CN114438117B - Application of GT1/HB13 gene in regulation and control of non-degradation and lodging resistance of maize female ear lower flowers - Google Patents
Application of GT1/HB13 gene in regulation and control of non-degradation and lodging resistance of maize female ear lower flowers Download PDFInfo
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Abstract
The invention discloses an application of GT1 and HB13 genes in regulating and controlling the degradation and lodging resistance of maize female ear lower flowers, and the invention utilizes CRISPR/Cas9 technology to edit genes of GT1 and HB13 targets in maize; the results show that compared with wild type and two single-mutant materials, the phenotype of the GT1/HB13 double-mutant plant is more severe, the tiller number is obviously increased, the root system is more developed, the number of ears is increased, a new female ear grows on the ear stem of a main ear, the multi-ear property is shown, axillary meristematic tissues at the lower part are also de-inhibited and developed into the female ear, the lower flowers in the female ear are not degraded, the carpals of the lower flowers continue to develop to form flowers, some lower flowers also form two flowers, the phenomenon of multi-flowers is shown, the later development continues to form additional small grains, and the corn mutant plant can show a strong female ear lower flower non-degradation phenotype and lodging resistance after knocking out GT1 and HB13 genes.
Description
Technical Field
The invention relates to the technical field of corn genetic breeding, in particular to application of GT1 and HB13 genes in regulation and control of female ear lower flowers without degradation and lodging resistance.
Background
Corn (Latin brand: zea mays L.) is the crop with highest economic value worldwide, is an important dual-purpose resource of grain, feed and biological energy, and is particularly important for guaranteeing the grain safety and economic development of China. The inflorescence is a reproductive organ of a plant and is a carrier of flowers and fruits, and the structure and the morphological formation of the inflorescence directly influence the number of flowers, the number of fruits, the size and a breeding system of the plant, so that the yield of crops is influenced. Early studies suggested that maize sexual differentiation involved fine regulation of various endogenous hormones, sex differentiation-related genes, external environmental factors, interactions between them, and the like. However, the molecular mechanisms of how the sex differentiation determinants of maize females and males and related hormones affect maize females and males formation are not completely understood. Therefore, the molecular mechanism of sex differentiation of corn is deeply analyzed, and particularly, a regulation network built by the corn inflorescences is revealed to provide a new target point for modern molecular genetic breeding of corn and a new view angle for the evolution of gramineous inflorescence structures.
HD-ZIP family proteins are a major class of transcription factors characteristic in plants, containing 60 (or 61) conserved amino acid Homeodomains (HD), are Homeobox (Homeobox) proteins, and 60 (or 61) conserved amino acids fold into a triple helix structure for specific binding to DNA. The HD-ZIP family comprises 4 subfamilies (HD-ZIP I-IV), wherein HD-ZIP I subfamilies members are involved in optical signal transduction and organ development, and are capable of integrating hormone signals such as abscisic acid and ethylene in response to abiotic stresses such as drought, low temperature and osmotic pressure. For example, atHB6, atHB7, atHB12 in Arabidopsis resist adverse reactions caused by drought and cold damage stress conditions through pathways such as stomatal closure, increased moisture uptake, and regulation of a series of stress-related genes. The rice HD-ZIP I subfamily gene Oshox22 has also been shown to affect abscisic acid synthesis and relies on abscisic acid signaling pathways to regulate plant response to drought and salt stress.
Chinese patent CN111909936A discloses that after mutation of HD-ZIP I subfamily gene Grassy tillers1 (GT 1) in corn, the carpels in the tassel florets of the plant are not degenerated and grow filaments, but the number of the filaments is not large, which indicates that the carpels of the tassel are abnormal in development, and the influence of mutation on the corn characters about GT1 and homologous gene HB13 thereof at the same time is not reported in the prior art.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide the application of the GT1/HB13 gene in regulating and controlling the female ear lower flowers of the maize inflorescence not to degenerate and resist lodging.
