CN112048512B - Forward regulation factor for regulating corn leaf included angle and application thereof - Google Patents

Forward regulation factor for regulating corn leaf included angle and application thereof Download PDF

Info

Publication number
CN112048512B
CN112048512B CN202010994830.6A CN202010994830A CN112048512B CN 112048512 B CN112048512 B CN 112048512B CN 202010994830 A CN202010994830 A CN 202010994830A CN 112048512 B CN112048512 B CN 112048512B
Authority
CN
China
Prior art keywords
gene
zmcla7
corn
promoter
leaf
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010994830.6A
Other languages
Chinese (zh)
Other versions
CN112048512A (en
Inventor
陈彦惠
库丽霞
任真真
胡冠军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zheng Zhoushizhongzizhan
Henan Agricultural University
Original Assignee
Zheng Zhoushizhongzizhan
Henan Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zheng Zhoushizhongzizhan, Henan Agricultural University filed Critical Zheng Zhoushizhongzizhan
Priority to CN202010994830.6A priority Critical patent/CN112048512B/en
Publication of CN112048512A publication Critical patent/CN112048512A/en
Application granted granted Critical
Publication of CN112048512B publication Critical patent/CN112048512B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to the field of crop genetic breeding, and relates to a method for regulating and controlling the included angle of corn leavesZmCLA7‑1Gene, in particular to a positive regulatory factor for regulating and controlling the leaf angle of corn and application thereof. The positive regulatory factor is a geneZmCLA7‑1Or a geneZmCLA7‑1The promoter of (1), the geneZmCLA7‑1The base sequence of (A) is shown as SEQ ID NO.1, the promoter sequence is shown as SEQ ID NO.5,ZmCLA7‑1the promoter of the gene has base mutation and insertion/deletion, and the invention utilizes RNAi technology to inhibit corn endogenesisZmCLA7‑1Expression of the gene. Specifically, the ZmCLA7-1 gene is fused with other regulatory elements, such as a constitutive promoter (such as CaMV35S promoter) or an organ-specific promoter to construct a gene suppression expression vector, and a maize inbred line with small leaf angle and compact plant type is created by a transgenic technology (such as antisense RNA or RNAi) and is used for cultivating a new maize density-tolerant variety.

