CN113151348A

CN113151348A - Application of rice OsTAM1 gene in regulation and control of plant insect resistance

Info

Publication number: CN113151348A
Application number: CN202110285091.8A
Authority: CN
Inventors: 颜健; 黎平; 白根祥; 杨忠艳
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-03-17
Filing date: 2021-03-17
Publication date: 2021-07-23
Anticipated expiration: 2041-03-17
Also published as: CN113151348B

Abstract

The invention discloses application of a rice OsTAM1 gene in regulation and control of plant insect resistance. The invention discovers that the overexpression of the rice OsTAM1 gene can accumulate the content of normal rice 3-amino-3- (4-hydroxyphenyl) propionic acid, thereby changing the metabolic flux of a phenylpropanoid metabolic pathway and enabling the normal rice seedling stage to have the biological activity of resisting rice planthopper and borer; in the rice storage process, whether the stored seeds are infected by the rice moth is positively correlated with whether the plants have 3-amino-3- (4-hydroxyphenyl) propionic acid. The content of the plant 3-amino-3- (4-hydroxyphenyl) propionic acid is increased, or the insect resistance of the plant can be increased by improving the expression of the OsTAM1 gene and promoting the synthesis of the 3-amino-3- (4-hydroxyphenyl) propionic acid, so that the breeding of insect-resistant rice is facilitated, the variety improvement of the plant can be carried out through molecular breeding, and the promotion effect on the breeding of insect-resistant plant varieties is greatly realized.

Description

Application of rice OsTAM1 gene in regulation and control of plant insect resistance

Technical Field

The invention relates to the field of plant genetic engineering and plant metabolism, in particular to application of a rice OsTAM1 gene in regulation and control of plant insect resistance.

Background

Plant Insect Resistance (Plant Insect Resistance) refers to the self-protective ability of plants to block Insect infestation using various defense mechanisms, and is a heritable biological property. Insect resistance in plants can be divided into constitutive resistance and induced resistance. Constitutive resistance refers to the physical and chemical factors originally present in plants that prevent phytophagous insects from feeding, such as trichome and waxy layers and pseudocortex on the surface of plants. The induced resistance refers to the induced defense response of the plant after being stimulated by insect feeding, mechanical damage or external adverse factors. The actions of feeding and spawning of insects activate the induced resistance of plants, and the generation of the induced resistance can be divided into direct defense and indirect defense. Direct defense means that plants induce the production of secondary metabolites harmful to insects, such as alkaloids, glucosinolates, lignins, etc., which can affect the growth and development of insects and even kill them. Indirect defence includes all the features which have no direct effect on the phytophagous animal itself and which attract natural enemies of the phytophagous animal, thereby reducing the loss of the plant. Common signals are Jasmonic Acid (JA), Salicylic Acid (SA), ABA, Ethylene (ET), Gibberellin (GA), etc., and these signal pathways further activate the expression of defense genes, the direct defense and the production of indirect defense compounds, so that plants show insect resistance. Common pests of rice include rice planthoppers, chilo suppressalis, sesamia inferens, cnaphalocrocis medinalis and the like, the yield and the quality of the rice can be influenced to different degrees, incomplete statistics is provided, and the yield reduction amount of the rice caused by the influence of the pests accounts for 20% -30% of the total yield of the rice every year. In rice production, the occurrence of plant diseases and insect pests is mainly controlled by chemical prevention, but the problems of environmental pollution, grain safety and the like are caused by the use of chemical pesticides without limitation. Therefore, the research of the self-generation resistance gene and the resistance substance of the rice has important significance for obtaining insect-resistant rice varieties.

With the rapid development of molecular biology and genetic engineering in the plant field, the molecular mechanism of plant insect resistance is clearer and clearer. Previous studies have shown that rice has broad-spectrum resistance mainly: in rice, over 30 brown planthopper resistant genes or Quantitative Trait Loci (QTL) have 8 genes cloned, namely BPH14, BPH26, BPH3, BPH29, BPH 9, BPH18, BPH32 and BPH3, and besides, after the rice OsPAL8 and the rice coding tryptamine 5-hydroxylase gene CYP71A1 are mutated, the resistance of the rice to the rice planthopper and chilo suppressalis is enhanced.