The above object of the present invention is achieved by the following technical solutions:
the invention utilizes CRISPR/Cas9 transgenic technology to edit genes of two targets of GT1 and HB13 genes in corn, then PCR amplifies GT1/HB13 genes of GT1/HB13 transgenic corn plants, and the PCR products are compared with wild type sequences after sequencing. The target site of the GT1 gene is shown to have base insertion and large fragment deletion, specifically shown as SEQ ID NO. 7, the 145bp position of the GT1 gene is shown to have 1bp base insertion, the 177 bp-408 bp base large fragment deletion leads to frame shift mutation, the HB13 gene is shown to have base deletion, specifically shown as SEQ ID NO. 8, the 307 bp-336 bp of the HB13 gene is shown to have base deletion, and the 728bp is shown to have 1 base insertion. The results of mutant plants show that the tillering number of GT1 and HB13 double mutant plants is increased, the root system is developed, the grabbing is stable, the lodging resistance of corn can be improved, the number of ears is increased, new female ears grow on the ear handles of main ears, the multi-ear property is shown, axillary meristematic tissues at the lower part are also de-inhibited and developed into female ears, and abnormal development of small flower barks of female ears of GT1 and HB13 double mutants is observed, normal filaments grow out without abortion of lower flower barks, and additional small grains continue to develop in the later period. Meanwhile, compared with two single-mutation materials of wild type and GT1 and HB13 genes, the phenotype of the GT1 and HB13 double-mutant plant is more intense, the number of tassel filaments is obviously increased, the tiller number is obviously increased, and the root system is more developed. The result shows that after the GT1 and HB13 genes are knocked out, the maize mutant plant can show a non-degenerate phenotype of the lower flowers of the female ears and stronger lodging resistance.
Therefore, the invention firstly protects the application of the GT1/HB13 gene in regulating and controlling the female ear lower flowers from degeneration and/or lodging resistance.
The invention also provides application of the GT1/HB13 gene in regulating and controlling corn tillering number, tassel femtocells and/or tassel multiple ears.
Specifically, the nucleotide sequence of the GT1 (Zm 00001d 028129) gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the HB13 (Zm 00001d 021934) gene is shown as SEQ ID NO. 2
Specifically, the double mutation of GT1 and HB13 genes is applied to the regulation of the female ear lower flowers of corn without degeneration, lodging resistance, corn tillering number, tassel femtization and/or female ear multiple ears.
Specifically, the double knockout mutation of GT1/HB13 genes, which is GT1 and HB13 genes, is applied to the regulation of the female ear lower flowers of corn not to degenerate, resist lodging, corn tillering number, male ear feminization and/or female ear multiple ear property.
Specifically, the GT1 gene mutant shown in SEQ ID NO. 7 and the HB13 gene mutant shown in SEQ ID NO. 8 are applied to the regulation of the female ear lower flowers of corn without degeneration, lodging resistance, corn tillering number, female tassel and/or female tassel multiple tassel.
The invention also provides application of the GT1/HB13 gene in cultivating maize varieties with no degradation, lodging resistance, increased tillering number, tassel feminization and/or tassel multiple-tassel female tassel.
Specifically, to knock out GT1 and HB13 genes in corn, GT1/HB13 double mutant transgenic corn plants were obtained.
Preferably, the knockout mutation is performed by using a CRISPR/Cas9 gene editing technology, specifically, a CRISPR/Cas9 double knockout vector of GT1 and HB13 genes is constructed, and corn is transformed, so that a GT1/HB13 double mutation transgenic corn plant is obtained.
Further preferably, the sgRNA sequence of the GT1 gene in the CRISPR/Cas9 gene editing vector is shown as SEQ ID NO 3-4, and the sgRNA sequence of the HB13 gene is shown as SEQ ID NO 5-6; specifically, a CRISPR/Cas9 gene editing vector containing sgRNA shown in SEQ ID NO 3-6 is used for transforming corn through an agrobacterium-mediated method to obtain a GT1/HB13 transgenic corn plant.
