CN114908117B

CN114908117B - Application of corn double-regulation module in regulation of plant growth and disease-resistant balance

Info

Publication number: CN114908117B
Application number: CN202210674892.8A
Authority: CN
Inventors: 苟明月; 于延文; 孙景凡; 张同祥; 李见坤; 母小焕; 徐莉萍; 汤继华
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2023-05-16
Anticipated expiration: 2042-06-15
Also published as: CN114908117A; NL2035090A

Abstract

The invention belongs to the technical field of agricultural biology, and in particular relates to application of a corn double-regulation module in regulating plant growth and disease-resistant balance, wherein the corn double-regulation module comprises a promoter of ZmTBF1a gene and a uORF sequence, namely pZmTBF1a: uORF ^ZmTBF1a The nucleotide sequence of which is shown in SEQ ID No.1; also included are the promoter and uORF sequence of the ZmTBF1b gene, i.e.pZmTBF 1b: uORF ^ZmTBF1b The nucleotide sequence of which is shown in SEQ ID No.2. On the basis, the invention clones the promoters pZmTBF1 and uORF upstream of the two genes ^ZmTBF1 The double regulating and controlling elements form a pathogen induction type gene regulating and controlling module suitable for corn; two plant expression vectors based on pCAMBIA3301 are constructed and used for regulating and controlling the induction expression of disease-resistant genes in corn, and the vector has application value in cultivating new corn transgenic varieties with high resistance and stable yield.

Description

Application of corn double-regulation module in regulation of plant growth and disease-resistant balance

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a corn double-regulation module in regulating plant growth and disease-resistant balance, in particular to application of a TBF1 gene promoter and a uORF element in regulating plant growth and disease-resistant balance.

Background

Corn is one of the main grain and feed crops in China and plays an important role in agricultural production. In recent years, with climate change and variety replacement, corn diseases have a trend of continuous aggravation, and seriously threaten the grain safety of China. Therefore, improvement of disease resistance of varieties becomes an important target of current corn breeding work. The molecular mechanism research of plant disease resistance has been advanced in the 21 st century, a basic framework of the plant immune system is established, and a good theoretical basis is laid for the improvement of corn disease resistance molecules. However, production practices and experimental researches find that a close antagonism relationship exists between plant growth and disease resistance, and introduction of disease resistance genes is an effective means for improving the disease resistance of corn, but unexpected growth defects are often caused when the disease resistance genes are excessively expressed, for example, the disease resistance genes often inhibit plant growth while improving crop resistance, so that crop yield is affected. Therefore, the balance problem of growth and disease resistance becomes a bottleneck for restricting the disease resistance genes in corn breeding application.

During long-term evolution and adaptation, plants develop complex regulatory networks and molecular mechanisms that balance their own growth and disease resistance. Among them, the Arabidopsis TBF1 gene is used as a key switch molecule, and plays an important role in the growth and disease-resistant transformation process of plants.

As a key switch molecule for growth and disease resistance switching, TBF1 expression is precisely regulated at the translational level by the upstream open reading frame (uORF, upstream Open Reading Frame). Studies have shown that the region upstream of the initiation codon of the TBF1 gene contains 2 uORFs rich in phenylalanine codons ^TBF1 . When grown normally, an appropriate amount of phenylalanine is able to meet normal metabolic demands, at which time the unphosphorylated translational initiator eIF2 alpha binds to the uORF ^TBF1 Where translation initiation of TBF1 is inhibited; in the case of invasion of pathogenic bacteria, however, the plant demand for phenylalanine increases, resulting in a large number of empty phenylalanine transport ribosomes (tRNA ^Phe ) And subsequent phosphorylation modification of eif2α facilitates binding to the translation initiation site of TBF1, activating TBF1 expression while facilitating plant switching to disease-resistant mode. Thus, uORF ^TBF1 The existence of the (2) can strictly inhibit the expression of TBF1 in the Arabidopsis thaliana which normally grows, and can be quickly started when pathogenic bacteria invade, thereby exerting the switch action of the growth and disease resistance conversion of the Arabidopsis thalianaWith (Pajerowska-Mukhtar, K.M., wang, W., tada, Y., et al (2012) The HSF-like transcription factor TBF1 is a major molecular switch for plant growth-to-sensitivity transition. Curr Biol 22, 103-112).

Based on the molecular mechanisms that are doubly regulated by plants at the level of TBF1 gene transcription and translation, the Dong Xin and Wang Danping teaching teams successfully overcome the growth defect caused by disease-resistant gene expression in arabidopsis and rice (Xu, g., yuan, m., ai, c., et al (2017) uof-mediated translation allows engineered plant disease resistance without fitness costs.nature 545, 491-494). However, the role of this mechanism in conservation in other crops, especially in corn against its own disease, is currently unclear.

In summary, the problem in the prior art is that the maize disease resistance gene can affect the yield while exerting disease resistance. Although it has been demonstrated in Arabidopsis and rice that the TBF1 gene regulatory module balances plant growth and disease resistance, the function of the TBF1 gene in maize is not yet known, and there has been no report on the balance of maize growth and disease resistance by using the maize TBF1 promoter and uORF dual regulatory module.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of a corn double-regulation module in regulating plant growth and disease-resistant balance.

