CN112048512A - 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 PDFInfo
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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 leavesZmCLA7‑1The gene, in particular to a positive regulatory factor for regulating 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, the present inventionSuppression of corn endogenesis using RNAi technologyZmCLA7‑1Expression 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
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 the average yield per mu of a hybrid (compact) containing the leafless tongue gene ligulless 2 is improved by 41.2% compared with the corresponding hybrid under the condition of close planting; 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 corn leaf angle are obtained by comparing genomics and mutant technologyAnd (5) researching. 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. Through cloning the corresponding gene, the gene is found,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. To pairyabbyThe 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 geneZmCLA7-1Is an unknown structure domain which is formed by 317 amino acids and contains a DUF822 family conservative function domain and is found by a map-based cloning technology to regulate the leaf angleGenes, the molecular biological functions of which have not been reported in plants at present. The invention researches the molecular biological function of the gene and the promoter thereof for regulating the included angle of the corn leaves, and provides a potential new gene resource for breeding a new variety of the close-planting type excellent high-yield corn.
The technical scheme of the invention is realized as follows:
forward regulatory factor for regulating and controlling leaf angle of corn, wherein 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) the product is isolated from Shen 137-NIL76 by using map-based cloning techniqueZmCLA7-1Genes, then constructing corn endogenesis by RNAi technologyZmCLA7-1An expression vector for the gene;
(3) endogenous 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 corn leaf angle is reduced, so that the cloning of the gene is helpful for understanding the molecular mechanism of corn leaf angle formation.
2. 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.
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.
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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 is a drawing bitMolecular identification results of cloned ZmCLA7-1 gene. In the figure, A: using BC3F2:3-8The 112 families of the population are positioned to position qLA7-1 into the intervals of the markers PZE-07122629825 and PZE-107077981; b: using BC4F28723 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 to a region of 43.6Kb by fine localization, which contained 1 candidate gene. D: the candidate geneZm00001d021927（ZmCLA7-1) 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 during 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 F1The 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 20141(ii) a Planting F in Hainan in winter in 20141The surrogate and acceptor parent sink 137, sink 137 and F1Backcrossing to obtain BC1F1；
According to BC1F1Selecting 13 plants with small leaf angle to form spikes in Zhengzhou in 2015 spring, and backcrossing with Shen 137 to obtain BC2F1(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 BC3F1；
2016 spring, mix BC3F1In Zhengzhou, the seeds grow into spikes and grow from BC3F1Carrying 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 BC4F1All are the same asTime-to-time crossover individual plants BC3F1-8 selfing to obtain BC3F2-8; 2016 in winter in Hainan, three, and mixing BC4F1And BC3F2-8 planting (simultaneously, using SNP corn 3K chip to pair BC3F2-Genotyping of 112 individuals of 8), according to BC4F1Phenotypic combination genotype of the population 11 crossover individuals were selected for selfing to obtain BC4F2；
2017 obtaining BC in Zhengzhou spring4F2Planting according to BC4F2Phenotype of the population combined with genotype analysis, 19 crossover individuals were obtained and backcrossed with Shen 137 to obtain BC5F2(ii) a 2017 winter Saiyan BC5F2Planting according to BC5F2And (3) combining the phenotype of the population with genotype analysis to obtain 9 crossover individuals, carrying out genetic background analysis on the 9 crossover individuals by adopting the SNP corn 3K chip, selecting 2 crossover individuals with high genetic background recovery rate from the 9 crossover individuals, and selfing to obtain a near-isogenic line which is homozygous and genetically stable at the site.
Example 2
Isolation by map-based cloningZmCLA7-1Gene
Using BC4F2The 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 restricted to marker SSR7-6 and SSR according to the marker genotype of the target segment of the exchanged individual7-10 (fig. 2A).
To further determine qLA7-1 as 1 or more candidate genes, BC was used5F2The 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 in the interval SSR7-15/SSR7-17 containing Yu 537A gene with the sinew 137 of the flattened inbred line, it is shown that the allele from Yu 537A reduces the upper leaf angle of the ear, while the allele from sinew 137 increases the upper leaf angle of the ear, and the interval SSR7-15/SSR7-17 may have a gene controlling the upper leaf angle of the ear. Combines with B73 genome sequence, marks 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 are free from 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 (ZmCL A4 gene map-based cloning and function analysis, Ph university of Henan agriculture, doctor's academic paper, 2014) was used to extract the 9-leaf-stage Shen 137 and Shen 137-NIL76 leavesmRNA 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 the sinker 137 and sinker 137-NIL76 is caused byZm00001d021927Is 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 levels at different times in Shen 137 were significantly higher than that of Shen 137-NIL76 (FIG. 3), indicating thatZmCLA7-1The leaf angle of the corn with high expression amount is relatively reduced, thereby providingZmCLA7-1The corn leaf included angle is regulated and controlled by the corn leaf extract as a positive regulation and control factor.
