and centrifuging at 12000rpm for 2min to collect the DNA solution.

(4) Purifying and recovering PCR products, performing enzyme digestion-connection, performing enzyme digestion connection at 37 ℃ for 2h, and performing enzyme inactivation at 80 ℃ for 10 min. And transforming the system into escherichia coli, sequencing after colony PCR, and carrying out plasmid miniextraction on the monoclonals with correct sequencing.

Transformation of ZmGAMYB1 Gene CRISPR/Cas9 knock-out vector

Crispr Cas9 MYB33& MYB65 is handed over to China seed group, Inc. to carry out the genetic transformation work of corn.

(1) Adding 100-200 ul of agrobacterium to 50ml of LB culture medium;

(2) carrying out shake culture at 28 ℃ and 200rpm overnight;

(3) when OD is reached₆₀₀Adding 50ul of As to the solution with the concentration of 0.4-0.6 to obtain the final solution with the concentration of 100 uM;

(4) when OD is reached₆₀₀Starting the conversion (about 2h) at 0.8-1.0;

(5) centrifuging at 3500rpm and 18-20 ℃ for 15 min;

(6) the supernatant was discarded and the pre-infected suspended cells OD were used₆₀₀Adding As to the solution of 0.2-0.4 until the final concentration is 100 uM;

(7) taking the pollinated ears 10-11 days, disinfecting with 70% alcohol, and airing and stripping embryos to pre-infection;

(8) Gently mixing the young embryos and the agrobacterium in a 2ml ep tube;

(9) swirling the EP tube for plus 40s and minus 40 s;

(10) standing for 10 min;

(11) sucking out the agrobacterium solution, placing the young embryos on sterilized filter paper, drying for 10-15 min, and culturing for 3 days in the dark;

(12) transferring to a resting culture medium for dark culture for 5-10 days;

(13) transferring to a screening culture medium, reducing the screening concentration by half (PPT is reduced to 0.4mg/L from the original 0.8mg/L, Hyg is reduced to 12.5-15 mg/L from the original 25-30 mg/L), and gradually increasing the concentration of the screening agent in the screening process, culturing for 2 months, and subculturing once every two weeks;

(14) subculturing the resistant callus to a regeneration culture medium, and performing illumination culture until leaves are formed;

(15) then subcultured to rooting culture medium;

(16) when the roots are formed (about 3-4 weeks), adding a small amount of sterile water for 3 days, and transplanting the roots into soil;

positive detection of ZmGAMBB 1 gene CRISPR/Cas9 knockout vector

(1) Screening of T1 generation positive plants

T1 generation seeds of all transgenic materials are grown and transplanted to a field, and meanwhile, DNA of leaves is extracted to identify the plants to be positive through PCR detection. Detection primers are designed on both sides of the editing site of GAMyb-Crispr/Cas9, the editing condition of the target site is detected, and an amplification product is detected by using 1.0% agarose gel. The T2 plants were treated identically to the T1 plants.

(2) Method for extracting corn leaf DNA by SLS (selective laser desorption ionization) method

When the corn seedlings reach 5 leaves, extracting the DNA of the leaves by an SLS method, and the operation steps are as follows:

a, taking a small amount of leaves, putting the leaves into a 2ml centrifuge tube, freezing the centrifuge tube with the leaves in liquid nitrogen by using a sample grinder, grinding, and adding 0.8ml of SLS-DNA extracting solution. Shaking for 5min to mix the solution and the sample;

b, adding equal volume of phenol prepared in advance: chloroform: shaking the isoamyl alcohol (25:24:1) solution for 5min, and mixing well;

c, placing the mixture into a centrifuge for centrifugation at 12000rpm for 10min, carefully sucking supernatant into a new 1.5ml centrifuge tube, adding precooled isoamyl alcohol with the same volume, shaking up, and incubating for 5min at the temperature of minus 20 ℃;

d, taking out the centrifuge tube, centrifuging at 12000rpm for 10min, pouring off the supernatant, and adding 500ml of 75% alcohol for rinsing;

e, pouring off the solution in the test tube, adding pure alcohol for rinsing, and airing;

f, adding water for dissolution, and adding sterilized water with proper volume according to the obtained DNA amount and application;

and g, detecting the quality of the DNA by adopting a spectrophotometer method, and storing the detected DNA in a refrigerator at the temperature of 20 ℃ below zero for later use.