The invention aims to provide an application of a corn double-regulation module in regulating plant growth and disease resistance balance, wherein the corn double-regulation module comprises a promoter of ZmTBF1a gene and a uORF sequence, namely pZmTBF1a: uORF ^ZmTBF1a (regulating the expression of ZmTBF1a gene), the nucleotide sequence of which is shown in SEQ ID No.1, wherein the 1-1565 positions are promoters, and the last 190-315 positions are uORF of the ZmTBF1a gene; the corn double regulation module also comprises a promoter of ZmTBF1b gene and a uORF sequence, namely pZmTBF1b: uORF ^ZmTBF1b (regulating the expression of ZmTBF1b gene) and its nucleotide sequence is shown in SEQ ID No.2, wherein positions 1-1576 are promoters and positions 155-280 are uORFs of the ZmTBF1b gene. The TBF1 gene comprises ZmTBF1a and ZmTBF1b, and the nucleotide sequence of the ZmTBF1a is shown in SEQ ID No.3, wherein the 1-234 st and the 1-663 st reciprocal are exons, and the ZmTBF1b nucleotide sequence is shown in SEQ ID No.4, wherein the 1-228 st and the 1-669 st reciprocal are exons.

Preferably, the corn double-regulation module is applied to regulating plant growth and disease resistance balance, and the corn double-regulation module inhibits the expression of corresponding genes under normal growth conditions, obviously induces the expression of the corresponding genes under disease conditions, ensures that the respective regulated genes are expressed only under disease conditions, and further balances the growth and disease resistance of plants, so that the plants have disease resistance and good growth characteristics.

Preferably, the corn double regulation module is applied to regulation of plant growth and disease resistance balance, wherein the plant is corn.

Preferably, the application of the corn double-regulation module in regulating plant growth and disease-resistant balance is that the corn double-regulation module (TBF 1 gene promoter and uORF) is used for regulating corn characters to enable corn to show disease resistance and stable yield; the disease resistance refers to disease and insect resistance.

Preferably, the application of the corn double-regulation module in regulating plant growth and disease and pest resistance balance is caused by rust fungus, curvularia lunata, fusarium graminearum, fusarium moniliforme, vermicular spore fungus, anthracnose graminearum, iris mosaic virus, corn budworm, armyworm or aphid.

Preferably, the corn double regulation module is applied to regulating plant growth and disease resistance balance, and uORF is amplified from corn DNA ^ZmTBF1 The elements and the corresponding ZmTBF1 promoter sequence are cloned into a plant expression vector pCAMBIA3301 respectively by using a seamless cloning method.

Preferably, the use of the above-described maize dual regulation module for regulating plant growth and disease resistance balance, said amplified uORF ^ZmTBF1 See table 5 for elements and primers for the corresponding ZmTBF1 promoter sequence.

Preferably, the application of the corn double-regulation module in regulating plant growth and disease resistance balance utilizes EcoRI and NcoI restriction enzymes to linearize pCAMBIA3301 vector, and utilizes a seamless cloning technology to double-regulate the amplified cornA control module sequence is connected into the plant expression vector; the corn double-regulation module comprises pZmTBF1a: uORF ^ZmTBF1a And pZmTBF1b: uORF ^ZmTBF1b 。

Preferably, the corn double regulation module is applied to regulating plant growth and disease resistance balance, ncoI and PmlI are utilized to carry out double enzyme digestion on the constructed vector, and GUS sequences in the vector are replaced between enzyme digestion sites by sequences of disease resistance genes (the disease resistance genes are correspondingly regulated and expressed by the corn double regulation module), so that the plant expression vector containing the corn double regulation module and the disease resistance genes is prepared.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention identifies two homologous genes (ZmTBF 1a and ZmTBF1 b) of TBF1 in corn, and finds that there is a conservative uORF upstream of the two genes and can regulate the initiation of gene translation ^ZmTBF1 A component. Further research results show that ZmTBF1 is induced by various corn pathogenic bacteria and insect pests, and the promoter has pathogen induction characteristics; and the over-expression of the gene can activate hypersensitivity reaction and promote the expression of immune response related gene ZmBiP 2. Shows that ZmTBF1 participates in the disease resistance reaction of corn and has the potential of regulating plant growth and disease resistance conversion. On the basis, the invention clones the promoters and uORF regulatory elements at the upstream of ZmTBF1a and ZmTBF1b genes to form a pathogen induction type gene regulatory module suitable for corn; two plant expression vectors based on pCAMBIA3301 are constructed and used for regulating and controlling the specific induction expression of disease-resistant genes in corn, so as to further cultivate a new transgenic corn variety with high resistance and stable yield.

2. The two pathogen induction type regulation and control modules designed by the invention can balance the influence of disease resistance genes on growth and disease resistance in corn application, thereby cultivating a new transgenic corn variety with high yield and strong disease resistance. In the future, the regulatory module designed by the invention can be introduced into the upstream of the corn disease-resistant gene by utilizing a gene editing technology, so that a high-yield disease-resistant new corn variety without a transgenic mark can be realized.