Description

Forward regulation factor for regulating corn leaf included angle and application thereof
Technical Field
The invention belongs to the field of crop genetic breeding, and relates to a method for regulating and controlling a corn leaf included angleZmCLA7-1The gene, in particular to a positive regulatory factor for regulating the leaf angle of corn and application thereof.
Background
Grain safety is a major problem related to the people's county. Irreversible factors such as continuous reduction of cultivated land, continuous increase of population, continuous increase of food demand and the like determine that food safety is a long-term and arduous task for ensuring the stable development of the society and economy in China. Therefore, the further breakthrough of the yield per unit level on the existing yield level is the core of the food yield increasing task in China. Domestic and foreign practices prove that increasing the planting density of the corn is an important technical approach for realizing continuous improvement of the unit yield. The yield of individual corn hybrids used in U.S. production has not improved significantly over the last 80 years, and continued improvements in the level of yield have been achieved by increasing plant density and increasing stress tolerance (Duvick, 1997, 2005). At present, the average yield per mu of developed countries such as the United states is 700 kilograms, and the planting density is about 5500 plants per mu; and the average yield per mu of the main yield area of the yellow-Huaihai corns in China is 400 kilograms, and the planting density is 3500 plus 4500 plants per mu. Therefore, the planting density and the yield of the corn in China still have further improved space at present.
The key for improving the field planting density is to select and popularize a density-resistant high-yield corn variety. Light is an energy source for photosynthesis of corn and an important environmental factor for regulating growth and development of the corn. How effectively corn can receive and utilize sunlight under high-density adverse conditions; the photosynthetic efficiency of the population can be improved only by depending on the regulation and control mechanism of the corns in the close planting environment; with the increase of population density, the leaves among individuals in the population are mutually overlapped to generate mutual shading, the lower leaves are shaded to influence the absorption and utilization of light energy, not only the photosynthetic efficiency is obviously reduced, but also the shading syndrome SAS (shade Avoidance Syndrome) is induced, the shading syndrome is a phenomenon which is often generated in high-density adverse circumstances, the generation of the shading syndrome often changes the original growth mode and the distribution mode of photosynthetic products, and the phenomenon is represented as stalk elongation, the vegetative growth period is shortened, the plants are induced to bloom and bear fruits in advance, and the early reproductive growth influences the development of seeds and fruits, thereby causing the reduction of yield (Franklin et al, 2005; Tao et al, 2008). Therefore, the new variety of the density-tolerant high-yield corn has to have the characteristics of density-tolerant plant type and density tolerance. Compact and reasonable plant type is an important extrinsic morphological index of high density and yield, and reasonable and efficient operation of a corn photosynthetic product source, flow and storehouse and the like is an important intrinsic physiological mechanism of high density and yield. The included angle of the stems and leaves is one of the most important traits in the plant type traits of the corns, the variety with small included angle of the stems and leaves is generally high in planting density, the absorption capacity of rhizome nutrients is strong, and the leaves of the plants are upright, so that more sunlight can be captured, the photosynthetic efficiency can be improved, and the population shading syndrome can be reduced. The influence of Pendleton et al (1968) on the corn leaf included angle and planting density on the yield is discovered by researches on the influence of the corn leaf included angle and planting density on the yield, and under the condition of close planting, the average yield per mu of a hybrid (compact) containing the leafless tongue gene ligulless 2 is improved by 41.2 percent compared with the corresponding hybrid; studies of Austin et al (1989) show that correlation exists between corn leaf included angle and yield, and the upright upper leaves and small included angle are beneficial to the middle leaves to receive illumination, enhance photosynthesis of the middle and upper leaves, and improve production and accumulation of dry matters, thereby improving yield. Therefore, the leaf angle is not only an important factor influencing the planting density and yield of the corn, but also a key character of the current and future high-yield breeding of the corn.
Although the QTL related to the leaf angle of corn is located, no corresponding candidate gene has been obtained by the map-based cloning technology so far. And some genes related to the included angle of the corn leaves are researched by comparing genomics and mutant technologies. Ku et al (2011) cloned a candidate gene for qLA2 on the second chromosome using comparative genomics methods. Research shows that the 5 ' -UTR end ' CTCC ' of the compact parent inbred line Yu 82 and the loose parent inbred line Shen 137 is changed into ' CCCC ', which influencesZmTAC1Thereby further influencing the size of the leaf angle. Thereby showing thatZmTAC1The level of gene expression is regulated by the change of 5 ' -UTR site sequence ' CTCC ' - ' CCCC '. Moreno et al (1997) pairslglThe research of the mutant shows that the mutant isligulelesslThe gene expression deletion mutant shows that the tongue and the leaf ear cannot be formed, and the joint of the leaf blade and the leaf sheath cannot be developed. Using activator (Ac) transposable element as a molecular taglglAllele of (2)lgl-mlIsolated and cloned from the culture medium, and the research proves thatLG1Genes function in a cell-autonomous manner. Juarez et al (2004) foundrld1Andlbl1the