CN113151348B - Application of rice OsTAM1 gene in regulation and control of plant insect resistance - Google Patents

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

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CN113151348B
CN113151348B CN202110285091.8A CN202110285091A CN113151348B CN 113151348 B CN113151348 B CN 113151348B CN 202110285091 A CN202110285091 A CN 202110285091A CN 113151348 B CN113151348 B CN 113151348B
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颜健
黎平
白根祥
杨忠艳
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South China Agricultural University
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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 3-amino-3- (4-hydroxyphenyl) propionic acid of the plant 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 method is not only beneficial to the breeding of insect-resistant rice, but also can improve the variety of the plant through molecular breeding, and has great promotion effect on the breeding of insect-resistant plant varieties.

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 a Plant to block Insect infestation using various defense mechanisms, and is a heritable biological property. The insect resistance of 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 the 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 on the self-generated resistance genes and resistance substances 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 more and more clear. 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 which are cloned and are BPH14, BPH26, BPH3, BPH29, BPH 9, BPH18, BPH32 and BPH3 respectively, and except that the resistance of the rice to rice planthoppers and chilo suppressalis is enhanced after OsPAL8 and the gene CYP71A1 of the tryptamine 5-hydroxylase gene of the rice are mutated.
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 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 rice beta-Tyr is not clear, so that the further excavation of the function of beta-Tyr in rice has important 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 rice OsTAM1 gene 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 C 9 H 11 NO 3 (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 through the following modes: constructing a rice OsTAM1 gene on a plant expression vector to obtain a super expression vector; then the overexpression vector is 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 EHA105.
The application of the rice OsTAM1 gene in regulating and controlling the insect resistance of plants.
The nucleotide sequence of the rice OsTAM1 (LOC _ Os12g 33610) is shown as SEQ ID NO:1 is shown.
The plant is a monocotyledon; preferably rice, wheat, corn or sorghum; more preferably rice.
The insect resistance is resistance to rice planthoppers, borers and/or rice moths.
The borers include Chilo suppressalis, cnaphalocrocis medinalis guenee or Cnaphalocrocis medinalis guenee.
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 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 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 borers include Chilo suppressalis, cnaphalocrocis medinalis guenee or Cnaphalocrocis medinalis guenee.
The application of the molecular marker detection primer of the rice OsTAM1 gene in screening and/or identifying insect-resistant plant varieties, wherein 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′(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 contain 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 genomic 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, wherein if a 750bp fragment is obtained by amplification, 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 a 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.
The borers include Chilo suppressalis, cnaphalocrocis medinalis guenee or Cnaphalocrocis medinalis guenee.
A method for improving insect resistance of plants is to increase the insect resistance of the plants by over-expressing rice OsTAM1 gene (so as to improve the accumulation of rice leaf 3-amino-3- (4-hydroxyphenyl) propionic acid).
The plant is a monocotyledon; preferably rice, wheat, corn or sorghum; more preferably rice.
The insect resistance is resistance to rice planthoppers, borers or rice moths.
The borers include Chilo suppressalis, cnaphalocrocis medinalis guenee or Cnaphalocrocis medinalis guenee.
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 the 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 a 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 the beta-Tyr is proved to endow the 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 the 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 the beta-Tyr and the secondary metabolites thereof, after the OsTAM1 gene is over-expressed, the 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 on the elucidation of the functions of the active substances containing the beta-Tyr framework, and the promotion effect on further digging functional rice is realized.
(4) Although some genes with broad-spectrum resistance are cloned at present, the reports of insect resistance research on the OsTAM1 gene of a rice PAL gene family member are less, the cloned OsTAM1 gene can improve the content of beta-Tyr, increase the rice planthopper and rice moth resistance of rice, and has great promotion effect on disclosing the rice insect resistance research, the influence on the rice insect resistance after the overexpression of the OsTAM1 gene, a further rice broad-spectrum resistance mechanism and great promotion effect on breeding insect-resistant rice varieties.