Example 4
ZmCLA7-1Verification of gene function by overexpression of transgenes
1. Construction of RNAi (RNA interference) vectors
Based on the structure of ZmCLA7-1 gene, we designed a pair of RNAi primers 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
Adopting an agrobacterium-mediated corn genetic transformation method (refer to Wangpian, creation of rough dwarf resistant transgenic corn material based on RNA interference, Henan agriculture)Master academic thesis 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 T0Seed generation, completing the transgenic T0And (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 provedZmCLA7-1The expression of the gene can be applied to a inbred line with compact plant type and small leaf included angle as a breeding basic material, and can be applied to the production popularization and application of breeding density-resistant hybrid seeds.
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 seed station of the university of agriculture in Henan
<120> forward regulatory factor for regulating corn leaf angle and application thereof
<141> 2020-09-21
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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 tcagaccatg acgtccgggg 1200
cggcggcggc ggcaggaggt ctggggcgga cgccgacgtg gaaggagcgg gagaacaaca 1260
agcgccggga gcgccggcgg agggccatcg ccgccaagat cttcacgggc ctgcgcgcgc 1320
tcggcaacta caagctgccc aagcactgcg acaacaacga ggtgctcaag gcgctctgcc 1380
gcgaggcggg gtgggtcgtc gaggacgacg gcaccaccta ccggaagggt tgcaggccgc 1440
cgccggggat gctgagcccg tgctcgtcgt cgcagctgct gagcgcgccg tcctcgagct 1500
tcccgagccc ggtgccgtcc taccacgcca gcccggcgtc gtcgagcttc ccgagcccga 1560
cgcgcctcga ccacagcagc ggcggcagca gcacccacaa ccccgccgcg gcggccgccg 1620
ccgccgcctc cctgctcccg ttcctccggg gcctgccgaa cctgccgccg ctccgcgtgt 1680
ccagcagcgc gcccgtcacg ccgccgctct cctctcccac ggccgcggcg gcggcgtcgc 1740
gaccgcccac caaggtccgc aggcccgact gggacgccgc cgccgacccc ttccggcacc 1800
ccttcttcgc ggtctccgcc cccgccagcc ccacccgcgc gcgccggcgc gagcacccgg 1860
acaccatccc ggagtgcgac gagtccgacg tctgctccgc ggccgactcc gcccggtgga 1920
tcagcttcca ggccaccacg gcgcccgcgt cgcccacgta caacctcgtc cacccggcct 1980
ccgactccat ggagctggac gggacgacgg cagccgtcga ggagttcgag ttcgacaagg 2040
gccgcgtcgt cacgccatgg gaaggcgagc ggatccacga ggtggccgcc gaggagctcg 2100
agctcacgct cggcgtcggc gccaagtga 2129
<210> 2
<211> 313
<212> PRT
<213> Zea mays
<400> 2
Met Thr Ser Gly Ala Ala Ala Ala Ala Gly Gly Leu Gly Ala Thr Pro
1 5 10 15
Thr Thr Leu Gly Ala Gly Ala Ala Leu Ala Ala Gly Ala Ala Ala Ala
20 25 30
Ala Ile Ala Ala Leu Ile Pro Thr Gly Leu Ala Ala Leu Gly Ala Thr
35 40 45
Leu Leu Pro Leu His Cys Ala Ala Ala Gly Val Leu Leu Ala Leu Cys
50 55 60
Ala Gly Ala Gly Thr Val Val Gly Ala Ala Gly Thr Thr Thr Ala Leu
65 70 75 80
Gly Cys Ala Pro Pro Pro Gly Met Leu Ser Pro Cys Ser Ser Ser Gly
85 90 95
Leu Leu Ser Ala Pro Ser Ser Ser Pro Pro Ser Pro Val Pro Ser Thr
100 105 110
His Ala Ser Pro Ala Ser Ser Ser Pro Pro Ser Pro Thr Ala Leu Ala
115 120 125
His Ser Ser Gly Gly Ser Ser Thr His Ala Pro Ala Ala Ala Ala Ala
130 135 140
Ala Ala Ala Ser Leu Leu Pro Pro Leu Ala Gly Leu Pro Ala Leu Pro
145 150 155 160
Pro Leu Ala Val Ser Ser Ser Ala Pro Val Thr Pro Pro Leu Ser Ser
165 170 175
Pro Thr Ala Ala Ala Ala Ala Ser Ala Pro Pro Thr Leu Val Ala Ala
180 185 190
Pro Ala Thr Ala Ala Ala Ala Ala Pro Pro Ala His Pro Pro Pro Ala
195 200 205
Val Ser Ala Pro Ala Ser Pro Thr Ala Ala Ala Ala Ala Gly His Pro
210 215 220
Ala Thr Ile Pro Gly Cys Ala Gly Ser Ala Val Cys Ser Ala Ala Ala
225 230 235 240
Ser Ala Ala Thr Ile Ser Pro Gly Ala Thr Thr Ala Pro Ala Ser Pro
245 250 255
Thr Thr Ala Leu Val His Pro Ala Ser Ala Ser Met Gly Leu Ala Gly
260 265 270
Thr Thr Ala Ala Val Gly Gly Pro Gly Pro Ala Leu Gly Ala Val Val