There are 9 TAM homologous genes in rice, but the functions and actions of these gene family members are currently poorly understood, and only a few genes have been reported. Researches show that the OsPAL4.1 gene encodes a broad-spectrum general resistance gene belonging to NPL family, and has resistance to rice blast and banded sclerotial blight, the expression of OsTAM1 catalyzes alpha-Tyr to form beta-Tyr, and the existence of beta-Tyr can inhibit the growth of dicotyledonous plants, but how the growth and development of rice are influenced after the accumulation of the rice beta-Tyr is not clear, so that the further excavation of the function of the beta-Tyr in the rice is of great significance for the breeding of functional rice varieties.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides the application of 3-amino-3- (4-hydroxyphenyl) propionic acid in regulating and controlling the insect resistance of plants.

The invention also aims to provide application of the rice OsTAM1 gene in regulation and control of plant insect resistance.

The purpose of the invention is realized by the following technical scheme:

application of 3-amino-3- (4-hydroxyphenyl) propionic acid in regulating and controlling plant pest resistance.

The regulation and control of the insect resistance of the plant is to improve the insect resistance of the plant by increasing the content of 3-amino-3- (4-hydroxyphenyl) propionic acid in the plant.

The increase of the content of the 3-amino-3- (4-hydroxyphenyl) propionic acid in the plant is realized by over-expressing the OsTAM1 gene of the rice and/or adding exogenous 3-amino-3- (4-hydroxyphenyl) propionic acid.

The English name of the 3-amino-3- (4-hydroxyphenyl) propionic acid is 3-amino-3- (4-hydroxyphenyl) propanoic acid, and the molecular formula is C₉H₁₁NO₃(CAS number: 6049-54-3), abbreviated as beta-Tyr, which can be isolated from plants or obtained commercially.

The plant is a monocotyledon; preferably rice, wheat, corn or sorghum; more preferably rice.

The insect resistance is resistance to rice planthopper, borer and/or rice moth.

The stem borers include Chilo suppressalis, Cnaphalocrocis medinalis, and the like.

The overexpression of the rice OsTAM1 gene is preferably realized by the following modes: constructing the rice OsTAM1 gene on a plant expression vector to obtain an over-expression vector; the overexpression vector is then transformed into a plant for expression.

The plant expression vector is preferably a pMDC32 vector.

The transformation can be carried out by transforming the obtained recombinant plant expression vector into plant cells or tissues by conventional biological methods such as agrobacterium mediation, plant viral vector, direct DNA transformation, conductance transformation and the like.

The agrobacterium is preferably agrobacterium EHA 105.

Application of rice OsTAM1 gene in regulation and control of plant insect resistance.

The nucleotide sequence of the rice OsTAM1(LOC _ Os12g33610) is shown as SEQ ID NO: 1 is shown.

The insect resistance of the plant is regulated and controlled by increasing the accumulation of leaf 3-amino-3- (4-hydroxyphenyl) propionic acid through over-expression of the OsTAM1 gene of the rice, so that the insect resistance of the plant is increased.

The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain containing the rice OsTAM1 gene are applied to regulation and control of plant insect resistance.

The application of the molecular marker detection primer of the rice OsTAM1 gene in screening and/or identifying insect-resistant plant varieties is characterized in that the molecular marker detection primer of the rice OsTAM1 gene comprises an upstream primer TAM1-F and a downstream primer TAM1-R for amplifying a TAM1 gene:

TAM1-F：5′-AGGCGGCGGAGGGTCTT-3′(SEQ ID NO：2)；

TAM1-R：5′-CCTCCAGGCGGGGTCGT-3′(SEQ ID NO：3)。

the molecular marker detection primer of the rice OsTAM1 gene can also comprise upstream and downstream primers Hyg-F and Hyg-R for amplifying hygromycin (Hyg) gene:

Hyg-F：5′-ACGGTGTCGTCCATCACAGTTTGCC-3′(SEQ ID NO：4)；

Hyg-R：5′-TTCCGGAAGTGCTTGACATTGGGGA-3′(SEQ ID NO：5)。

the application of the molecular marker detection primer of the rice OsTAM1 gene in screening or identifying insect-resistant plant varieties is realized by any one of the following modes:

(1) amplifying the genome DNA of a plant variety to be detected by using the molecular marker detection primers TAM1-F and TAM1-R of the rice OsTAM1 gene, and if a 750bp fragment is obtained by amplification, indicating that the plant variety has insect resistance;

or

(2) The molecular marker detection primers TAM1-F and TAM1-R of the rice OsTAM1 gene are used for amplifying the genome DNA of the plant variety to be detected to obtain a 750bp fragment, and the molecular marker detection primers Hyg-F and Hyg-R are used for amplifying to obtain a 280bp fragment, so that the plant variety has insect resistance.