(3) Positive identification of transgenic PCR

The DNA extracted from the SLS is used for Bar gene detection, the reaction system is Bar-F/Bar-R each 1 μ l, the primer sequence is shown in table 1, 2 xTaq Master Mix 5 μ l, DNA template 1 μ l, ddH is used ₂O is supplemented to 10 μ l, and the reaction program is referred to. The results of the detection by electrophoresis on 1% agarose are shown in FIG. 1.

(4) Target site editing detection

Specific primers at two ends of the target site are used, the target site is amplified by high-fidelity enzyme, and then the system is subjected to gel recovery. The recovered gel product was TA cloned with pMD19-T Simple Vector 1. mu.l, the recovered PCR product, Solution I5. mu.l, ligated at 16 ℃ for 30min, and transformed into E.coli, 10 single clones were picked for each individual strain and sequenced, compared to wild type C01, and the results are shown in FIG. 2.

Phenotypic identification of CRISPR/Cas9 knock-out vector positive transformation event for ZmGAMYB1 gene

The grain length, grain width and hundred grain weight of C01 and transgenic plant grains are measured respectively, and variance analysis is carried out by a t-test. The results are shown in fig. 3, with significant increases in grain length, grain width and hundred grain weight of the transgenes compared to the wild type.

Example 2

Evolutionary conservation of corn grain filling gene ZmGAMYB1

As shown in FIG. 4, the results of the evolutionary tree show that MYB transcription factors have unity of conservation and diversity during the evolution process, and are inferred to be a protein with elastic regulatory function generated by plants for adapting to and responding to the change of environment.

Example 3

Corn grain filling gene ZmGAMYB1 influences expression of downstream gene

The DAP-seq technology is utilized to research the whole genome binding site of the GAMYB transcription factor and analyze the genes which are possibly regulated and controlled at the downstream. The operation mainly comprises the following steps: (1) in vitro translation of transcription factors, (2) construction of DAP-library, (3) binding reaction, and (4) data analysis. The specific operation is as follows:

2-3 mu g pFN-19k-GAMyb1, pFN-19k-GAMyb2 and pFN-19k-GAMyb3 are mixed with 30 mu l TnT SP6 High-Yield Wheat Protein Expression System (Promega) cell-free Expression System, and then the tag fusion Protein can be expressed after 2h of Expression at 25 ℃.

Ultrasonic disruption of genomic DNA

1) The extracted genomic DNA was disrupted (average length 200bp) as follows:

ultrasonic setting:

Sample volume(μl)：130

Peak Incident Power(W)：50

Duty Factor：20％

Cycles per Burst：200

Treatment Time(s)：420

Temperature(℃)：11

2) DNA samples of 5. mu.g or less were diluted into 130. mu.l of TE buffer, disrupted according to the above procedure, and verified by 2% agarose gel electrophoresis, the results are shown in FIG. 5 a.

The Reaction systems were added to each other, 3. mu.l of Adapter, 5. mu.l of Quick T4 DNA library, 10. mu.l of 5 Xquick library Reaction Buffer, and ddH was used₂O to 50. mu.l, 5. mu.l, 10 XDA-Tailing Reaction Buffer 10. mu.l, End Repaired DNA 42. mu.l, dA-Tailed DNA 25. mu.l, with ddH ₂O was supplemented to 50. mu.l. The reaction system is reacted at 20 ℃ for 1h, A is added at the tail end of a DNA fragment, then 1.8 Xbeads are used for purification and recovery, finally 55 mu l of 0.1 XTE is added to elute the target DNA from the beads, namely DAP-library, and 1 mu l of detection concentration is taken.

DAP-library detection

Mu.l of the constructed DAP-library was PCR amplified with primers of unannealed DAP-seqA1 and DAP-seqB1 and detected by 2% agarose gel electrophoresis, the result is shown in FIG. 5 b.