mutants exhibited a leaf morphology in the paraxial/superior direction. By cloning the corresponding geneNow, the process of the present invention,rld1encodes an HD-ZIPIII protein, and limits the spatial near axial end expression through the transcriptional cleavage of miR166-directed at the far axial end. Semi-dominantRldl-OOne single nucleotide substitution of the mutant at the complementary site of miR166 results in sustained expression of the mutant transcript at the distal axis, which causes leaves to be biased towards the proximal axis. The genetic analysis shows thatlbl1AndRldl-Oinhibit each other, indicating that these 2 genes function in the same pathway. For is toyabbyThe gene research finds that the gene directly causes the lateral organ to grow outwards. Compared with the identified QTL, the number of identified genes related to the leaf angle is far insufficient, which indicates that a large number of candidate genes related to the leaf angle are to be further identified.
Disclosure of Invention
In order to solve the problems of breeding a new variety of close-planting high-yield corn in the prior art, the invention provides a forward regulatory factor for regulating and controlling the leaf angle of corn and application thereof, and a related geneZmCLA7-1An unknown gene which is discovered by a map-based cloning technology and can regulate the leaf angle size, consists of 317 amino acids and comprises a DUF822 family conservative function structural domain, and the molecular biological function of the unknown gene is not reported in plants at present. The invention researches the molecular biological function of the gene and the promoter thereof for regulating and controlling the corn leaf angle, and provides a potential new gene resource for breeding a close-planting type excellent high-yield corn new variety.
The technical scheme of the invention is realized as follows:
forward regulatory factor for regulating and controlling leaf angle of corn, and forward regulatory factor is geneZmCLA7-1Or a geneZmCLA7-1The promoter of (1), the geneZmCLA7-1The base sequence of (A) is shown as SEQ ID NO.1, the promoter sequence is shown as SEQ ID NO.5,ZmCLA7-1the promoter of the gene has base mutation and insertion/deletion as shown in FIG. 5.
The geneZmCLA7-1The amino acid sequence of the encoded protein is shown in SEQ ID NO.2, and comprises a DUF822 family conserved functional structural domain.
The application of the positive regulation factor in regulating the corn leaf included angle comprises the following steps:
(1) using compact selfing line Yu 537A as a donor parent, using loose corn selfing line Shen 137 as a receptor parent, and adopting backcross transformation combined with molecular marker to assist in selecting and constructing a near isogenic line Yu Shen 137-NIL76 of the major qLA7 located on the seventh chromosome;
(2) isolation from Shen 137-NIL76 by using map-based cloning techniqueZmCLA7-1Genes, then constructing the corn endogenesis by RNAi technologyZmCLA7-1An expression vector for the gene;
(3) endogenously transforming cornZmCLA7-1The expression vector of the gene is used for transforming agrobacterium-infected cells for transforming a maize inbred line B104 to obtain transgenic positive plants with leaf included angle expression reduced to different degrees.
In the step (2), the corn is endogenousZmCLA7-1The expression vector of the gene was obtained using cDNA from selfed line 537A as template, according to B73CLA7-1The primer pair designed by the cDNA sequence is obtained by constructing the primer.
The primer pair is RNAi-F and RNAi-R, wherein the sequence of RNAi-F is shown as SEQ ID NO.3, and the sequence of RNAi-R is shown as SEQ ID NO. 4.
The invention has the following beneficial effects:
1. the invention aims to provide a gene ZmCLA7-1 for controlling the leaf angle of corn and a promoter thereof, and a forward genetics method (preliminary QTL determination and fine positioning of the leaf angle by constructing a mapping population) is adopted to accurately separate candidate genes. The gene has a DNA fragment shown as SEQ ID NO.1, and the promoter is shown as SEQ ID NO. 5. The invention also comprises a protein sequence shown as SEQ ID NO.2, the accumulation amount of mRNA of the gene is reduced, and the maize leaf angle is reduced, so that the cloning of the gene is helpful for understanding the molecular mechanism of maize leaf angle formation.
2. The invention utilizes RNAi technology to inhibit corn endogenesisZmCLA7-1Expression of the gene. Specifically, ZmCLA7-1 gene is fused with other regulatory elements, such as constitutive promoter (such as CaMV35S promoter) or organ-specific promoter to construct gene suppression expression vector, transgenic technology (such as antisense RNA or RNAi) is used to create compact-plant-type maize inbred line with small leaf angle, and the maize inbred line is used to control the plant growth of maizeThe method is used for cultivating new density-resistant corn varieties.
3. The invention transforms the gene containing the sequence shown in SEQ ID NO.1, introduces the gene into corn plants, can create corn breeding materials with compact plant type and small leaf included angle, and has important practical application significance for corn breeding.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a field profile of Shen 137-NIL76 and Shen 137. In the figure, A: plant morphology of Shen 137-NIL76 (left) and Shen 137 (right) in the stationary phase (10 days after maize flowering); b: shen 137-NIL76 (left) and Shen 137 (right) have the average angle between the leaves on the upper part of the ear.
FIG. 2 shows the molecular identification result of the map-based clone ZmCLA7-1 gene. In the figure, A: using BC 3 F 2:3-8 The 112 families of the population are positioned to position qLA7-1 into the intervals of the markers PZE-07122629825 and PZE-107077981; b: using BC 4 F 2 8723 individuals are selected in a group, 19 cross-over individuals are screened, and fine positioning is carried out on the 19 cross-over individuals, wherein qLA7-1 reduced marker SSR7-35 and SSR7-157 intervals are obtained; c: the interval of qLA7-1 was narrowed down by fine localization to a region of 43.6Kb containing 1 candidate gene. D: the candidate geneZm00001d021927ZmCLA7-1) The expression level (mRNA accumulation level) in deposit 137-NIL76 and deposit 137.