Drawings
FIG. 1 is a diagram showing the positive identification result of OsTAM1 gene overexpression 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 phenotype of PI (control plant) and OE-1 (overexpression line of the OsTAM1 gene) after being attacked by rice planthopper; wherein; a represents the 10d,15d,20d and 30d phenotypes after the rice planthopper harms the rice PI and OE-1; b is a graph showing the change of the number of deaths from rice planthopper damaging rice PI and OE-1 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 (3 lines over-expressed 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 4 control OsTAM1 gene over-expressed plants at the 10 th day after Chilo suppressalis damage (in the figure, PI is a control, and OE-1, OE-2, OE-3 and OE-4 are over-expressed 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 and OsTAM1 gene over-expressed plants (in the figure, PI is a 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 involved in the examples of the present invention is disclosed in the literature of reference (Qiuhuang, chenxionghui, liujian, penghai, wanbanhui. PI312777 antigen rice new strain potential field evaluation and yield comparison [ J ]. Yunnan university of agriculture (Nature science), 2012,27 (04): 461-466+474 ]);
the rice variety 9311, yellow flower occupation, R2821, meixiang occupation, 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 disclosed 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, xiaowuming, huangming. Construction of rice RIL population genetic map [ J ] based on whole genome sequencing, university of south China school report 2021,42 (02): 44-50.).
Example 1 construction of OsTAM1 Gene overexpression vector
1.1 overexpression of the OsTAM1 gene:
taking Rice leaves of Rice variety Nipponbare (Ni) at tillering stage, using RNA extraction reagent of Takara and extracting total RNA, and using reverse transcription kit of Takara to reverse transcribe total RNA into cDNA, inputting gene landing number (LOC _ Os12g 33610) of OsTAM1 into Rice Genome inhibition Project (http:// Rice. Plant biology. Msu. Edu /), obtaining CDs sequence (SEQ ID NO. 1) of the gene, designing synthetic primer pair (33610F.
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 is ground by liquid nitrogen, leached by 10% (w/w) hydrochloric acid, subjected to ice bath ultrasound for 15min, filtered to a sample injection bottle by a filter membrane of 2 mu m, and 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.250mm, 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
Figure BDA0002980122170000071
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 T3 homozygous plant through subculturing for 3 times, and taking OE-1, OE-2 and OE-3 homozygous plants for subsequent experiments.
1.3 Experimental results: 12 OsTAM1 overexpression strains are obtained through genetic engineering and rice genetic transformation, and homozygotes are obtained through separation of the strains through successive generation culture 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 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 rice planthopper resistance 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 repeated line is sowed with 60 grains, 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 FIGS. 3A and 3B.
2.2 Chilo suppressalis resistance test:
seeds of homozygous lines of PI, OE-1, OE-2, OE-3 and OE-4 (obtained in example 1) are taken respectively, 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, respectively planting rice, as shown in figure 4, six plants of each line, culturing until the tillering stage, inoculating chilo suppressalis second-instar insects to the leaf sheaths of the rice, inoculating two plants to each line, covering with a net cover, 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 (-): the rice moth is completely absent; insect-sensing (+): 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 verify the influence of the accumulation of beta-Tyr and the occurrence of rice seed with rice moth. We used 15 conventional rice varieties for validation, 11 of which were indica varieties: PI312777, 9311, HUANGHUAZHAN, MDS, R2821, MEIXIANGZHAN, R281, R321, R315, IR64, east orchid fructus Zizaniae Caduciflorae, chen morning glutinous No. 1; japonica rice varieties: nipponbare, 02428, and Zhonghua11. The specific insect-susceptible conditions are shown in table 2, 30 seeds of all varieties are taken for germination acceleration and seedling raising, then the seeds are transplanted to a field, 5 samples are taken from each variety in the flowering period, each sample is 100mg, after grinding, 10% (w/w) hydrochloric acid is used for leaching, after ice-water bath ultrasonic vibration is carried out for 15min, 2um filter membranes are used for filtering to a sample injection bottle, and 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, harvesting and drying the seeds of a single plant. 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 the rice planthoppers show that the death number of the rice PI is obviously more than that of an over-expressed plant and the leaf withering is more obvious along with the increase of the damage time, which shows that the rice planthoppers resistance is enhanced after the OsTAM1 is over-expressed (figure 2 and figure 3).