The plant described in the modes (1) and (2) is a monocotyledon; preferably rice, wheat, corn or sorghum; more preferably rice.

The insect resistance described in the modes (1) and (2) is resistance to rice planthopper, borer or rice moth.

A method for improving insect resistance of plants is to increase the insect resistance of the plants by over-expressing the OsTAM1 gene of rice (so as to improve the accumulation of 3-amino-3- (4-hydroxyphenyl) propionic acid in rice leaves).

The insect resistance is resistance to rice planthopper, borer or rice moth.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention takes PAL gene family member OsTAM1 of rice as an object, clones the CDs full-length sequence of OsTAM1 gene from rice Ni (Nipponbare), and accumulates the content of normal rice beta-Tyr (namely 3-amino-3- (4-hydroxyphenyl) propionic acid) after overexpression, thereby changing the metabolic flow of phenylpropane metabolic pathway, leading the normal rice seedling stage to have the biological activity of resisting rice planthopper and chilo suppressalis, and leading the rice storage process to show the biological activity of resisting rice moth. The accumulation of beta-Tyr is proved to endow rice with broad-spectrum insect resistance.

(2) After the OsTAM1 gene cloned by the invention is over-expressed, the content of beta-Tyr in leaves is increased, the rice planthopper resistance in the rice production process is increased, and the rice moth resistance in the rice storage process is increased, so that the insect resistance of rice can be increased by improving the expression of the OsTAM1 gene through a genetic engineering technology, which is not only beneficial to the breeding of insect-resistant rice of rice, but also can be used for improving the variety of plants through molecular breeding.

(3) The research on the insect resistance of the OsTAM1 gene and the further confirmation of the functions of beta-Tyr and secondary metabolites thereof, after the OsTAM1 gene is over-expressed, the leaf beta-Tyr is accumulated, further participates in the synthesis of downstream metabolites and the synthesis of active substances thereof, so that the insect resistance of rice is increased, the research has important significance for the elucidation of the functions of active substances containing beta-Tyr frameworks, and the promotion effect is great for further digging functional rice.

(4) Although some genes with broad-spectrum resistance are cloned at present, reports on rice PAL gene family member OsTAM1 gene in pest resistance research are less, the cloned OsTAM1 gene can improve the content of beta-Tyr, increase rice planthopper and rice moth resistance of rice, and has great promotion effect on revealing rice pest resistance research, the influence on rice pest resistance after overexpression of the OsTAM1 gene, a further rice broad-spectrum resistance mechanism and great promotion effect on breeding of pest resistance rice varieties.

Drawings

FIG. 1 is a diagram showing the positive identification result of OsTAM1 gene over-expressed plants; wherein; a is the result of positive plant PCR identification (T: amplified fragment of aminotransferase gene (TAM 1); H: amplified fragment of hygromycin resistance gene (Hyg)); b is the detection result of the corresponding plant beta-Tyr.

FIG. 2 is a phenotypic plot of PI (control plant) and OE-1 (overexpression line of OsTAM1 gene) after damage by rice planthopper; wherein; a represents the 10d, 15d, 20d and 30d phenotypes after the rice planthopper damages PI and OE-1 of rice; and B is a graph showing the change of the death numbers of rice planthoppers after damaging PI and OE-1 of rice at 10d, 15d, 20d and 30 d.

FIG. 3 is a graph of the phenotypic changes before and after the hazard of rice planthoppers to PI (control plants) and OE-1, OE-2 and OE-3 (over-expressed 3 lines of the OsTAM1 gene); wherein A is a phenotype before the rice planthopper is damaged; b is the phenotype after rice planthopper hazard (28 days).