Specifically, in order to delete the 1bp base insertion at 145bp of the GT1 gene shown in SEQ ID NO. 1 in corn, 177 bp-408 bp base large fragment deletion and 307-336 bp base deletion of the HB13 gene shown in SEQ ID NO. 2, 728bp 1 base insertion are carried out, thereby obtaining the GT1 and HB13 gene double mutation corn plants shown in SEQ ID NO. 7 and SEQ ID NO. 8.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an application of GT1/HB13 genes in regulating and controlling maize female ear lower flowers not to degenerate, lodging-resistant, maize tillering number, male ear feminization and/or female ear multiple ears. The invention utilizes CRISPR/Cas9 transgenic technology to edit genes of two targets of GT1 and HB13 in corn, so that the target site of GT1 gene has base insertion and large fragment deletion, and HB13 gene has base deletion, thus obtaining GT1 and HB13 double mutant plants; the results show that the tillering number of the GT1 and HB13 double mutant plants is increased, the root system is developed, the grabbing is stable, the lodging resistance of corn can be improved, the number of ears is increased, new female ears grow on the ear handles of main ears, the multi-ears are displayed, the axillary meristem at the lower part is also de-inhibited to develop into female ears, the female ears of the GT1 and HB13 double mutant are observed to have small flower-core skin dysplasia, the lower flower-core skin is not aborted and normal flowers grow, and the later stage continues to develop to form extra small grains. Meanwhile, compared with two single-mutation materials of wild type and GT1 and HB13 genes, the phenotype of the GT1 and HB13 double-mutant plant is more intense, the number of tassel filaments is obviously increased, the tiller number is obviously increased, and the root system is more developed. After the GT1 and HB13 genes are knocked out, the corn mutant plant can show a non-degradation phenotype of flowers at the lower position of female ears and stronger lodging resistance, the corn mutant plant can show a pericardium dysplasia phenotype, and the transgenic corn plant is cultivated; and simultaneously, a theoretical basis is provided for revealing a regulation network built by the maize inflorescences.
Drawings
FIG. 1 shows the genotyping of GT1/HB13 transgenic plants, and the sequencing of PCR products followed by alignment with the wild type sequence.
FIG. 2 shows the phenotype of GT1/HB13 for regulating the tiller number, the female tassel, the multi-tassel property of the female tassel, the non-degeneration of the flowers at the lower position of the female tassel and the like of corn plants.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 construction of CRISPR/Cas9 transgenic plants of maize GT1 and HB13 genes
1. Method of
The construction process of the CRISPR/Cas9 knockout vector of the GT1 (SEQ ID NO: 1) and HB13 genes (SEQ ID NO: 2) is as follows: firstly, designing and screening GT1/HB13 gene specific target sequences (sgRNA) by utilizing SnapGene Viewer software and homologous sequence alignment, and selecting two optimal target sequences for each gene in order to ensure the gene editing efficiency:
GT1：GACGCCCCGCAAGGTGCAGCTGG(SEQ ID NO:3)，
GCTGAAGATGAAGGACAGGCTGG(SEQ ID NO:4)；
HB13：AGCAGGTCGCCGTCTGGTTC(SEQ ID NO:5)，
TCTGCAGCGGGAGCCCGAGC(SEQ ID NO:6)；
these target sequences were then introduced into the sgRNA expression cassette. At the same time, the hSpCas9 sequence in humans was commercializedThe PCR Cloning Kit is cloned into a pCPB vector to construct a pCPB-ZmUbi: hSpCas9 vector. Next, the two sgRNA expression cassettes were passed +.>The HDCloning Kit is inserted into the H of pCPB-ZmUbi: hSpCas9And (3) between indIII cleavage sites. The finally constructed CRISPR/Cas9 gene editing vector is used for subsequent genetic transformation after the PCR sequencing verification.
The constructed CRISPR/Cas9 vector converts a maize inbred line KN5585 by an agrobacterium-mediated method to obtain a GT1/HB13 transgenic maize plant.
2. Results
(1) Genotyping
The GT1/HB13 gene of the GT1/HB13 transgenic corn plant is amplified by PCR, and the PCR product is compared with the wild type sequence after sequencing. The result is shown in figure 1, which shows that the target site of the GT1 gene has the insertion of a base and the deletion of a large fragment, specifically the insertion of 1bp base at 145bp of the GT1 gene shown in SEQ ID NO. 1, and the deletion of a large fragment of 177 bp-408 bp base, which leads to the frame shift mutation; the HB13 gene has the base deletion, specifically the 307-336 bp of the HB13 gene shown in SEQ ID NO. 2 has the base deletion and the 728bp has the 1 base insertion; the obtained mutant GT1 gene sequence is shown as SEQ ID NO. 7, and the mutant HB13 gene sequence is shown as SEQ ID NO. 8.