Drawings

FIG. 1 shows blast results of the protein sequence of Arabidopsis TBF1 in the maize genome;

FIG. 2 is an evolutionary analysis of protein sequences of Arabidopsis HSF family genes and TBF1 candidate homologous genes in maize; the evolutionary tree is constructed by adopting a maximum likelihood method;

FIG. 3 is a comparison of the TBF1 gene proteins, promoters and 5' -UTR sequences of Arabidopsis and maize;

a, amino acid sequence alignment of the TBF1 proteins of arabidopsis and corn; b, comparing the promoter sequences of the TBF1 genes of the arabidopsis and the corn; c, 5' -UTR sequence alignment of Arabidopsis thaliana and corn TBF1 genes;

FIG. 4 is uORF ^TBF1 Sequence and evolution analysis of (a);

DNA (A) and amino acid (B) sequences of the uORF of the Arabidopsis and maize TBF1 genes are aligned; c, different species uORF2 ^TBF1 Alignment of homologous sequences; d, different species uORF2 ^TBF1 Evolution analysis of homologous sequences; at, arabidopsis thaliana arabidopsis, pv, phaseolus vulgaris kidney bean, gm, glycine max soybean, gr, gossypium raimondii cotton, nb, nicotiana benthamiana the present smoke, ca, cicer arietinum chickpea, pd, phoenix dactylifera date, ma, musa acuminata subsp.

FIG. 5 is uORF ^ZmTBF1 Inhibition of downstream gene translation levels;

A，pZmTBF1:uORF ^ZmTBF1 schematic of vector driving LUC reporter; b, uORF ^ZmTBF1a Effects on downstream gene protein levels; c, uORF ^ZmTBF1a Influence on the transcription level of downstream genes; d, uORF ^ZmTBF1b Effects on downstream gene protein levels; e, uORF ^ZmTBF1b Influence on the transcription level of downstream genes; in fig. 5B-E, I, II, III, IV corresponds to the vector of fig. 5A, respectively;

FIG. 6 shows the expression analysis of the ZmTBF1 gene of the present invention;

a, tissue expression characteristics of ZmTBF1a gene; b, tissue expression characteristics of ZmTBF1B gene; c, processing the up-regulated expression of ZmTBF1a gene; d, processing the up-regulated expression of ZmTBF1b gene; e, mutant of ZmTBF1a gene up-regulated expression; f, mutant of ZmTBF1b gene up-regulated expression; in FIG. 6, F.graminearum, fusarium graminearum; verticilliides, fusarium moniliforme; MIMV, iran mosaic virus; graminicola, anthrax gramicis; turcium, helminthiasis helminth; SA, salicylic acid; s.exigua, corn earworm; separating a, corn armyworm; aphid, aphid; t. urectae, tetranychus urticae;

FIG. 7 is a graph showing rapid induction of ZmTBF1 by maize disease;

a, zmTBF1a and ZmTBF1b genes are induced by southern rust pathogenic bacteria of corn; b, the ZmTBF1a and ZmTBF1B genes are induced by pathogenic bacteria of corn curvularia leaf spot; c, expression pattern of ZmPR1 gene after corn inoculation with Curvularia lunata; d, expression mode of ZmTBF1a gene after corn inoculation of Curvularia lunata; e, expression pattern of ZmTBF1b gene after corn inoculation with Curvularia lunata. P, polysora, puccinia multicastus; lunata, curvularia lunata;

FIG. 8 is a graph showing the modulating effect of ZmTBF1 on immune response;

a, the overexpression of ZmTBF1a and ZmTBF1b genes induces hypersensitivity; b, a vector schematic diagram of ZmTBF1 overexpression and a ZmBiP2 promoter-driven LUC reporter gene; c, zmTBF1 induces the up-regulated expression of a LUC reporter gene driven by a ZmBiP2 promoter;

i, II, III, IV in FIG. 8C corresponds to the vector in FIG. 8B, respectively, "+" indicates that both vectors have been co-transformed;

FIG. 9 shows that the gene contains ZmTBF1a promoter and uORF ^ZmTBF1a Element (A) and promoter and uORF containing ZmTBF1b gene ^ZmTBF1b Construction of plant expression vector of element (B);

FIG. 10 is a diagram of the operational modes of two pathogen-inducible plant expression vectors designed based on the ZmTBF1 gene.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described with reference to specific examples and drawings.

In the description of the present invention, unless otherwise specified, reagents and vectors are commercially available, and methods (e.g., methods of constructing vectors, methods of PCR amplification, etc.) are employed using techniques conventional in the art.

1. Identification and sequence analysis of maize TBF1 homologous genes

The Arabidopsis thaliana TBF1/HSF4 gene belongs to the HSF heat shock protein family. However, functional studies have shown that AtTBF1 is not involved in the plant heat shock response. The invention firstly searches the protein sequence of AtTBF1 by utilizing the BLAST tool of Gramen (https:// www. Gramen. Org /) website, and discovers that more sequences similar to the AtTBF1 protein exist in corn, but the consistency is relatively low (figure 1). Thus, the present invention further constructs a phylogenetic tree of these genes and the Arabidopsis HSF family genes (FIG. 2). The results indicate that there are two candidate genes (Zm 00001eb314890 and Zm00001eb 100770) very closely related to the evolution of the arabidopsis TBF1 gene, most likely homologous genes whose functions are similar, and thus named ZmTBF1a and ZmTBF1b, respectively (fig. 2).