FIG. 3 is a drawing showingZmCLA7-1Expression patterns of genes, qRT-PCR detectionZmCLA7-1The expression level (mRNA accumulation level) of gene from 7-9 leaf stage in Shen 137 and Shen 137-NIL76 in leaf, 18S gene as internal control;
FIG. 4 shows the use of RNAi technique, ZmCLA7-1The transgene (c) confers a reduced leaf angle plant phenotype on inbred line B104. In the figure, A: overexpression in stationary phaseZmCLA7-1Morphology of control plants (right) and positive plants (left); b: overexpression during stationary phaseZmCLA7-1Average value of the included angle of the upper leaves of the ears of the control plants (right) and the positive plants (left).
FIG. 5 is a drawing showingZmCLA7-1The base mutation and insertion/deletion of the promoter of the gene result in the change of the expression level of the gene. In the figure, A: the gene promoter between Shen 137 and Shen 137-NIL76 was more differentially compared between Shen 137 and Shen 137-NIL 76; b: the relative expression levels of the reporter gene driven by the Shen 137 and Shen 137-NIL76 promoters; the two grey boxes represent the start and stop codons, respectively.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
The invention utilizes compact corn inbred line Yu 537A bred by Henan agricultural university as female parent and loose corn inbred line Shen 137 (figure 1) bred by Shenyang city agricultural academy as male parent to obtain F 1 The recombinant inbred line population obtained by multi-generation continuous inbred is a positioning population, QTL positioning is carried out on the leaf angle through the phenotype identification of the leaf angle of 3 places, and 2 QTLs related to the leaf angle are positioned, wherein the effect value of the main effect qLA7-1 on the seventh chromosome is the maximum. Therefore, the inbred line 537A is used as a donor parent, and the inbred line Shen 137 is used as a receptor parent to construct qLA7-1 near isogenic line. The constructed near-isogenic gene is Shen 137-NIL76 (FIG. 1), and the following example 1 will describe the specific technical implementation steps of the present invention in detail for further understanding the contents and purposes of the present invention.
Example 1
Construction of near isogenic lines
A near isogenic line is constructed by taking a maize inbred line 537A with a small leaf angle and a compact plant type as a donor parent and taking a maize inbred line Shen 137 with a large leaf angle and a loose plant type as a receptor parent through backcross transformation combined with a molecular marker-assisted selection technology (figure 1).
Obtaining F by combining two parents in Zhengzhou spring in 2014 1 (ii) a Planting F in Hainan in winter in 2014 1 The surrogate and acceptor parent sink 137, sink 137 and F 1 Backcrossing to obtain BC 1 F 1
According to BC 1 F 1 Selecting 13 plants with small leaf angle to form spikes in Zhengzhou in 2015 spring, and backcrossing with Shen 137 to obtain BC 2 F 1 (ii) a Selecting 15 single plants with small leaf included angle from the plants, carrying out three-seed ear-forming in 2015 winter in Hainan, and carrying out backcross with Shen 137 to obtain BC 3 F 1
2016 spring, mix BC 3 F 1 In Zhengzhou, the seeds grow into spikes and grow from BC 3 F 1 Carrying out genotype analysis according to the size of the included angle of the plant leaves and the combination of qLA7-1 bilateral markers to obtain 16 crossover single plants, and continuously backcrossing the 16 crossover single plants with Shen 137 to obtain BC 4 F 1 Simultaneously for exchanging individual plants BC 3 F 1 -8 selfing to obtain BC 3 F 2 -8; 2016 winter in Hainan, three, BC 4 F 1 And BC 3 F 2- 8 planting (simultaneously, using SNP corn 3K chip to pair BC 3 F 2- Genotyping of 112 individuals of 8), according to BC 4 F 1 Phenotypic combination genotype of the population 11 crossover individuals were selected for selfing to obtain BC 4 F 2
2017 obtaining BC in Zhengzhou spring 4 F 2 Planting according to BC 4 F 2 Phenotype of the population combined with genotype analysis, 19 crossover individuals were obtained and backcrossed with Shen 137 to obtain BC 5 F 2 (ii) a 2017 winter in Hainan Jianjiang BC 5 F 2 Planting according to BC 5 F 2 The phenotype of the colony is combined with genotype analysis to obtain 9 crossover individuals, genetic background analysis is carried out on the 9 crossover individuals by adopting the SNP corn 3K chip, 2 crossover individuals with high genetic background recovery rate are selected from the 9 crossover individuals for selfing, and then the homozygous crossing at the site is obtainedGenetically stable near isogenic lines.
Example 2
Isolation by map-based cloningZmCLA7-1Gene
Using BC 4 F 2 The strain 8723 is used as fine positioning population, and the single strain 812 with leaf angle less than or equal to 20 deg is selected from the fine positioning populationqLA7-1The two-sided markers PZE-07122629825 and PZE-107077981 were genotyped, from which 12 strains were selected from which recombinant crossover occurred at PZE-07122629825 or PZE-107077981; meanwhile, 673 single plants with leaf included angles larger than 25 degrees are selected, and 7 exchange single plants with large leaf included angles are selected and screened by combining with molecular marker selection. The genotype of the crossover individual paper strain is analyzed by using newly developed 12 pairs of primers SSR7-1, SSR7-2, SSR7-3, SSR7-4, SSR7-5, SSR7-6, SSR7-8, SSR7-9, SSR7-10, SSR7-11 and SSR7-12 (the marker development method refers to ZmCLA4 gene map-position cloning and function analysis related to ZmCLA and maize leaf angle, doctor class of university of agriculture in Henan, 2014). qLA7-1 can be defined between markers SSR7-6 and SSR7-10 based on the marker genotype of the target segment of the crossover individual (FIG. 2A).