FIG. 4 is a phenotype diagram of control, over-expression plants of OsTAM1 gene at 10 days after Chilo suppressalis damage (in the figure, PI is control, OE-1, OE-2, OE-3 and OE-4 are over-expression plants of OsTAM1 gene); wherein A is a phenotype of PI, OE-1 and OE-2 overexpression plants 10 days after chilo suppressalis damage; b is the phenotype of PI, OE-3 and OE-4 overexpression plants at the 10 th day after the chilo suppressalis damage.

FIG. 5 is a graph showing the occurrence of rice moth after storage for 60 days in 3 lines of control, OsTAM1 gene over-expressed plants (in the figure, PI is control, and OE-1, OE-2 and OE-3 are OsTAM1 gene over-expressed plants).

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental procedures used are conventional and can be carried out according to the recombinant techniques already described (see molecular cloning, A laboratory Manual, 2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.) and (modern techniques of plant physiological and biochemical research, gas phase Press); the materials, reagents, etc. used are commercially available.

The rice variety PI312777 related to the embodiments of the present invention is disclosed in the literature references (Qiu Shang, Cheng Xionghui, Liu Yang Qiang, Penghai, Wan bang. PI312777 antigen rice new strain potential field evaluation and yield comparison [ J ]. Yunnan agriculture university report (Nature science), 2012,27(04):461- > 466+ 474.);

the rice variety 9311, daylily, R2821, Meixiangzhan, R281, R321, IR64, Donglan black rice, Chen early glutinous No.1, Nipponbare (Nipponbare), 02428 and Zhonghua11 (Zhonghua No. 11) related in the embodiment of the present invention are published in the national rice data center (https:// www.ricedata.cn /);

the rice varieties R315 and MDS related in the embodiment of the invention are disclosed in references (Wangchahuan, Song Bo Wen, Yunjia, Shouwu, Huangming. construction of rice RIL population genetic map [ J ] based on whole genome sequencing, proceedings of southern China agricultural university, 2021,42(02): 44-50.).

Example 1 construction of OsTAM1 Gene overexpression vector

1.1 overexpression of OsTAM1 Gene:

taking Rice leaves of a Rice variety Nipponbare (Ni) at tillering stage, using an RNA extraction reagent of Takara and extracting total RNA, using a reverse transcription kit of Takara to reverse transcribe the total RNA into cDNA, inputting a gene login number (LOC _ Os12g33610) of OsTAM1 in Rice Genome Annotation Project (http:// Rice. plant biology. msu. edu /), obtaining a CDs sequence (SEQ ID NO.1) of the gene, designing a synthetic primer pair (33610F: 5'-ATGAGGCTTGCCAAGGAGCA-3'; 33610R: 5'-GCAGCGCTAGCAGATCGTC-3') of a target gene according to the sequence, obtaining cDNA of the OsTAM1 gene by PCR amplification, connecting the target fragment to a pMDC32 vector (pMDC32 vector is purchased from Botany Bio Inc) by a seamless cloning method, determining that the vector construction is successful by PCR amplification and sequencing, introducing the constructed overexpression vector OsTAM1-pMDC 105, verifying the correct PCR amplification result, the overexpression vector is introduced into a normal indica rice variety PI312777 (PI for short) by an agrobacterium-mediated genetic transformation method, and finally an overexpression T0 transgenic seedling of the gene is obtained and planted in a greenhouse.

1.2 transgenic plant positive identification: taking T0 rice leaves 15 days after transplantation, extracting total DNA from a part of the leaves by a CTAB method to be used as a template for identification, and respectively carrying out PCR identification by using a hygromycin (Hyg) synthetic gene and a TAM1 target gene, wherein two PCR identification primers are as follows