Through further protein sequence alignment, the amino acid sequence identity of ZmTBF1a and ZmTBF1b proteins was very high, reaching 83.61%, but there was a large difference from the sequence of the arabidopsis TBF1 protein, with only 38.87% and 37.42% identity, respectively (fig. 3A). Promoters are important for the function of genes as important regulatory regions for gene expression. The promoters of the AtTBF1, zmTBF1a and ZmTBF1B genes all differ significantly, with a consistency of around 35% (FIG. 3B). However, there was a very high region of identity in the 5' -UTR region of the ZmTBF1a and ZmTBF1b genes (FIG. 3C).

2. uORF in maize ^ZmTBF1 The element being capable of controlling translation initiation of a downstream gene

It was found by sequence analysis that ZmTBF1a and ZmTBF1b contain a sequence which is highly identical to uORF2 in the 5' -UTR region ^AtTBF1 Open reading frames of similar sequence (FIGS. 4A and 4B). Studies in Arabidopsis have shown that the function of TBF1 depends on two phenylalanine-rich open reading frames upstream of its gene (uORF 1 ^TBF1 And uORF2 ^TBF1 ) However, the function of the uORF element present in the 5' -UTR region of the maize ZmTBF1 gene is not yet known. Search results in different species indicate that uORF2 ^TBF1 Is a widely occurring conserved open reading frame (FIG. 4C). Currently, uORF2 is not yet known ^TBF1 During evolution, whether functional diversity is generated for sequence variation. Evolutionary analysis showed that this element was closely related in maize and rice (fig. 4D).

Previous studies have shown that an important function of the uoorf is to regulate the level of translation of downstream genes. Thus, the present invention amplifies two uORFs from the DNA of maize B73 inbred line ^ZmTBF1 The elements and corresponding ZmTBF1 promoter sequences were cloned separately into the vector of the dual luciferase reporter system using a seamless cloning method (completed using the ClonExpress Ultra One Step Cloning Kit product of nuuzan biotechnology company) (fig. 5A). At the same time, the uORF is destroyed by introducing a mutant base (ATG to CTG) into the primer ^ZmTBF1 Is a function (uf) ^ZmTBF1 Fig. 5A). The primers constructed from the above vectors are shown in Table 1. These vectors were transiently transformed into maize protoplasts by PEG-mediated methods and luciferase activity was assayed. As a result, it was found that two uORFs ^ZmTBF1 There was no significant difference in the transcriptional level expression of downstream gluc after mutation, but the expression level was significantly increased relative to the enzyme activity, i.e., protein level (fig. 5B-E). These results demonstrate that these two uofs in maize ^ZmTBF1 Has inhibiting effect on the translation of downstream genes. Primers for gene expression assays are shown in Table 2.

TABLE 1 uORF ^ZmTBF1 Carrier construction primer for function verification

TABLE 2 uORF ^ZmTBF1 Carrier construction primer for function verification

3. ZmTBF1 gene is induced by various corn pathogens

The arabidopsis TBF1 gene is used as a switching factor for growth and disease resistance conversion, and the transcription and translation levels of the arabidopsis TBF1 gene are strictly regulated. The expression levels of the ZmTBF1a and ZmTBF1b genes in a public database were analyzed by means of the PPRD (http:// ipf. Sustech. Edu. Cn/pub/plantra /) website. The results indicate that the expression level of ZmTBF1 gene was maintained at a relatively low level in tissues such as roots, leaves and stamens of maize (fig. 6A and 6B). And when corn pathogenic bacteria (such as fusarium graminearum and fusarium moniliforme which are pathogenic bacteria of ear rot and stem rot, and anthracnose gramicifuga which is pathogenic bacteria of large spot disease, and anthracnose gramicifuga which is pathogenic bacteria of anthracnose) are treated, the expression of the two genes is remarkably increased (figures 6C and 6D). Similar to the results in arabidopsis, the expression level of ZmTBF1 was also significantly induced under SA treatment (fig. 6C and 6D). In addition, the Iran leaf virus and some common corn pests such as corn green worms, armyworms and aphids can also induce the gene expression of ZmTBF1, which suggests that ZmTBF1 is widely involved in the pest response reaction of corn, and the promoter of the gene has the characteristic of pathogen-induced expression (FIGS. 6C and 6D).

Rp1-D21 mutant is a spot-like mutant caused by R gene recombination and shows broad-spectrum rust resistance. In the transcriptome data of the public database, the expression level of both ZmTBF1 genes was significantly increased in the Rp1-D21 mutants (fig. 6E and 6F). A plurality of lesion-like mutants (les) were collected in the early stage of the subject group, and transcriptome sequencing was performed on materials exhibiting an improvement in disease resistance. The results showed that the ZmTBF1 gene was also significantly increased in the expression levels in these les mutants (fig. 6E). The above results suggest that the promoter of ZmTBF1 gene is regulated by invasion of pathogens, resulting in an increase in the expression level of the gene.