To further determine qLA7-1 as 1 or more candidate genes, BC was used 5 F 2 The population 6851 strain is taken as a fine positioning population, 452 strains are selected from the population, the genotype of each single strain is analyzed by using markers SSR7-6 and SSR7-10 on both sides of qLA7-1, and 12 strains of the single strain are selected from the population, wherein the single strain has a leaf included angle of less than or equal to 20 degrees, and the single strain is recombined and exchanged at SSR7-6 or SSR 7-10; meanwhile, 386 single plants with leaf included angles larger than 25 degrees are selected and 5 exchange single plants with large leaf included angles are selected and screened by combining molecular marker selection. Then, the genotype of the crossover individual is analyzed by using a 10 marker (SSR 7-7, SSR7-8, SSR7-9, SSR7-13, SSR7-14, SSR7-15, SSR7-16, SSR7-17, SSR7-18 and SSR 7-19) with strong polymorphism. qLA7-1 can be defined between markers SSR7-15 and SSR7-17 based on the marker genotype of the target segment of the crossover individual (FIG. 2B).
By comparing the homozygous recombinants marked with SSR7-15/SSR7-17 interval containing Yu 537A gene with Shen 137 which is a flattened selfing line, it is shown that the allele from Yu 537A reduces the upper leaf angle of the ear, and the allele from Shen 137, the upper leaf angle of the panicle is increased, and genes for controlling the upper leaf angle of the panicle may exist in the interval of SSR7-15/SSR 7-17. Combined with B73 genome sequence, the marker SSR7-15/SSR 7-17. Using the MaizeGBD database search, it was shown that there was a predicted gene within this interval,Zm00001d021927and (fig. 2C). The mRNA of Shen 137 and Shen 137-NIL76 was reverse-transcribed into cDNA as a template, and the candidate gene was divided, and the promoter sequence of the candidate gene was isolated using the DNA of the above 2 materials as a template. The results of sequence variation analysis showed that the promoter sequence of the candidate gene was different between Shen 137 and Shen 137-NIL76, and thus it was found thatZm00001d021927The coding region and 3' -UTR region of the gene do not have important base mutations and insertions/deletions. The difference between the leaf angle of the sinker 137 and that of sinker 137-NIL76 is explainedZm00001d021927Differences in the sequence of the promoter region. To further verify the conclusion, qTR-PCR technology (ZmCL A4 gene map-based cloning and function analysis related to ZmCL, corn leaf angle, Ph university of Henan agriculture, academic thesis 2014) is adopted, Shen 137 and Shen 137-NIL76 leaves at 9 leaf stage are taken as materials, mRNA is extracted and is reversely transcribed into cDNA; these cDNAs were used as templates for analysisZm00001d021927The results show that the expression level of (A),Zm00001d021927there was a significant difference in the 2 materials (fig. 2D). These results indicate that the difference between the leaf angles of Shen 137 and Shen 137-NIL76 is caused by the difference between the leaf angles of Shen 137 and Shen 137-NIL76Zm00001d021927Is caused by different expression amounts, thereby illustratingZm00001d021927Namely a candidate gene for regulating and controlling the corn leaf angle in the qLA7-1 region, which is named asZmCLA7-1
Example 3
ZmCLA7-1Forward regulation and control of corn leaf angle
ZmCLA7-1Since the leaf angle was increased in Shen 137 and decreased in Shen 137-NIL76, we examined the expression of ZmCL 7-1 gene. The qRT-PCR detection result shows that the leaf blade (figure 3) of 6-12 leaf stage of the growth and development of the corn is efficiently expressed. In the case of a different material, the material,ZmCLA7-1the expression level in Shen 137 at different time periods was significantly higher than that in Shen 137-NIL76 (FIG. 3), indicating thatZmCLA7-1The leaf angle of the corn with high expression amount is oppositeIs enlarged, thereby proposingZmCLA7-1The corn leaf included angle is regulated and controlled by the corn leaf extract as a positive regulation and control factor.
Example 4
ZmCLA7-1Transgenic verification of gene function
1. Construction of RNAi (RNA interference) vectors
Based on the structure of ZmCLA7-1 gene, we designed a pair of RNAi primer RNAi-F sequence in the coding region of ZmCLA7-1 gene as shown in SEQ ID NO.3, wherein-actagtggatcc-The restriction site and the RNAi-R sequence are shown in SEQ ID NO.4, wherein-agatctggtacc-The DNA fragment is amplified from the gene sink 137 as a cleavage site, and the hairpin structure formed by the sequence and the reverse sequence thereof is inserted into pJL1460 vector, thereby forming pZmCLA7-1-RNAi vector. The recombinant plasmid is transformed into agrobacterium-infected cells for transforming a maize inbred line B104.
2. Genetic transformation
An agrobacterium-mediated corn genetic transformation method is adopted (refer to Wangpian, creation of rough dwarf resistant transgenic corn material based on RNA interference, Master academic thesis of Henan university of agriculture, 2011). Using young embryos of 1.2-1.8mm as receptor materials, infecting the young maize embryos by agrobacterium LBA4404 under the conditions that the OD600 value is 0.5 and the infection time is 10 minutes, and obtaining regeneration plants through co-culture, resting culture, callus induction, callus low-pressure screening (PPT concentration is 3mg/L) and high-pressure screening (PPT concentration is 6 mg/L); screening the concentration phenotype of 200 mg/L herbicide (PPT) of regenerated plants, removing the seedlings sensitive to the herbicide, extracting the total DNA of leaves of the seedlings insensitive to the herbicide, carrying out PCR (polymerase chain reaction) detection on plant marker genes (Bar) and target genes, transplanting the detected positive plants into the field, and selfing to obtain T 0 Seed generation, completing the transgenic T 0 And (4) identifying the generations.
In this example, 7 independent transgenic positive plants were obtained, which showed a different degree of reduction in leaf angle, while the wild-type control plant showed no significant change in leaf angle (FIG. 4). It can be seen that endogenous sources in maize are promoted by RNAI technologyZmCLA7-1The expression of the gene is reduced, so that the included angle of the corn leaves is obviously reduced, and the control is provedZmCLA7-1Expression of the genes can be usedThe inbred line with compact plant type and small leaf included angle is used as a breeding base material, and can be used for breeding density-resistant breeding hybrids to be popularized and applied in production.
Example 5