(2) Phenotype observation of transgenic plants
As shown in fig. 2, compared with the wild type, the GT1 and HB13 double mutant plants have increased tillering number, developed root system and stable grasping, can improve lodging resistance of corn, increase the number of ears, grow new female ears on the ear handles of main ears, show multi-ear property, the axillary meristematic tissue at the lower part also takes off inhibition and grows into female ears, lower flowers in female ears do not degenerate, the carpel of lower flowers continues to develop to form filaments, some lower flowers also form two filaments, the phenomenon of multi-filament is shown, and the later development continues to form extra small grains. Meanwhile, compared with two single-mutation materials of wild type and GT1 and HB13 genes, the phenotype of the GT1 and HB13 double-mutant plant is more intense, the number of tassel filaments is obviously increased, the tiller number is obviously increased, and the root system is more developed.
Sequence listing
<110> agricultural university of south China
Institute of Biotechnology, Chinese Academy of Agricultural Sciences
<120> application of GT1/HB13 gene in regulation and control of non-degradation and lodging resistance of maize female ear lower flowers
<141> 2022-01-28
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<213> corn (Zea mays L.)
<400> 2
ggtacttcgt ctctgcaact gcgcagctca tcatcagtac cggcctaccc gctacctgca 60
ggacatgcac ctgctacatg aatggcaatt gatgtgtatt ctgtgcggtt tgcaggcact 120
gcgcaggagg agagaccgag agcgcggcgc aggaggcgtc gagcagcgag gtgcggcggc 180
ggcggcggcg agctggacgg aggaggggac cacaagaagc ggcggctgac cgacgagcag 240
gtagagatgc tggagctgag cttccgggag gagcggaagc tggagaccgg ccggaaggtg 300
cacctggccg ccgagctcgg gctcgacccc aagcaggtcg ccgtctggtt ccagaaccgc 360
cgcgctcgcc acaagagcaa gctgctcgag gaggagttcg ccaagctcaa gcaggcacac 420
gacgccgcca tcctccacaa atgccacctt gagaacgagg tacatgccat cctacttctc 480
actctttgcc tgtgctccaa tcctcttctc tgttcatccc catgcgccat ggatcatgga 540
cggcgttgca gacttgcagt atatatatat aatactgagc atgcatttcg tgggcacatg 600
cacggctggc aggtgatgag gctgaaggac aagctggtgc tcgccgagga ggagctgacg 660
cgtttcagat ccgcgggcaa ccacgcggtc tccggtgacg gcggagacgt catggcccgt 720
gccgtctgca gcgggagccc gagctcatcg ttctcgactg gcacctgcca gcagcccgga 780
ggaggcggcg gcggcggcga tcacctgggg gacgacgacc tgctctatgt tcctgactat 840
gcctacgctg acagcagcgt ggtcgagtgg tt 872
<210> 3
<211> 23
<212> DNA
<213> corn (Zea mays L.)
<400> 3
gacgccccgc aaggtgcagc tgg 23
<210> 4
<211> 23
<212> DNA
<213> corn (Zea mays L.)
<400> 4
gctgaagatg aaggacaggc tgg 23
<210> 5
<211> 20
<212> DNA
<213> corn (Zea mays L.)
<400> 5
agcaggtcgc cgtctggttc 20
<210> 6
<211> 20
<212> DNA
<213> corn (Zea mays L.)
<400> 6
tctgcagcgg gagcccgagc 20
<210> 7
<211> 372
<212> DNA
<213> corn (Zea mays L.)
<400> 7
gggttcggag cgcagccgcg gccggcggag ggcggcgagg gcgcggacga gcaggccagg 60
aagcggcggc tgagcgacga ccaggcgcgg ttcctggagc tcagcttcag gaaggagcgc 120
aagctggaga cgccccgcaa ggtgctagct ggccgccgag ctgggcctcg acgccaaggc 180
tggcggaggt ggaggaggag aagacgaagc tcgtcgcggc ggcggcggcg gcggcggccg 240
gcggcggagg agcgggcgga aggagcagcc ccccgacctc gtcgtcctcc tcgacgacga 300
cccacccccc ggccgcgctg ctggaggggg ggcacgtcgg ggaggagccg gaggacgccg 360
acgacccgca ca 372
<210> 8
<211> 754
<212> DNA
<213> corn (Zea mays L.)