2.4.2 Chilo suppressalis treatment experiments show that the leaf sheath of the PI plant has obvious hazard after ten days of Chilo suppressalis inoculation (figure 4), the heart leaves of the PI plant are withered, the heart leaves are extracted by hands, obvious chewing traces exist at the stem cut part, old leaf blades turn yellow and the like, but the over-expressed plant has no phenomena, and the fact that the OsTAM1 has a certain resistance effect on the Chilo suppressalis after over-expression is shown.
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, the 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 OsTAM1 overexpression seed and PI moth-sensing statistics
Number of Name(s) 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
Figure BDA0002980122170000091
Figure BDA0002980122170000101
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 modifications are intended to be included in the scope of the present invention.
Sequence listing
<110> southern China university of agriculture
Application of rice OsTAM1 gene in regulation and control of plant insect resistance
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2043
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Rice OsTAM1 Gene
<400> 1
atggagtgtg agaccggtct ggtcgaccgt cccctcaacg gcgacccctt gtactggggc 60
aaggcggcgg agggtcttgc ggggagccac ctcgacgagg tgaagaggat ggtggtggag 120
taccgcgcgc cgctggtgaa gatcgacggc gccatgctca gcgtcgccaa ggtggcagcc 180
gtcgctggcg aggccgcccg ggtgcaggtg gtgctggacg aatccgcacg accccgcctg 240
gaggctagtc gcgagtgggt cttcgacagc accatgaacg gcaccgacac gtacggcgtc 300
accaccggct tcggaggtgc cgcccaccgc cgcaccaagg agttcgccgc gctccagaaa 360
gagctgatcc gccgcagcga cggctacacg ctgccgacgg aggtcactcg cgcagccatg 420
ctggtgcgca tcaacaccct cacccagggc tactcgggca tccgcttcga gatcctcgag 480
gccatcgcca agctgctcaa cgccaacgtg acgccgtgcc tgccgctccg gggcaccatc 540
accgcgtccg gcgacctggt cccgctgtcc tacatcgccg gcctcatcac cggccgccag 600
aactccgcgg cggtggcccc ggacggcagc aaggtggacg ccgctgaggc gttcaggatc 660
gccggcatcg agcacgggtt cttcgcgttg cagcccaagg aagggctcgc catcgtcaac 720
ggcacggccg tgggctccgg cctcgcggcg atcgtgctct tcgaggccaa cgtcctggcc 780
gtccttgccg aggtcctctc ggcggtgtac tgcgaggtga tggccggcaa tccggagtac 840
accgaccacc tcatccacgc gctgaagcac caccctggac agatcgaagc tgcggccatc 900
atggagcaca tactggaagg cagctcctac atgaggcttg ccaaggagca gggcgagctt 960
gaccggttga cgaagctgag gcaggacagg tacgccatcc gcacggcgcc gcagtggctc 1020
ggcccgcagg tcgaggtcat ccgcttcgcc accaagtcga tcgagcggga gatcaactcc 1080
gtcaacgaca acccggtcat cgacgtcgcc cgccgcaagg cgctccacgg cggcaacttc 1140
cagggcactc ccatcggggt gtccatggac aacactcgtc tcgccatcgc tgccatcggc 1200
aggctcatgt tctcgcagtt ctccgagctt gccagtagct tctacagcaa cggccttccc 1260
tccaatctgt ccggcgggcg caacccgagc ttggactacg gtttcaatgg cgccgaggtc 1320
gccatggcgt cctactgctc tgagctgcag ttcctcgcca acccggtgac caaccatgtg 1380
cagaccgcgg agcagcacaa ccagagcgtc aactctctcg gcctcatctc ctccaggatg 1440
acagcagagg ccgtcaccat cctgaagctc atgtcctcta ctttcctcat cgcactgtgc 1500
caggccgtcg acctgcgcca actcgaggag agcatcaagg ccgctgttaa caagtgcgtg 1560
acaaatgtcg ccaagaaatc cctggccatg gacgatgacg atctgctagc gctgctcggc 1620
gctgctatcg accgcgtggc ggtgttcacg tacgcagaag acccatgcag atccagcttg 1680
ccactcatac agaagctccg cgcggtgctc atggaccatg cgctggccaa cggtgacaac 1740
cagctggcca aggtggctga gttcgagcag cagctccgcg cggtgctcca cgacgaggtg 1800
gaggccgcac gcgcggctgt ggagagcggc acggccccga acaggatcac ccagtgtcgc 1860
tcgtacccgc tgtacaggtt cgtgcgcaag gagctcggcg ccgagtacct gaccggcgag 1920
aagacgcggt cgcccgggga ggaggtggac aaggtggtga tcgccatgaa ccagcacaag 1980
cacatcaacc cactgctgga gtgcctcagc gagtggaaag gcgcgcccct gccacttaat 2040
tag 2043
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<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 (8)

  1. The application of 3-amino-3- (4-hydroxyphenyl) propionic acid in regulating and controlling plant pest resistance is characterized in that:
    the insect resistance of the plant is regulated and controlled by increasing the content of 3-amino-3- (4-hydroxyphenyl) propionic acid in the plant so as to improve the insect resistance of the plant;
    the increase of the content of 3-amino-3- (4-hydroxyphenyl) propionic acid in plants is achieved by over-expressing riceOsTAM1The way of gene realization;
    the plant is rice;
    the insect resistance is resistance to rice planthopper, borer and/or rice moth;
    the rice isOsTAM1The nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.
  2. 2. Rice (RICE)OsTAM1The application of the gene in regulating and controlling the insect resistance of plants is characterized in that:
    the insect resistance of the plants is regulated and controlled by over-expressing riceOsTAM1The gene improves the accumulation of 3-amino-3- (4-hydroxyphenyl) propionic acid on leaves, thereby increasing the insect resistance of plants;
    the plant is rice;
    the insect resistance is resistance to rice planthopper, borer and/or rice moth;
    the rice isOsTAM1The nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.
  3. 3. Contains riceOsTAM1The application of the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria of the gene in regulating and controlling the insect resistance of plants is characterized in that:
    the insect resistance of the regulating plant is controlled by over-expressing riceOsTAM1The gene improves the accumulation of 3-amino-3- (4-hydroxyphenyl) propionic acid in leaves, thereby increasing the insect resistance of plants;
    the plant is rice;
    the insect resistance is resistance to rice planthoppers, borers and/or rice moths;
    the rice isOsTAM1The nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.
  4. 4. Use according to any one of claims 1 to 3, characterized in that: the stem borer is Chilo suppressalis, ostrinia nubilalis or Cnaphalocrocis medinalis.
  5. 5. Rice (Oryza sativa L.) with improved resistance to stressOsTAM1The application of the molecular marker detection primer of the gene in screening and/or identifying insect-resistant plant varieties is characterized in that:
    the rice isOsTAM1The molecular marker detection primer of the gene contains the primer for amplificationOsTAM1Upstream and downstream primers for genes TAM1-F and TAM1-R:
    TAM1-F：5′-AGGCGGCGGAGGGTCTT-3′；
    TAM1-R：5′-CCTCCAGGCGGGGTCGT-3′；
    the plant is rice;
    the insect resistance is resistance to rice planthopper, borer and/or rice moth;
    the rice isOsTAM1The nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.
  6. 6. Use according to claim 5, characterized in that:
    the rice isOsTAM1The gene molecular marker detection primer also contains upstream and downstream primers Hyg-F and Hyg-R for amplifying the hygromycin gene:
    Hyg-F：5′-ACGGTGTCGTCCATCACAGTTTGCC-3′；
    Hyg-R：5′-TTCCGGAAGTGCTTGACATTGGGGA-3′。
  7. 7. the use according to claim 6, characterized in that, the method is realized by any one of the following methods:
    (1) Using riceOsTAM1Gene molecular marker detection primers TAM1-F and TAM1-R for genomes of plant varieties to be detectedAmplifying the DNA, and if a 750bp fragment is obtained by amplification, indicating that the plant variety has insect resistance;
    or
    (2) Using riceOsTAM1The gene molecular marker detection primers TAM1-F and TAM1-R 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.
  8. 8. A method of increasing insect resistance in a plant, comprising: by overexpressing riceOsTAM1The gene increases the insect resistance of the plant;
    the plant is rice;
    the insect resistance is resistance to rice planthoppers, borers or rice moths;
    the rice isOsTAM1The nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.
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