Enrichment of DNA

Incubating GAYB 1-HaloTag fusion protein translated in vitro with Magne HaloTag Beads, fixing transcription factors in a capture label mode, and reacting with constructed DAP-library. The DNA fragment specifically binding GAYB 1-HaloTag was then eluted, and the long linker for high throughput sequencing was added by means of PCR, the results are shown in FIG. 5 c. Analysis of motif from GAMYB1

GAYB binding motifs (motifs) were analyzed from 6094peaks and it was found that the GAYB 1 transcription factor bound to the motif TAACTTCCTA (FIG. 6), which was detected in the promoter region of 357 genes obtained by sequencing.

GAMYB direct binding to motif was detected in the promoter region of 357 genes, including 14 non-coding genes, and 244 genes with functional annotations (excluding those with unknown function). To further reveal the function of downstream genes regulated by GAMYB1, Gene Ontology (GO) annotation was performed on these 244 functional genes, and the results showed (fig. 7) that it was significantly enriched in the cellular components that were plastids (24%), chloroplasts (23%), chloroplast fractions (15%); significantly enriched in molecular function are lyase activity (10%), carbon-carbon lyase activity (5%), catalytic activity (6%); significant enrichment in biological processes is stress response (23%), cell wall biosynthesis (6%), response to stimuli (29%), root development (5%), aerobic acid metabolism (11%).

Example 4

Gene ZmGAYB 1 gene internal marker for corn grain filling and breeding practice for enhancing arsenic heavy metal stress resistance of corn

Selecting seeds C01 and Cas9-ZmGAMYB1 which have plump seeds and uniform size and are free from insect pests, placing the seeds and the Cas9-ZmGAMYB1 in a culture dish with wet filter paper for accelerating germination for 72 hours, wherein the germination condition is a biochemical culture box with the temperature (25 +/-1) DEG C and the relative humidity of 75%. After corn seedlings emerge, selecting healthy plants with consistent growth vigor, transplanting the plants into nutrient solution containing 1/2Hoagland (pH 6.0), and conventionally culturing in a phytotron under the culture conditions that: the illumination/dark time is 16h/8h, the day and night temperature is 28 ℃/20 ℃, the illumination intensity is 110 mu mol.m < -2 > s < -1 >, and the relative humidity is 70%. During the culture period, continuously pumping gas into the nutrient solution by using a vacuum air pump, replacing the nutrient solution every 3 days, and respectively setting As with the concentration of 0 and 20mg/L to treat seedlings in a 4-leaf one-heart period, wherein each concentration treats 30 plants with consistent growth vigor.

10 days of hydroponics, WT and Cas 9-ZmGAMBB 1 were not treated with arsenic, no phenotypic difference, left in FIG. 8; after treatment with 20mg/L concentration of As, the material of Cas9-ZmGAMYB1 showed a significant increase in resistance, right in FIG. 8.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations.

Sequence listing

<110> Henan university of agriculture

<120> gene for controlling corn grain filling, and coding product, primer, vector and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2333