Hyg-F：5′-ACGGTGTCGTCCATCACAGTTTGCC-3′；

Hyg-R：5′-TTCCGGAAGTGCTTGACATTGGGGA-3′；

TAM1-F：5′-AGGCGGCGGAGGGTCTT-3′；

TAM1-R：5′-CCTCCAGGCGGGGTCGT-3′。

Meanwhile, 100mg of leaf liquid nitrogen is ground, is leached by 10 percent (w/w) hydrochloric acid, is subjected to ultrasonic treatment in ice bath for 15min, is filtered to a sample injection bottle by a 2-micron filter membrane, and is subjected to LC-MS quantitative detection to determine whether plant leaves contain beta-Tyr, wherein the LC-MS quantitative detection conditions are as follows:

a chromatographic column: zorbax XDB-C18(4.6 mm. times.250 mm, 5 μm); mobile phase A: 0.1% (v/v) aqueous acetic acid, mobile phase B: 0.1% (v/v) acetonitrile acetate solution at a flow rate of 300. mu.L/min, the gradient elution procedure is shown in Table 1; the column temperature is 42 ℃, and the sample injection amount is 1 mu L;

mass spectrum conditions: electrospray ion source (ESI), negative ion scanning mode, multi-reaction monitoring, sample chamber temperature of 16 ℃, capillary voltage of 3.5kV, taper hole voltage of 15V, ion source temperature of 150 ℃, desolvation temperature of 100 ℃, desolvation gas flow of 800L/h, and taper hole flow of 50L/h.

TABLE 1 gradient elution procedure

If the PCR identification can amplify 280bp fragments and 750bp fragments, as shown in FIG. 1A (T: an amplified fragment of an aminotransferase gene (TAM 1); H: an amplified fragment of a hygromycin resistance gene (Hyg)), and the beta-Tyr can be detected by LC-MS, the plant is indicated to be a positive plant; if the two fragments are not expanded and the beta-Tyr is not detected, the transgenic negative plant is indicated. And finally, identifying 13 strains to obtain 12 positive plants, wherein each plant is regarded as 1 strain and is respectively marked as OE-1 to OE-13, and the identification result of the positive plants is shown in figure 1B (No. 11 is false positive). And (3) harvesting and subculturing a single positive plant, obtaining a homozygous plant of T3 through subculturing for 3 times, and taking an OE-1, OE-2 and OE-3 homozygous plant line for subsequent experiments.

1.3 Experimental results: 12 OsTAM1 overexpression strains are obtained through genetic engineering and rice genetic transformation, and homozygote is obtained through separation of each strain through successive subculture and can be used for subsequent insect-resistant function verification. After the OsTAM1 gene is over-expressed, the leaves can synthesize a large amount of beta-Tyr, the accumulation of the beta-Tyr and secondary metabolites thereof influence the phenylpropanoid metabolic pathway.

Example 2 functional verification of OsTAM1 Gene

2.1 anti-rice planthopper experiment:

2.1.1 taking homozygous OE-1 and PI (control) seeds in example 1, sowing the seeds after pregermination in a well-partitioned plug tray, sowing 60 seeds of each of PI and OE-1, inoculating 120 rice planthopper second-instar nymphs to rice seedlings after 15 days of cultivation, covering the rice seedlings with a net (which can be covered with a baby mosquito net), counting the survival rate of the rice and photographing the recorded phenotype at 10d, 15d, 20d and 30d respectively, and repeating for 3 times, wherein the results are shown in fig. 2A and 2B.

2.1.2 to further verify the anti-planthopper function of the OsTAM1 gene of rice, seeds of PI and OE-1, OE-2 and OE-3 homozygous lines (obtained in example 1) are taken to accelerate germination, then sowed in a hole tray divided into blocks, the steps are repeated for three times, each line is sowed with 60 grains for each repetition, after 15 days of cultivation, 300 rice planthopper second-instar nymphs are inoculated to rice seedlings, the rice seedlings are covered with a net cover, the change condition of the rice is observed after 28 days, and the effects before and after treatment are shown in the figure 3A and the figure 3B.

2.2 Chilo suppressalis resistance test:

seeds of homozygous lines of PI and OE-1, OE-2, OE-3 and OE-4 (obtained in example 1) are respectively taken, immersed in a constant temperature box at 30 ℃ for pregermination, sowed in a hole tray containing nutrient soil, cultured for 15 days, and then transplanted to a field, and one part is used for Chilo suppressalis resistance experiments. The experimental process for the chilo suppressalis resistance is as follows: taking blue pots with the specification of 38cm multiplied by 24cm, planting six rice plants in each line respectively as shown in figure 4, inoculating chilo suppressalis second instar insects to leaf sheaths of the rice plants after the tillering stage, inoculating two rice plants in each line, covering the rice plants with a net, observing the phenotype of the plants after 10 days, repeating for 3 times, and obtaining the treatment effect as shown in figure 4.