Promoter strength analysis
In order to determine whether the difference in the expression level of the gene is due to the change in the promoter or coding region, the inventors performed an alignment analysis on the base sequence of the gene as shown in FIG. 5A, wherein the coding region of the present application contains a DUF822 family conserved functional domain, the proteins expressed in Shen 137 and Shen 137-NIL76 of the differential strain are structurally identical, the promoter sequence is largely deleted/inserted, as shown in FIG. 5A, and the two gray boxes represent the start codon and the stop codon, respectively; to further clarify that the promoter difference is critical to cause the change in the expression level of the gene, the inventors have used the promoters of Shen 137 and Shen 137-NIL76 to promote the firefly luciferase reporter gene (LUC) (FIG. 5B), and have used Renilla luciferase promoted by the 35S promoter as an internal reference, and have examined the relative expression level of the reporter gene to show the promoter strength. The tobacco leaf is infected by agrobacterium, tobacco protein is extracted, and the relative expression level of the firefly luciferase is detected by a multifunctional enzyme-linked immunosorbent assay, and the result shows that the expression level of the firefly luciferase reporter gene initiated by the promoter of the sinker 137 is remarkably higher than that of the sinker 137-NIL76 (shown in figure 5B), which indicates that the capability of the promoter of the sinker 137 for initiating the gene expression is remarkably higher than that of the sinker 137-NIL 76.
In this way, it can be seen that,ZmCLA7-1base mutation and insertion/deletion of a gene promoter cause a change in the expression amount of a gene.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> Zhengzhou city seed station of Henan university of agriculture
<120> forward regulatory factor for regulating corn leaf angle and application thereof
<141> 2020-09-16
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 942
<212> DNA
<213> Zea mays
<400> 1
atgacgtccg gggcggcggc ggcggcagga ggtctggggc ggacgccgac gtggaaggag 60
cgggagaaca acaagcgccg ggagcgccgg cggagggcca tcgccgccaa gatcttcacg 120
ggcctgcgcg cgctcggcaa ctacaagctg cccaagcact gcgacaacaa cgaggtgctc 180
aaggcgctct gccgcgaggc ggggtgggtc gtcgaggacg acggcaccac ctaccggaag 240
ggttgcaggc cgccgccggg gatgctgagc ccgtgctcgt cgtcgcagct gctgagcgcg 300
ccgtcctcga gcttcccgag cccggtgccg tcctaccacg ccagcccggc gtcgtcgagc 360
ttcccgagcc cgacgcgcct cgaccacagc agcggcggca gcagcaccca caaccccgcc 420
gcggcggccg ccgccgccgc ctccctgctc ccgttcctcc ggggcctgcc gaacctgccg 480
ccgctccgcg tgtccagcag cgcgcccgtc acgccgccgc tctcctctcc cacggccgcg 540
gcggcggcgt cgcgaccgcc caccaaggtc cgcaggcccg actgggacgc cgccgccgac 600
cccttccggc accccttctt cgcggtctcc gcccccgcca gccccacccg cgcgcgccgg 660
cgcgagcacc cggacaccat cccggagtgc gacgagtccg acgtctgctc cgcggccgac 720
tccgcccggt ggatcagctt ccaggccacc acggcgcccg cgtcgcccac gtacaacctc 780
gtccacccgg cctccgactc catggagctg gacgggacga cggcagccgt cgaggagttc 840
gagttcgaca agggccgcgt cgtcacgcca tgggaaggcg agcggatcca cgaggtggcc 900
gccgaggagc tcgagctcac gctcggcgtc ggcgccaagt ga 942
<210> 2
<211> 313
<212> PRT
<213> Zea mays
<400> 2
Met Thr Ser Gly Ala Ala Ala Ala Ala Gly Gly Leu Gly Arg Thr Pro
1 5 10 15
Thr Trp Lys Glu Arg Glu Asn Asn Lys Arg Arg Glu Arg Arg Arg Arg
20 25 30
Ala Ile Ala Ala Lys Ile Phe Thr Gly Leu Arg Ala Leu Gly Asn Tyr
35 40 45
Lys Leu Pro Lys His Cys Asp Asn Asn Glu Val Leu Lys Ala Leu Cys
50 55 60
Arg Glu Ala Gly Trp Val Val Glu Asp Asp Gly Thr Thr Tyr Arg Lys
65 70 75 80
Gly Cys Arg Pro Pro Pro Gly Met Leu Ser Pro Cys Ser Ser Ser Gln
85 90 95
Leu Leu Ser Ala Pro Ser Ser Ser Phe Pro Ser Pro Val Pro Ser Tyr
100 105 110
His Ala Ser Pro Ala Ser Ser Ser Phe Pro Ser Pro Thr Arg Leu Asp
115 120 125
His Ser Ser Gly Gly Ser Ser Thr His Asn Pro Ala Ala Ala Ala Ala
130 135 140
Ala Ala Ala Ser Leu Leu Pro Phe Leu Arg Gly Leu Pro Asn Leu Pro
145 150 155 160
Pro Leu Arg Val Ser Ser Ser Ala Pro Val Thr Pro Pro Leu Ser Ser
165 170 175
Pro Thr Ala Ala Ala Ala Ala Ser Arg Pro Pro Thr Lys Val Arg Arg
180 185 190
Pro Asp Trp Asp Ala Ala Ala Asp Pro Phe Arg His Pro Phe Phe Ala
195 200 205
Val Ser Ala Pro Ala Ser Pro Thr Arg Ala Arg Arg Arg Glu His Pro
210 215 220
Asp Thr Ile Pro Glu Cys Asp Glu Ser Asp Val Cys Ser Ala Ala Asp
225 230 235 240
Ser Ala Arg Trp Ile Ser Phe Gln Ala Thr Thr Ala Pro Ala Ser Pro
245 250 255
Thr Tyr Asn Leu Val His Pro Ala Ser Asp Ser Met Glu Leu Asp Gly
260 265 270
Thr Thr Ala Ala Val Glu Glu Phe Glu Phe Asp Lys Gly Arg Val Val
275 280 285
Thr Pro Trp Glu Gly Glu Arg Ile His Glu Val Ala Ala Glu Glu Leu
290 295 300
Glu Leu Thr Leu Gly Val Gly Ala Lys
305 310
<210> 3
<211> 31
<212> DNA
<213> Unknown
<400> 3
actagtggat cccaacaagc gccgggagcg c 31
<210> 4
<211> 31
<212> DNA
<213> Unknown
<400> 4
agatctggta ccgtagttgc cgagcgcgcg c 31
<210> 5
<211> 1187
<212> DNA
<213> Zea mays
<400> 5
atcgtgacct cagaggcgac atggctcagg acaggacttt ttcccccgtg actggttggt 60
taactacggg ggagcgacga acgactgagg acatcagccg tactaggatc tccaacaggg 120
caatgctgcg tcgtcttcac ctggcaactg atggtggcgg agcgccgtgg acgcgccatt 180
gttggagttg gaactgtgga gcactggagc tcggcctgcg gtcatcaaaa tccccgtgtg 240
aatactccag tgtggagtac acacactgcg gcagcgcata aagtgcctag tacgtacgta 300
gtacgccact ttgcaaaaag aagcgtacca gcacagctac tcgctcttgc agacatcaca 360
cgacgcttgc attgctggac gcctacgacg gtggaatcgc cgccgtgaag ctcgccgtcg 420
cagatcccaa ggcacatact acccgcgcac cggccctaat caactgcact cgcctcgctc 480
acgcacagca gcacactctc gtctggcgct ctcaccccaa aagccgagcc gagccgagac 540
ccaggctcca gggaggtgag gtaaaagtag gccacgcaca cccaccatag ccgtgccagt 600
agaatctacc cctgctaaag tttagtccgg gtcacatcaa acgtttgact ttcaaataag 660
agtatgaaat atagacccaa ccaactgaac tagattcgtc tcgtctttta atattcggct 720
gacaaattag ttttataatc cgactacatt taatacccgg aacggaggtt caaacattcg 780
atgtgataag agctaaagtt tagtttggga taaccaacca cccctcacag ttgcaatata 840
aagtaatata agcttacggt accaccctcg tgatcgtgag cccggcgggg cccgcgaggg 900
agaaagctca aagcagctcg cacgccatgc catgacataa caacactcta ctcatactct 960
acactctact actagtctac tactactaca gcgcgccacc aggcagtaca ctgcagccag 1020
ccagccacct ccctcctctc agttcagttg gctccctccc ggctcccgcc ccactagtcg 1080
ttgtagtcaa tactgtgcag tacgccaccc accacctctc caagtctcca ttaaagaaaa 1140
ggggaggcaa gtcggggcct cggggagaag gggctgagca tcagacc 1187