<212> DNA

<213> wheat

<400> 1

ctctttctct ttctctctct ctctctcctg aaatctctct ctcgctgacc tgagagttat 60

ccaaatactc tggtatggcg agatccccct cctcgggttc ccactcaact gctgtatcgt 120

agtagtcgac tacttccctg caatccgttg gtcagctcac aatagcagat gagtccattg 180

gaggagttcg actagtctcc accgctgctg cgagattccg ccagattata aaacttctgc 240

ccgagcaaag cggccgacca agttaccaca gagcaagtca gcgaggcgaa ataggcgacc 300

aagtcccaac cgagtaagaa agggagacga aactgccgac caagtctcta ggcccacaag 360

gttccgcgca taccctgacg gcaagccatg taccgggtga agagccaggc ggagggcgag 420

ggcgagggca aggacgagat gatgtcgcag gaccagatgg actcgccggt ggacaacgat 480

gtcagcagca gccgcaggtc gcctcgcagg ggcgtcgggg cgcccctgaa gaaagggccc 540

tggacggacg cggaggacgc catcctgata gactacgtta agaagcacgg cgtgggcaac 600

tggaacgcgg tgcggaagaa caccgagcta ttgcgctgcg gcaagagctg ccgcctcagg 660

tgggcgaacc acctcaggcc caacctcaag aaggaggcct tcaccccgga ggaggagcgc 720

ctcatcatcc agctccacgc caagctgggg aacaagtggt cgaggatggc tattcatttg 780

ccagggcgta ctgacaacga aataaaaaac tactggaaca cacgaaaaaa gagatgtgaa 840

cgagctagcc ttcctatcta tcctgctggt gtacgtaatc aatcttcaaa tgaagaccag 900

caattgtctg gtgatttgaa cggtggcgag aacatgtcca atgatcttct atccggaaac 960

agcctttgtc taccagattt taacaatgac agtttccgtg cgaaactgaa ggctttacca 1020

ccacagctgc cagctgtttc aataagcaat ttgctcggcc aaagctttgc atcaaaaggt 1080

tgtagcttca tggatcaggt agaccaagca gggatgctga aacaatctgg cagtgcgctt 1140

cctacattga gcgatgccat tgacgatgtg atttcctcgg ttgatcaatt ttcaaatgac 1200

tctgagaagc tcatgcagac tttaggtttt ggttatctca atgaagccaa cgctaccagc 1260

aagagtattg cgccttttgg ggttgcactt actggcagcc atgccccttt aaatggtatt 1320

ttctctgcat ctaggctcac aaatggtcct tcgaagatgg agcccccttc agtccaaaat 1380

agcaggctca agtatactgt ggatcctgca atgcagccta ctgagttagt agatccttac 1440

atgcagtctc tatcagcgac cccttcagtg aaatcagagt gtgcatcgcc gagaaacagt 1500

ggtcttttcg aagagctgct tcatgaacct catgcactaa gatctgggaa gagccaacaa 1560

ccatcggtcc gaagttcaag ttcttctgct ggcacacctt atgggactat ggttagctca 1620

gaatttgata tgggtcagga atattgggaa gaacagcccg gttctctcct cagcgaatat 1680

gctcacttca gtgggaatta tttggctgaa tgcgctcctc ctgttagcgc tgcatcaact 1740

gatatctttc cgctccccaa gatttctcct gcagaaagcc cttcaatggg ctctggcgag 1800

caggcgttag agcctaaaca tgagtcagca gcttcacgta cgtcatcttg gaaacttgag 1860

gcatgatgca ttattctctg ggaacacagc ccattcatcc agtttcaacg ataccatagc 1920

aatgctcatc ggcgatgtac ctgttcttgg tgatggaatt gtgctcgatt cttcctcatg 1980

ggacaacatg ccacatgctt ttcaaatggc ggaattcaaa tgagttccat ataatttctt 2040

tgtgatgctg aagggcttct tcctgcttgt tttggggtca atatcaggga acgccccact 2100

gattgtgacg ctgcattcct gacagagatc cttgtgatct tcatgcggat agtccttttg 2160

tctaattaag tgcatggaag ataacctgga tattattttt aattttttgc tttgtattag 2220

agaaccgttt ttgccatccc tgacagcatt tctgttggaa acgtgtcatg acttttgttt 2280

gaacaattta aatatcggtt atagataatg tgatgtgtgc catcccttga tgc 2333

<210> 2

<211> 492

<212> PRT

<213> wheat

<400> 2

Met Tyr Arg Val Lys Ser Gln Ala Glu Gly Glu Gly Glu Gly Lys Asp

1 5 10 15

Glu Met Met Ser Gln Asp Gln Met Asp Ser Pro Val Asp Asn Asp Val

20 25 30

Ser Ser Ser Arg Arg Ser Pro Arg Arg Gly Val Gly Ala Pro Leu Lys

35 40 45

Lys Gly Pro Trp Thr Asp Ala Glu Asp Ala Ile Leu Ile Asp Tyr Val

50 55 60

Lys Lys His Gly Val Gly Asn Trp Asn Ala Val Arg Lys Asn Thr Glu

65 70 75 80

Leu Leu Arg Cys Gly Lys Ser Cys Arg Leu Arg Trp Ala Asn His Leu