2.3 anti-rice moth experiment:

2.3.1 transplanting the other part of the rice seedlings in the 2.2 into a field, and harvesting and drying seeds of a single plant after the seedlings are mature. 30g of each of PI and seeds of three plants (OE-1, OE-2 and OE-3) of 3 over-expression lines are taken and packed in a self-sealing bag of 10 x 6cm, marked and placed in a dry and ventilated seed cabinet together with a tray for storage. After 60 days, the situation of rice moth infection is observed, and the situation of rice insect infection is counted (3 times of repetition): insensitivity to insects (-): is completely free of rice moth; sensing insects (+): there is a development of rice moth. The respective species were quantitatively detected by LC-MS (detection method same as 1.2). The results are shown in FIG. 5 and Table 1 (PI-1, PI-2 and PI-3 in Table 1 represent the results of 3 replicates, and so on).

2.3.2 further verifies the influence of the accumulation of beta-Tyr and the occurrence of rice moth on rice seeds. We used 15 conventional rice varieties for validation, 11 of which were indica varieties: PI312777, 9311, HUANGHUAZHAN, MDS, R2821, MEIXIANGZHAN, R281, R321, R315, IR64, Donglan black rice, Chen morning glutinous No. 1; japonica rice variety: nipponbare, 02428, Zhonghua 11. The specific pest-sensing condition is shown in (table 2), 30 seeds of all varieties are taken for accelerating germination and seedling raising, the seeds are transplanted to a field, 5 samples are taken from each variety in the flowering period, each sample is 100mg, the ground seeds are leached by 10% (w/w) hydrochloric acid, after ultrasonic vibration is carried out in ice-water bath for 15min, a 2um filter membrane is used for filtering the samples into a sample injection bottle, and the LC-MS quantitative detection is carried out (the detection method is the same as 1.2). The results are shown in Table 2 as to whether or not β -Tyr is contained. After each variety is mature, the single plant is harvested and dried in the sun. Taking 30g of seeds of 3 plants of each variety, packaging in a self-sealing bag of 10 multiplied by 6cm, marking, placing in a dry and ventilated seed cabinet together with a tray for storage, and repeating for 3 times. And (5) observing the occurrence of the rice moth at 60d, and counting: insensitivity to insects (-): is completely free of rice moth; sensing insects (+): there is a development of rice moth.

2.4 Experimental results:

2.4.1 Rice plantlets treated by rice planthoppers show that the death number of the rice PI is obviously more than that of over-expressed plants and the leaf withering is more obvious along with the increase of the damage time, which shows that the rice planthopper resistance is enhanced after the over-expression of the OsTAM1 (figure 2 and figure 3).

2.4.2 Chilo suppressalis treatment experiments show that the leaf sheath of PI plants ten days after Chilo suppressalis inoculation has obvious hazard (figure 4), the heart leaves of the PI plants are withered, the heart leaves are extracted by hands, obvious chewing traces exist at stem cuts, old leaf blades turn yellow and the like, but the over-expressed plants have no phenomena, which indicates that the OsTAM1 has certain resistance effect on the Chilo suppressalis after over-expression.

2.4.3 experiments against rice moth show that whether the stored seeds feel rice moth is positively correlated with whether the plant has beta-Tyr (Table 1 and Table 2), and rice has synthesized beta-Tyr, which shows that no rice moth occurs, otherwise, rice moth occurs in storage.

In conclusion, after the OsTAM1 is over-expressed, rice can synthesize a large amount of beta-Tyr, the accumulation of the beta-Tyr and secondary metabolites of the beta-Tyr can participate in the broad-spectrum insect resistance of the rice.