275 280 285
Thr Pro Thr Gly Gly Gly Ala Ile His Gly Val Ala Ala Gly Gly Leu
290 295 300
Gly Leu Thr Leu Gly Val Gly Ala Leu
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 (5)
1. A positive regulatory factor for regulating and controlling the leaf angle of corn is characterized in that: 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, and the promoter sequence is shown as SEQ ID NO. 5.
2. The positive regulator of claim 1, wherein the positive regulator is selected from the group consisting of: the geneZmCLA7-1The coded amino acid sequence is shown in SEQ ID NO.2 and is a protein containing a DUF822 family conserved functional structural domain.
3. The use of the forward regulatory factor of claim 1 for regulating maize leaf angle, comprising the steps of:
(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) the product is isolated from Shen 137-NIL76 by using map-based cloning techniqueZmCLA7-1Genes, then constructing corn endogenesis by RNAi technologyZmCLA7-1An expression vector for the gene;
(3) endogenous 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.
4. Use according to claim 3, characterized in that: in the step (2), the corn is endogenousZmCLA7-1The expression vector of the gene takes cDNA of the self-bred line 537A as a template, andaccording to B73CLA7-1The primer pair designed by the cDNA sequence is obtained by constructing the primer.
5. Use according to claim 4, characterized in that: 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.
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| CN202010994830.6A CN112048512B (en) | 2020-09-21 | 2020-09-21 | Forward regulation factor for regulating corn leaf included angle and application thereof |
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| CN112048512A true CN112048512A (en) | 2020-12-08 |
| CN112048512B CN112048512B (en) | 2022-09-02 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736914A (en) * | 2022-06-15 | 2022-07-12 | 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 |
| CN114736277A (en) * | 2022-04-24 | 2022-07-12 | 河南农业大学 | Forward regulation factor for regulating salt tolerance of corn, InDel molecular marker and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480121A (en) * | 2014-12-17 | 2015-04-01 | 河南农业大学 | ZmCLA1 gene capable of controlling sizes of included angles of maize leaves as well as method and application of ZmCLA1 gene in breeding high-density-tolerant maize |
| CN111118030A (en) * | 2020-01-22 | 2020-05-08 | 华南农业大学 | DNA sequence for regulating and controlling corn leaf included angle, mutant, molecular marker, detection primer and application thereof |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480121A (en) * | 2014-12-17 | 2015-04-01 | 河南农业大学 | ZmCLA1 gene capable of controlling sizes of included angles of maize leaves as well as method and application of ZmCLA1 gene in breeding high-density-tolerant maize |
| CN111118030A (en) * | 2020-01-22 | 2020-05-08 | 华南农业大学 | DNA sequence for regulating and controlling corn leaf included angle, mutant, molecular marker, detection primer and application thereof |
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| MING-YI BAI等: "Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice", 《PROC NATL ACAD SCI U S A》 * |
| NCBI: "EU969826.1", 《GENBANK》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736277A (en) * | 2022-04-24 | 2022-07-12 | 河南农业大学 | Forward regulation factor for regulating salt tolerance of corn, InDel molecular marker and application thereof |
| 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 |
| CN114736914A (en) * | 2022-06-15 | 2022-07-12 | 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112048512B (en) | 2022-09-02 |
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