85 90 95

Arg Pro Asn Leu Lys Lys Glu Ala Phe Thr Pro Glu Glu Glu Arg Leu

100 105 110

Ile Ile Gln Leu His Ala Lys Leu Gly Asn Lys Trp Ser Arg Met Ala

115 120 125

Ile His Leu Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn

130 135 140

Thr Arg Lys Lys Arg Cys Glu Arg Ala Ser Leu Pro Ile Tyr Pro Ala

145 150 155 160

Gly Val Arg Asn Gln Ser Ser Asn Glu Asp Gln Gln Leu Ser Gly Asp

165 170 175

Leu Asn Gly Gly Glu Asn Met Ser Asn Asp Leu Leu Ser Gly Asn Ser

180 185 190

Leu Cys Leu Pro Asp Phe Asn Asn Asp Ser Phe Arg Ala Lys Leu Lys

195 200 205

Ala Leu Pro Pro Gln Leu Pro Ala Val Ser Ile Ser Asn Leu Leu Gly

210 215 220

Gln Ser Phe Ala Ser Lys Gly Cys Ser Phe Met Asp Gln Val Asp Gln

225 230 235 240

Ala Gly Met Leu Lys Gln Ser Gly Ser Ala Leu Pro Thr Leu Ser Asp

245 250 255

Ala Ile Asp Asp Val Ile Ser Ser Val Asp Gln Phe Ser Asn Asp Ser

260 265 270

Glu Lys Leu Met Gln Thr Leu Gly Phe Gly Tyr Leu Asn Glu Ala Asn

275 280 285

Ala Thr Ser Lys Ser Ile Ala Pro Phe Gly Val Ala Leu Thr Gly Ser

290 295 300

His Ala Pro Leu Asn Gly Ile Phe Ser Ala Ser Arg Leu Thr Asn Gly

305 310 315 320

Pro Ser Lys Met Glu Pro Pro Ser Val Gln Asn Ser Arg Leu Lys Tyr

325 330 335

Thr Val Asp Pro Ala Met Gln Pro Thr Glu Leu Val Asp Pro Tyr Met

340 345 350

Gln Ser Leu Ser Ala Thr Pro Ser Val Lys Ser Glu Cys Ala Ser Pro

355 360 365

Arg Asn Ser Gly Leu Phe Glu Glu Leu Leu His Glu Pro His Ala Leu

370 375 380

Arg Ser Gly Lys Ser Gln Gln Pro Ser Val Arg Ser Ser Ser Ser Ser

385 390 395 400

Ala Gly Thr Pro Tyr Gly Thr Met Val Ser Ser Glu Phe Asp Met Gly

405 410 415

Gln Glu Tyr Trp Glu Glu Gln Pro Gly Ser Leu Leu Ser Glu Tyr Ala

420 425 430

His Phe Ser Gly Asn Tyr Leu Ala Glu Cys Ala Pro Pro Val Ser Ala

435 440 445

Ala Ser Thr Asp Ile Phe Pro Leu Pro Lys Ile Ser Pro Ala Glu Ser

450 455 460

Pro Ser Met Gly Ser Gly Glu Gln Ala Leu Glu Pro Lys His Glu Ser

465 470 475 480

Ala Ala Ser Arg Thr Ser Ser Trp Lys Leu Glu Ala

485 490

<210> 3

<211> 36

<212> DNA

<213> Artificial sequence

<400> 3

aataatggtc tcaggcgagc aggctcaagt atactg 36

<210> 4

<211> 36

<212> DNA

<213> Artificial sequence

<400> 4

attattggtc tctaaacttt tctggtattc caaaag 36

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence

<400> 5

gagcaggctc aagtatactg gttttagagc tagaaatagc 40

<210> 6

<211> 33

<212> DNA

<213> Artificial sequence

<400> 6

ttttctggta ttccaaaagc gcttcttggt gcc 33

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence

<400> 7

acccacgtca tgccagttc 19

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

ctgcaccatc gtcaaccact a 21

Claims (3)

1. A method of increasing the size of a corn kernel, comprising:

knock-out in maizeZmGAMYB1A gene;

the above-mentionedZmGAMYB1The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. A method for increasing arsenic resistance in corn, comprising:

knock-out in maizeZmGAMYB1A gene;

the above-mentionedZmGAMYB1The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

3.ZmGAMYB1The application of the gene in breeding new corn variety with improved grain size and arsenic resistance is characterized in that the new corn variety has in vivoZmGAMYB1Low or no expression of the gene;

the above-mentionedZmGAMYB1The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

Images