<400> 8
cgctagggta cttcgtctct gcaactgcgc agctcatcat cagtaccggc ctacccgcta 60
cctgcaggac atgcacctgc tacatgaatg gcaattgatg tgtattctgt gcggtttgca 120
ggcactgcgc aggaggagag accgagagcg cggcgcagga ggcgtcgagc agcgaggtgc 180
ggcggcggcg gcggcgagct ggacggagga ggggaccaca agaagcggcg gctgaccgac 240
gagcaggtag agatgctgga gctgagcttc cgggaggagc ggaagctgga gaccggccgg 300
aaggtgcacc tggtcgccgt ctggttccag aaccgccgcg ctcgccacaa gagcaagctg 360
ctcgaggagg agttcgccaa gctcaagcag gcacacgacg ccgccatcct ccacaaatgc 420
caccttgaga acgaggtaca tgccatccta cttctcactc tttgcctgtg ctccaatcct 480
cttctctgtt catccccatg cgccatggat catggacggc gttgcagact tgcagtatat 540
atatataata ctgagcatgc atttcgtggg cacatgcacg gctggcaggt gatgaggctg 600
aaggacaagc tggtgctcgc cgaggaggag ctgacgcgtt tcagatccgc gggcaaccac 660
gcggtctccg gtgacggcgg agacgtcatg gcccgtgccg tctggcagcg ggagcccgac 720
tctctccccg ggaggggggg ccccggggaa accg 754
Claims (8)
1.GT1AndHB13the application of the gene double mutation in regulating and controlling the non-degradation and/or lodging resistance of the lower flowers of the female ears of the corn; the saidGT1The nucleotide sequence of the gene is shown as SEQ ID NO. 1,HB13the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
2.GT1AndHB13the application of the gene double mutation in regulating corn tillering number, tassel femtocells and/or tassel multifilance; the saidGT1The nucleotide sequence of the gene is shown as SEQ ID NO. 1,HB13the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
3. The use according to claim 1 or 2, characterized in that theGT1AndHB13double mutation of genes intoGT1AndHB13double knockout mutation of genes.
4. The use according to claim 3, wherein theGT1AndHB13the double mutation of the gene is described as SEQ ID NO. 7GT1Gene mutation and SEQ ID NO. 8HB13Mutation of the gene.
5.GT1AndHB13the application of the gene double mutation in cultivating corn varieties with no degradation, lodging resistance, increased tillering number, tassel feminization and/or tassel multiple-tassel female tassel; the saidGT1The nucleotide sequence of the gene is shown as SEQ ID NO. 1,HB13the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
6. The use of claim 5, wherein the corn is knocked outGT1AndHB13genes, getGT1/HB13Double mutant transgenic maize plants.
7. The use according to claim 6, characterized in that for the constructionGT1AndHB13CRISPR/Cas9 double-knockout vector of gene and transformed corn to obtainGT1/HB13Double mutant transgenic maize plants.
8. The use according to claim 7, wherein,GT1the sgRNA sequence of the gene is shown in SEQ ID NO 3-4,HB13the sgRNA sequence of the gene is shown in SEQ ID NO 5-6.
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| CN202210109362.9A CN114438117B (en) | 2022-01-28 | 2022-01-28 | Application of GT1/HB13 gene in regulation and control of non-degradation and lodging resistance of maize female ear lower flowers |
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| CN110577964A (en) * | 2019-08-28 | 2019-12-17 | 华南农业大学 | Application of UB2/UB3 gene in regulation and control of multiple-ear development of corn |
| CN111909936A (en) * | 2020-07-08 | 2020-11-10 | 华南农业大学 | Application of GT1 gene in regulation and control of maize male inflorescence sex determination and/or multi-spike development |
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| CN110577964A (en) * | 2019-08-28 | 2019-12-17 | 华南农业大学 | Application of UB2/UB3 gene in regulation and control of multiple-ear development of corn |
| CN111909936A (en) * | 2020-07-08 | 2020-11-10 | 华南农业大学 | Application of GT1 gene in regulation and control of maize male inflorescence sex determination and/or multi-spike development |
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