Claims (3)

1. A promoter, characterized in that: the promoter is a geneZmCLA7-1The promoter of (1), the geneZmCLA7-1The sequence is shown as SEQ ID No.1, and the promoter sequence is shown as SEQ ID No. 5.
2. A method for regulating and controlling the included angle of corn leaves is characterized by comprising the following steps:
(1) the gene according to claim 1ZmCLA7-1,Construction of corn endogenesis by RNAi technologyZmCLA7-1An interfering vector for the gene;
(2) endogenous cornZmCLA7-1The interference vector of the gene is used for transforming agrobacterium-infected cells for transforming a maize inbred line B104 to obtain transgenic positive plants with leaf included angle expression reduced to different degrees.
3. The method of claim 2, wherein: the primer pair for constructing the interference vector of the corn endogenous ZmCLA7-1 gene is RNAi-F and RNAi-R, wherein the RNAi-F sequence is shown as SEQ ID NO.3, and the RNAi-R sequence is shown as SEQ ID NO. 4.
CN202010994830.6A 2020-09-21 2020-09-21 Forward regulation factor for regulating corn leaf included angle and application thereof Active CN112048512B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010994830.6A CN112048512B (en) 2020-09-21 2020-09-21 Forward regulation factor for regulating corn leaf included angle and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010994830.6A CN112048512B (en) 2020-09-21 2020-09-21 Forward regulation factor for regulating corn leaf included angle and application thereof