In order to further analyze the relationship between the gene and corn diseases, the invention detects the expression level of the ZmTBF1 gene after inoculation of pathogenic bacteria of southern rust, namely Puccinia striolata and Curvularia lunata of Curvularia lunata by a qRT-PCR method, and the used primers are shown in Table 3. The results indicate that inoculation of both pathogens significantly induced the expression of ZmTBF1a and ZmTBF1B (fig. 7A and 7B). In addition, the invention monitors the expression changes of ZmTBF1 gene and disease course related gene ZmPR1 for one week after corn is inoculated with Curvularia lunata. As a result, it was found that the expression level of ZmPR1 gene began to be up-regulated 4 days after inoculation with the pathogen, and the expression levels of ZmTBF1a and ZmTBF1b genes reached a peak at day 4, followed by rapid recovery to the pre-inoculation level (FIGS. 7C-E). Thus, the ZmTBF1 gene is very sensitive to the response of the disease.

Table 3 quantitative primers for ZmTBF1 and ZmPR1 genes

4. ZmTBF1 activates hypersensitivity reaction and promotes expression of immune response related gene ZmBiP2

Plants often elicit hypersensitivity reactions after activation of the immune system, leading to cell death against invasion by pathogenic bacteria. To verify the function of maize ZmTBF1, the present invention constructed the vector for genomic overexpression (fig. 8B), transiently transformed into tobacco by agrobacterium-mediated methods. The results indicate that overexpression of both ZmTBF1a and ZmTBF1b resulted in a distinct hypersensitivity phenotype (fig. 8A), indicating that the ZmTBF1 gene was active for the immune response. Previous studies have shown that the Arabidopsis TBF1 protein can bind to the TL1 element of the BiP2 gene promoter and participate in the immune response process of plants. Sequence analysis revealed that the promoter region of the maize BiP2 homologous gene also contains a typical TL1 element. Thus, the present invention constructs a reporter vector for the ZmBiP2 promoter driving LUC and co-transformed with an overexpression vector for ZmTBF1 into maize protoplasts (fig. 8B and 8C). The relevant vector construction primers are shown in Table 4. Since overexpression of ZmTBF1 causes a strong hypersensitivity reaction, the enzyme activity assay for the reporter LUC was performed 8 hours after transformation. The results indicate that both ZmTBF1a and ZmTBF1b promote LUC gene expression driven by the ZmBiP2 promoter (fig. 8C).

Table 4 primers for ZmTBF1 overexpression and ZmBiP2 promoter report vector construction

5. Cloning of ZmTBF1 Gene promoter and uORF and construction of plant expression vector

The above experimental results show that the promoters and uORFs of the two ZmTBF1 genes ^ZmTBF1 The regulation and control module formed by the elements can sensitively respond to plant diseases (such as southern rust and curvularia leaf spot) of corn, and has higher application potential in the aspect of balancing the growth and disease resistance of the corn. Thus, the present invention uses two ZmTBF1 promoters and uORF _ZmTBF1 The sequence-composed regulatory modules were cloned into the plant expression vector pCAMBIA3301 (FIGS. 9-10). The vector construction primers are shown in Table 5. In the subsequent application process, ncoI and PmlI are utilized to carry out double enzyme cutting, and GUS sequences in the vector are replaced by sequences of disease resistance genes between enzyme cutting sites, so that a disease resistance gene plant expression vector containing a pathogen induction module is constructed. The mode of operation of the pathogen-inducible regulatory module is shown in FIG. 10.

Table 5 primers for ZmTBF1 overexpression and construction of ZmBiP2 promoter reporter vector

It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

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ccagaaataa tcataatgat actcagattc tcgccacaac cagattctcc tcacagccag 600

attttcagaa aagctggtca gaaaaaagct gaaccaaaca ggccccatgt caagcgagcc 660

tgaccgcttt agggctactt tgggaacctg agatacacct tatgattaaa tggtattggg 720

atagaaataa tgtaattttc tttctaatct ctttcaattt tgaagtggat ttaagtttcc 780

aaactagcct taagtgatcc tcatggtggc ggtagccaaa catcttgttc gcacctacct 840

cgaacctttt ttttttggtt tcacggagaa gaaactaaca atggctggtg aaattatagg 900

tatacacact atgatggcta gctaagagtg cgatggatca aagcagcaca gcgtcagcgt 960

caccggccta ctagattgga ctgcaagcat catcgaccgt cgaccgacgg cgcgtgcgcc 1020

tctctcgttc ttcgtagtcc gagaatttcg cgtacgtgta gtagggacgg acttcgcgtt 1080

tgcccaccca ccaccaaccc atccaacttc tgggttgggg cgacgacgtc cgtggaagca 1140

agatgtggcg ccgcagtggg agtgcaaacg agcgcgcaag ccaccggtgg tgggtctttg 1200

ggttttctgg aacgcgcgga ctgcggccac ggtacggtgc gcgcggaccc aaaggacatt 1260

caactcgtcc tcgaccgcgg gaggcgctcg cgcgggccct cttctgcgtg ccgtgcaccc 1320

gtcccaccgc gaggctacca ggccaggagc ggcgggcccc gctcgcgcgc gacgattttt 1380

gactcggttc acggtgggag gggtacaggg tcgcgcccac tgccagctgt ggaagcttct 1440

attcggaccg gagcggctga aagggcgagc atacgtacta acgtacgtat accgaagaaa 1500

cacacaagac tgacccgcgc gaagaaatag tcgcagctca cgctgccaga cgaaagcaac 1560

cgcctcgctg gtctatcgcc gcaaagccat tccatccttt aaatcgcccg aggcgacggg 1620

gcgagcgact catctccctc cccagacaag cggcgacacg ccagggcgga gagcacacac 1680

cgaagcgggc cgcggcggcg gcgcaggcgc aggcgcttac atgggagtag aggcgggcgg 1740

gtgcgggcgg agggcggtcg tgaccgggtt ctacgtctgg ggctgggagt tcctcaccgc 1800

cctcctgctc ttctcggccg ccgccgccgc cgcagactct tactagctag ctagccacct 1860

cttcttcctc ctccttgtag gtagtagtag gctacacaac aaccacatat atacatacta 1920

ggtactagca ggcgagcgag attcgtgcaa taatcaaggc agccagttat taatagttaa 1980

tcgtcggcgc cgagcgagag 2000

<210> 3

<211> 2928

<212> DNA

<213> corn

<400> 3

atgggagaag cggccgcggc cgtggcggcg tcgaagaggg gcggcgggcc ggcgccgttc 60

ctgaccaaga cgcaccagat ggtggaggag cggggcacgg acgaggtgat ctcgtgggcg 120

gagcagggcc gctccttcgt ggtgtggaag cccgtggagc tggcgcgcga cctcctcccg 180

ctccacttca agcactgcaa cttctcctcc ttcgtccgcc agctcaacac ctacgtgagt 240

acactacgcc gccgctccgg ccatcatctc ttctactacg atcgatgcaa tatatcacct 300

gtcgtcgtcg tttagtgatt gcaaaacaca tacacttggt ttccgtatta aattaatcag 360

ctagctagct agatgatcgt tctctgctct atgatctgtt agttctgaag catgttgttg 420

ttttcgtctg tgctcgataa attaagctat gttatgtggt cgacgagcga gccttccagg 480

cagctaccgt accgtcttcc aaggagtata tgcgtgtgag cgtgtcacgg ttcgtaggaa 540

ggagtgcgtc agtcatgaca catctctacc accctttaat tcctttccca cgcaaagcat 600

gcttgtcgtt tcagagctag ctgaagagga atgacctgcg ataacacttg aagattaggg 660

tgccggtgcg ggtctgaaat tacacctgtg ggtacgatcg tgatttagat agacgacttc 720

acggatgtga ttacaggagt tttttttttt ctctacctga tctaaggccc tgtttgggaa 780

cacagttttt tcaaactgca gtttttcaaa tactaaagta tactttagtc atgacattac 840

tacagtttac aatgcttcag ttttcgaata caacagtatt caatacatca aggtgtttgg 900

gaaaaacttt ggttgagacc aatcagccag agcgggacca agctggcact ctctttacag 960

agaaaaactt tggctgagac caaagtttcc aaaactgcaa aacaagtgca gtatttgcaa 1020

tactacagtt tagtatacag agatttcaga tgagtttcca aacacctcaa agtatataat 1080

accacagtat tgctcaatac tacagtattg cttcaatact gcagaaaaac tttgttccca 1140

aacaccccct aaactgccat ctctaactac tatatatgta tagagcaagg tgcacgggga 1200

aattgaataa gcaaagcaaa tcaggtcggt tgacacgcca cggtattgta gtggcgacag 1260

aagcatggta ttctatggaa cagttaaggc cctgtttggg aacaaagttt ttgaaaacca 1320

cagtttttga aatactatac tatactttag ttatgacaat accgtagttt ataataccgc 1380

agttttgaaa actgaggtcc agagctaagt ttagaatgcc ttaaaacaac tatagtattt 1440

gcaatacttc agttttgaaa acagagattt tacctagctt gccaaacacc attatgtata 1500

taatactgca gtatttgaga atactgcagt attcttccaa aactgcagaa aaactttgtt 1560

cccaaacacc ccctaagaag cttcggatgg acgagctttt cagggctagc tcttctgcgt 1620

ggcctacaag aaggttaatt tagctaggaa ttggatgcta ttagctgagc aagcaatata 1680

atcatccaag gcatccagca agtatactaa tcttttgttg cctcttccat ctattagctg 1740

ggatacgaaa tcgctcaaga aattgacttg gaagttagga tgatgattta ggccctgttt 1800

gtagtttctc caacagctag cttcataatt tgtttttgtt ttttggctgg atagtatttt 1860

ccaaaatagc ttcatggtat ttggtaaagc ttcttctttt tttctctctc tcaagccaaa 1920

ggaaagtgat gcagggatac gaatagctga aacacgagta gcttattcta gcgcagtcaa 1980

agattcacac tgacttgggt tcgttctcac tgaaccttaa tctattaatc agagggagag 2040

agagctagct tctctaaatc aatgtgtgaa cagctataag gcgttatctg accatgtgag 2100

cgacgtatgg tggtcaaagt agacaggcct gacgtgttca tttcggcgtt tgtttaggga 2160

ctggctgaat aggacactgt gtcgaatgca gctcttgttc tttttgccgc attggatact 2220

tacgtcgacg gcgaccatgg cgcatgcgca tccatatcca tgcagggttt ccgaaaggtg 2280

gtgccggacc ggtgggagtt cgcgaacgac aacttccgtc gaggcgagca gggtctcctg 2340

tccggcatcc gccgccgcaa gtcaacggcg ctgcagatgt ccaagtccgg atccggcggc 2400

agcggcggcg tgaacgccac gttccccccg cctctgcccc ctccgcctcc cgcgtcggcc 2460

accacgtccg gcgtccacga gcgcagctcg tcgtcggcgt cgtcgccacc gcgggcgccc 2520

gacctggcca gcgagaacga gcagctcaag aaggacaacc acacgctgtc cgccgagctg 2580

gcgcaggcgc gccggcactg cgaggagctc ctgggcttcc tctcgcgctt cctcgacgtc 2640

cggcagctcg acctccggct gctcatgcag gaggacgtgc gagcgggggc aagcgacgac 2700

ggcgcacagc gccgcgcgca cgcagtggcc agccagctgg agcgcggcgg cggcgaggag 2760

gggaagagcg tgaagctgtt cggcgtactc ttaaaggacg ccgcgaggaa gaggggccgg 2820

tgcgaggaag cggcggccag cgagcggccc atcaagatga tcagggtcgg cgagccgtgg 2880

gtcggcgtcc cgtcgtcggg cccgggccgg tgcggcggcg agaattaa 2928

<210> 4

<211> 3253

<212> DNA

<213> corn

<400> 4

atggtggagg aaggcgccac cgcggcggcg tcgaggagcg ggccggcgcc gttcctgagc 60

aagacgcacc agatggtgga ggagcggggc acggacgagg tgatctcgtg ggcggagcaa 120

ggccgctcct tcgtggtgtg gaagcccgtg gagctggcgc gcgacctcct cccgctccac 180

ttcaagcact gcaacttctc ctcctttgtc cgccagctca acacctacgt gagtactact 240

acgtacactc ctccatctat acatcatctg ttctctgttc agttgatgac tagactagat 300

agctgcctca taaaacaaag gtctgctttg gttcgatctg tgtgtgtgtc ttttgtttat 360

gtccaacgaa actgatggga gaagaaagcc tttcgaccgg cgtcgtcgtc agagagttgc 420

cgaacgtgcg tgtgtggtcg gcgagtctag aaaccttcca caaccttccg agagcgagcg 480

aggagacgta cacgtaacgt aaggccgccg gtcgaaacgt agaaactgag caaggcggcg 540

gccagggctt cttcctttcc tttccttgtg tcgtttgtgc aacgacctgg ctatcggttt 600

ccgtttcccg gtgtgtgtag tgtaccccta cgcacgtagc acaacacctg tactacgcca 660

cctgtcacct gtgtgacctt tgcttcgaga tgtacaacat ggtacagcag acagtcaatc 720

agagtacgcg tttaattagt agtataggaa gtggtggctt cattcgtcct cgtcgcgata 780

cacctagcag caacacgtac gcgcgcaaaa aggtgctgcg actgcgaggt gggagctgtt 840

ccgtggaata agcaaagcaa agcaaagcaa attaggcgcg cgcctgacac gccacagcgt 900

tctagtgcgc ctgcggtcga ttatcaatgg aacagttagg aatctcaagg aagcttcgcg 960

cgcgtggatg agcctttctg ggctcttcta tgcgtgtggt ctactactag aagctaattt 1020

agctaggaac tgctggacgt gtgtgtatat gctagcagct agctgaacaa gcaatattat 1080

catcgtccaa tgcaaggcac ccagcgaata tatatactga ccttttgttg ccttttagtt 1140

gggctcagaa atcgggtcaa taaataaatt gattttgaag ttagaatgaa tgaatgggag 1200

atttattgta gccaaacaac cgaacatgcg cgcaagcgat tgagtcttgt acggagacgc 1260

atgatgtgag tatgtgacga cgacttgccc actcccacag tcaaagcttc acacgcacag 1320

ctggtttctt tttgttctca ccggtccatc atcattcaac tattccatct atatagttag 1380

atggggagct agcttccgtg aataatgtct aaaaagaaaa agcgaaggcg acgcgcggta 1440

tctgacactg cgagcaacat gttctattca aagtagtata ggtaacactg ttagcaaaaa 1500

ataattttag gagataggcc aaaatatgtg ttttagttta tagattctgc agttcagagt 1560

ttatataatt ctgaccttaa atgttcgtat agtaaaaaaa ctgtagtgct cgatccatgc 1620

tgcatgcatg tttgctgttt acaattaata acttctttat ttgaaatcgt ttacagcctt 1680

taatattttt tttaaaaaaa attgtatagg agatttgttt tgttcaaaac gtacagagac 1740

acgcatccta tttcaaactt tttaaaactt tgtagttact tttcttttgt ttcagttcat 1800

ttattaccca agatatttag agtcggtttg gtttaggtcg tagctgtgaa aaaaaccatt 1860

gtaagccgtg agttgtgaaa aaaaagatgt tatgggctgt gagctgttta aaattttaaa 1920

ccatttggta ggtagaacct actaaaagtc gttcaaagtt ctttcatata tatttttatg 1980

gttgcatctg caagctgcta aaaacaggta aaaaacactt tcaattttgc actcagagaa 2040

agtttgcttt tagaaagaaa aaaaaaactg gtttctcgat tcaggccttt aatatggctt 2100

ttagctttta gggagcaaaa ggcaaaacca aaagtcaagc taaacacata cttattatct 2160

acaaaaaagt aacatactat atattgtaga gaggtgcgaa tctttgttaa cctattactg 2220

tgtctggctc cggaagatct aactaaccta gacattgtga tggagccacg gaggtggcgt 2280

tgtgcgtgtc tccattaata aaacggtcaa caatgattta tttgctagtg cacgcctcac 2340

atgtaccgta taatatatca gggacgtatc aaatgtttac tcatataagt acagatttgc 2400

acacgatttt attccgtctc tgaaattgtc gtcttagaca aaacaagaat tggttagggg 2460

cctgccgcct gcttaatggg acagtgatga atgctgcaca tttgatcact tttgccgctg 2520

ttttttttta gcatggatct gcattccaca tttccactta cgtcgatggc gacaatccat 2580

gcagggtttc cggaaggtgg tgccggaccg gtgggagttc gcgaacgaga atttccggcg 2640

aggcgagcaa ggcctcctgt ccggcatccg tcgccgcaag tcaacgacgc cgcagccatc 2700

caagtacggc ggcggcagcg tcgtgaacac cgcgttccct ccgccgttgc cccttccgcc 2760

tcccgcgtcg gtcaccacgt ctggcggtgg cggtgccggt ggcgctggca acgagcgcag 2820

ctcctcgtcg gcatcgtcgc cgccacggac agacgacctg accagcgaga acgagcagct 2880

caagaaggac aaccgcacgc tgtccaccga gctggcgcag gcgcgtcggc actgcgagga 2940

gctcctgggc ttcctctcgc gcttcctcga cgtccggcag ctcgaccttg ggctgctcat 3000

gcaggaagac gtgcgagcgg gtgccggtga cgacgccgcg ccgcgccgcg caatggtcag 3060

ccagctagag cgcggcggcg aggaggggaa gagcgtgaag ctgttcggcg tactcctcac 3120

ggatgccgca aggaagaggg cccggtgcga ggaggcggcg gccagcgagc ggcccattaa 3180

gatgatcaga atcggcgagc cgtggatcgg cgtcccgtcg tcgggcccgg ggcggtgcgg 3240

cggcgggaat taa 3253

Claims

1. The application of the corn double-regulation module in regulating plant growth and disease-resistant balance is characterized in that the corn double-regulation module comprisesZmTBF1aGene promoteruORFSequences, i.e.pZmTBF1a:uORF ^ZmTBF1a The nucleotide sequence of which is shown in SEQ ID No.1; the corn double-regulation module further comprisesZmTBF1bGene promoteruORFSequences, i.e.pZmTBF1b:uORF ^ZmTBF1b The nucleotide sequence of which is shown in SEQ ID No.2;

the application includes the following stepsZmTBF1aIs connected topZmTBF1a:uORF ^ZmTBF1a Downstream of (a) the (c) is,ZmTBF1bis connected topZmTBF1b:uORF ^ZmTBF1b Downstream of (2);ZmTBF1athe nucleotide sequence is shown in SEQ ID No.3,ZmTBF1bthe nucleotide sequence is shown in SEQ ID No.4;

wherein the plant is maize;

the disease resistance refers to disease resistance caused by Puccinia striolata and Curvularia lunata.

2. The use of a maize dual regulatory module of claim 1 for regulating plant growth and disease resistance, wherein the uORF is amplified from maize DNA ^ZmTBF1 Element and correspondingZmTBF1Promoter sequence, using seamless gramThe cloning method is respectively cloned into a plant expression vector pCAMBIA 3301.

3. The use of a maize dual regulatory module according to claim 2 for regulating plant growth and disease resistance, wherein the amplified uoorf ^ZmTBF1 Element and correspondingZmTBF1The primers for the promoter sequence are as follows:

ZmTBF1a-3301：

Forward：5’-atgaccatgattacgaattcCATCGCCATCCATTTAAACATT-3’

Reverse：5’-taccctcagatctaccatggCCCTCGCCGCCGGCTATGTATA-3’

ZmTBF1b-3301：

Forward：5’-atgaccatgattacgaattcGCTCGCTCGATCGTCCGCCCTT-3’

Reverse：5’-taccctcagatctaccatggCTCTCGCTCGGCGCCGACGATT-3’。

4. use of a maize double regulatory module according to claim 2 for regulating plant growth and disease resistance, characterized in that the pCAMBIA3301 vector is linearized with EcoRI and NcoI restriction enzymes and the amplified maize double regulatory module sequence is linked to the plant expression vector using seamless cloning technology.