TABLE 1 statistics of OsTAM1 overexpression seeds and PI-sensitive moth

Numbering	Name (R)	Situation of moth planting	Whether or not it contains beta-Tyr
				1	PI-1	+	Whether or not
2	PI-2	+	Whether or not
				3	PI-2	+	Whether or not
4	OE-1-1	-	Is that
				5	OE-1-2	-	Is that
6	OE-1-3	-	Is that
				7	OE-2-1	-	Is that
8	OE-2-2	-	Is that
				9	OE-2-3	-	Is that
10	OE-3-1	-	Is that
				11	OE-3-2	-	Is that
12	OE-3-3	-	Is that

TABLE 2 statistics of beta-Tyr content and moth-resistant phenotype of cultivars in different places

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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<220>

<223> TAM1-F

<400> 2

aggcggcgga gggtctt 17

<210> 3

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TAM1-R

<400> 3

cctccaggcg gggtcgt 17

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Hyg-F

<400> 4

acggtgtcgt ccatcacagt ttgcc 25

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Hyg-R

<400> 5

ttccggaagt gcttgacatt gggga 25

Claims

Application of 3-amino-3- (4-hydroxyphenyl) propionic acid in regulating and controlling plant pest resistance.
2. Use according to claim 1, characterized in that:

the regulation and control of the insect resistance of the plant is to improve the insect resistance of the plant by increasing the content of 3-amino-3- (4-hydroxyphenyl) propionic acid in the plant;

the increase of the content of the 3-amino-3- (4-hydroxyphenyl) propionic acid in the plant is realized by over-expressing the OsTAM1 gene of the rice and/or adding exogenous 3-amino-3- (4-hydroxyphenyl) propionic acid;

the plant is a monocotyledon;

the insect resistance is resistance to rice planthopper, borer and/or rice moth.
3. Application of rice OsTAM1 gene in regulation and control of plant insect resistance.
4. The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain containing the rice OsTAM1 gene are applied to the regulation and control of the insect resistance of plants.
5. Use according to claim 3 or 4, characterized in that:

the insect resistance of the plant is regulated and controlled by increasing the accumulation of leaf 3-amino-3- (4-hydroxyphenyl) propionic acid through over-expression of a rice OsTAM1 gene, so that the insect resistance of the plant is increased;

the plant is a monocotyledon;

the insect resistance is resistance to rice planthopper, borer and/or rice moth.
6. Use according to claim 2 or 5, characterized in that:

the plant is rice, wheat, corn or sorghum;

the stem borer is Chilo suppressalis, Ostrinia nubilalis or Cnaphalocrocis medinalis.
7. The application of the molecular marker detection primer of the rice OsTAM1 gene in screening and/or identifying insect-resistant plant varieties is characterized in that:

the molecular marker detection primer of the rice OsTAM1 gene comprises an upstream primer TAM1-F and a downstream primer TAM1-R for amplifying the TAM1 gene:

TAM1-F：5′-AGGCGGCGGAGGGTCTT-3′；

TAM1-R：5′-CCTCCAGGCGGGGTCGT-3′。
8. use according to claim 7, characterized in that:

the molecular marker detection primer of the rice OsTAM1 gene also comprises upstream and downstream primers Hyg-F and Hyg-R for amplifying the hygromycin gene:

Hyg-F：5′-ACGGTGTCGTCCATCACAGTTTGCC-3′；

Hyg-R：5′-TTCCGGAAGTGCTTGACATTGGGGA-3′。
9. the use according to claim 8, characterized in that, the method is realized by any one of the following methods:

(1) the molecular marker detection primers TAM1-F and TAM1-R of the rice OsTAM1 gene are used for amplifying the genome DNA of the plant variety to be detected, and if a 750bp fragment is obtained by amplification, the plant variety is proved to have insect resistance;

or

(2) Amplifying the genomic DNA of the plant variety to be detected by using molecular marker detection primers TAM1-F and TAM1-R of the rice OsTAM1 gene to obtain a 750bp fragment, and amplifying by using molecular marker detection primers Hyg-F and Hyg-R to obtain a 280bp fragment, which indicates that the plant variety has insect resistance;

the plant described in the modes (1) and (2) is a monocotyledon;

the insect resistance described in the modes (1) and (2) is resistance to rice planthopper, borer or rice moth.
10. A method of increasing insect resistance in a plant, comprising: increasing the insect resistance of the plant by over-expressing the OsTAM1 gene of the rice;

the plant is a monocotyledon;

the insect resistance is resistance to rice planthopper, borer or rice moth.