Publications (2)

Publication Number Publication Date
CN112048512A CN112048512A (en) 2020-12-08
CN112048512B true CN112048512B (en) 2022-09-02

Family

ID=73604081

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010994830.6A Active CN112048512B (en) 2020-09-21 2020-09-21 Forward regulation factor for regulating corn leaf included angle and application thereof

Country Status (1)

Country Link
CN (1) CN112048512B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114736277B (en) * 2022-04-24 2023-05-12 河南农业大学 Forward regulatory factor for regulating salt tolerance of corn, inDel molecular marker thereof and application of forward regulatory factor
CN114736914B (en) * 2022-06-15 2022-08-23 中国农业科学院作物科学研究所 ZmTGA4 gene and application thereof in adjusting and controlling corn leaf angle and increasing density and yield

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104480121B (en) * 2014-12-17 2017-06-16 河南农业大学 Control ZmCLA1 genes and its method in the seed selection type of resistance to more seedlings corn and the application of maize leaves corner dimension
CN111118030B (en) * 2020-01-22 2022-07-01 华南农业大学 DNA sequence for regulating and controlling corn leaf angle, mutant, molecular marker, detection primer and application thereof

Also Published As

Publication number Publication date
CN112048512A (en) 2020-12-08

Similar Documents

Publication Publication Date Title
CN111902547B (en) Methods for identifying, selecting and producing disease-resistant crops
CN110139872A (en) Plant seed character-related protein, gene, promoter and SNP and haplotype
CN109022450B (en) ZmCL 2-1 gene for regulating and controlling included angle of corn leaves and application thereof
CN112351679B (en) Methods for identifying, selecting and producing southern corn rust resistant crops
CN110938120B (en) StSCI protein for changing self-incompatibility of diploid potato material
US20220396805A1 (en) Dna sequence for regulating maize leaf angle, and mutant, molecular markers, detection primers, and use thereof
MX2013015338A (en) Methods and compositions for selective regulation of protein expression.
CN112390865B (en) Application of Zm5008 gene in regulating and controlling plant height and internode distance of corn
WO2015143972A1 (en) Rice panicle traits regulatory gene pt2 and application thereof
CA3175033A1 (en) Autoflowering markers
CN104480121B (en) Control ZmCLA1 genes and its method in the seed selection type of resistance to more seedlings corn and the application of maize leaves corner dimension
CN108291234A (en) Multiple sporinite forms gene
CN112048512B (en) Forward regulation factor for regulating corn leaf included angle and application thereof
WO2023065966A1 (en) Application of bfne gene in tomato plant type improvement and biological yield increase
WO2019129145A1 (en) Flowering time-regulating gene cmp1 and related constructs and applications thereof
WO2019068647A1 (en) Complete resistance to downy mildew in basil
CN109456396A (en) A kind of protein, molecular labeling and the application of Senescence of Rice and fringe type controlling gene HK73 and its coding
CN112457385B (en) Application of gene LJP1 for controlling rice growth period
CN114946639A (en) Application of two wheat induction systems in wheat haploid immature embryo and seed identification
CN110642930B (en) Gene for regulating and controlling tillering number of corn, and encoded protein and application thereof
CN115216554A (en) Plant pathogen effector and disease resistance gene identification, compositions, and methods of use
CA2547514A1 (en) Method for screening genomic dna fragments
CN108315336B (en) Application of gene PIS1 for controlling development of rice spikelets
CN114516906B (en) Corn and mycorrhizal fungi symbiotic related protein, and coding gene and application thereof
CN114907461B (en) Gray spot resistance related protein ZmPMT1, encoding gene and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant