WO2022134132A1

WO2022134132A1 - Rice striped stem bore damage-inducible promoter posisa1 and application thereof in breeding smart stem borer-resistant rice

Info

Publication number: WO2022134132A1
Application number: PCT/CN2020/139903
Authority: WO
Inventors: 陈浩; 凃巨民; 都浩; 曹方彬; 梅磊
Original assignee: 浙江大学
Priority date: 2020-12-22
Filing date: 2020-12-28
Publication date: 2022-06-30
Also published as: CN112522270A; CN112522270B

Abstract

A rice striped stem bore damage-inducible promoter pOsISA1, at least comprising a nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence as shown in SEQ ID NO. 2. The sequence of a gene OsISA1 is as shown in SEQ ID NO. 3. The rice striped stem bore damage-inducible promoter pOsISA1 and the gene OsISA1 can be used to breed striped stem bore-resistant rice. New uses of the rice striped stem bore damage-inducible promoter pOsISA1 and the gene OsISA1 in the breeding of striped stem bore-resistant rice are discovered, and important gene resources are provided for the breeding of a striped stem bore-resistant rice variety.

Description

Rice borer damage-inducible promoter pOsISA1 and its application in breeding smart borer-resistant rice

technical field

The invention relates to the technical field of plant genetic engineering, in particular to a rice borer damage-inducible promoter pOsISA1 and its application in cultivating intelligent borer-resistant rice.

Background technique

Insect pests are one of the most important factors in reducing rice production, and statistics show that the annual production reduction caused by this is staggering. At present, the control of pests in agricultural production mainly relies on chemical pesticides. The extensive use of chemical pesticides not only increases production costs, but also causes environmental pollution and even threatens human health. There is an urgent need to cultivate insect-resistant rice varieties to improve the rice's own insect resistance. However, conventional rice breeding technology not only takes a long time to cultivate new insect-resistant varieties, but also has no effective resistance to rice borers, the most important pests of rice, such as Diploid chinensis, Trilophos chinensis and Rice leaf roller. Therefore, it is still not feasible to improve the insect resistance of rice through conventional breeding. Therefore, the most direct method is to use transgenic technology to introduce exogenous insect-resistant genes into the recipient rice to create new insect-resistant varieties. So far, many useful insect-resistant genes have been identified and cloned in plants, animals and even microorganisms, some insect-resistant genes have been transferred into rice to obtain transgenic insect-resistant lines, and some field experiments have been carried out to prevent the above-mentioned pests. Shows good resistance.

From the perspective of the development of transgenic insect-resistant rice, from the initial stage, constitutive expression promoters such as 35sCaMV, Actin I, and Ubiquitin were used to drive the expression of insect-resistant genes, and many new transgenic rice lines with better resistance to borers were obtained. . With the development of transgenic technology and detection methods, it was found that the use of constitutive expression promoters exposed some problems in the process of experiments, such as excessive consumption of substances and energy in plant cells, increased metabolic burden of rice, and changes in agronomic traits. At the same time, there are also excessive public concerns about food safety and so on. The main reason for these problems is that the use of constitutive expression promoters to drive the target gene cannot effectively regulate the expression of the target gene from time and space, but can only be continuously and constantly expressed in various tissues and organs of transgenic recipient plants. . In addition, when the same constitutive expression promoter is repeatedly used to drive the expression of two or more exogenous genes, it may cause transgene silencing or co-suppression, which brings a lot of trouble to multigene transformation. Therefore, with the development of plant genetic engineering, it is urgent to find more effective tissue- and organ-specific expression promoters or inducible expression promoters to replace constitutive expression promoters, in order to better regulate time and space. The gene of interest is expressed in the target tissue or organ.

An inducible promoter means that under the stimulation of certain physical or chemical signals, the transcription level of the target gene can be greatly increased. Among them, there are mainly hormone-inducible promoters, such as SAUR15 (Small Auxin-up RNA15) gene promoter is induced by auxin and brassinolide and up-regulated expression (Walcher, C.L., Nemhauser, J.L., Bipartite promoter element required for auxin response [ J].Plant physiology, 2012, 158 (1), pp 273-282.); secondly, there is a type of abiotic stress inducible promoter, rice Wsi18 gene can be significantly induced by NaCl, ABA and drought stress and up-regulated expression , but no obvious response to low temperature stress. The promoter was isolated and cloned, and it was found that the background activity of this promoter in transgenic rice is very low, but it can be induced to increase expression by the above-mentioned stress (Yi, N., Oh, S.-J., Kim, Y.S., etc. , Analysis of the Wsi18, a stress-inducible promoter that is active in the whole grain of transgenic rice[J]. Transgenic research, 2011, 20(1), pp 153-163.). There is also a type of biotic stress inducible promoter, such as the PmTNL1 gene promoter can be specifically and efficiently induced by white rust (Liu, J.-J., Ekramoddoullah, A.K., Genomic organization, induced expression and promoter activity of a resistance gene analog (PmTNL1)in western white pine(Pinus monticola)[J].Planta,2011,233(5),pp 1041-1053.), the promoter of rice Xa13 gene can be specifically induced by bacterial blight (Yuan, T. ,Li,X.,Xiao,J.,etc.,Characterization of Xanthomonas oryzae-responsive cis-acting element in the promoter of rice race-specific susceptibility gene Xa13[J].Molecular plant,2011,4(2),pp 300-309.). Hua et al. (Hua, H., Lu, Q., Cai, M., etc., Analysis of rice genes induced by striped stemborer (Chilo suppressalis) attack identified a promoter fragment highly specifically responsive to insect feeding[J].Plant molecular biology, 2007, 65(4), pp 519-530.) was isolated and cloned into a specific inducible expression promoter fragment of Diploss spp., which has a very promising prospect in insect-resistant transgenic research.

However, there are still few studies on pest damage-inducible promoters, and there are fewer studies on the inducible promoters of rice borer damage. It is necessary to do further in-depth research to find more Promoters and genes.

SUMMARY OF THE INVENTION

The invention provides a new rice borer damage-inducible promoter pOsISA1, gene OsISA1 and its new use in cultivating borer-resistant rice, and provides a basis for cultivating intelligent borer-resistant rice.

The specific technical solutions are as follows:

The present invention provides a rice borer damage-inducible promoter pOsISA1, which at least comprises the nucleotide sequence shown in SEQ ID NO.1.

Further, the nucleotide sequence of the rice borer damage-inducible promoter pOsISA1 is shown in SEQ ID NO.2.

The promoter pOsISA1 is a predicted cytochrome P450-like gene promoter with a full length of 2360bp, of which the last 650bp region is predicted to contain the core element of the promoter by bioinformatics, that is, the nucleotide sequence shown in SEQ ID NO.1. Promoters such as New PLACE (https://www.dna.affrc.go.jp/PLACE/?action=newplace) and plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) The predicted website is predicted to contain core elements such as TATA box and CAAT box that initiate transcription, as well as the predicted stress response element CATTTG. The above-mentioned promoter can rapidly respond to the damage of Dillite borer and initiate the transcription of downstream genes.

According to the transcriptome sequencing data of rice before and after inoculation with S. chinensis, the mRNA transcription level of the gene OsISA1 in various tissues and parts was extremely low and almost undetectable before being inoculated by S. chinensis. However, the transcription level was sharply up-regulated in the damaged tissues such as stems after the damage of Dichilla borer, and the up-regulation fold was more than 500 times. It is indicated that the gene can quickly and efficiently respond to the damage of Diploxin, and its promoter is shown as a Diploid damage-inducible promoter. Therefore, the above-mentioned promoter and insect-resistant element are cloned from the japonica rice variety Nipponbare to form an insect-resistant module and introduced into the chassis crop rice, which can realize the intelligent expression of the insect-resistant gene. The specific performance is that the anti-insect module does not work when the pests are not harmed, and there is no expression of the anti-insect protein, which does not affect the growth and development of rice. Finally, ensure that no anti-insect protein is produced and accumulated in the edible part. The invention also discloses the cloning of the above promoter and the construction method of the corresponding expression vector, as well as the genetic transformation method of rice mediated by Agrobacterium. The above characteristics of the promoter have strong application value in cultivating new intelligent insect-resistant rice.

The invention also provides the application of the rice borer damage-inducible promoter pOsISA1 in cultivating intelligent borer-resistant rice.

The borers include lepidopteran pests such as S. chinensis, S. chinensis, and rice leaf roller.

Further, described application is characterized in that, comprises the following steps:

(1) construct a recombinant vector containing the rice borer damage-inducible promoter pOsISA1 described in any one of

claims

1 or 2;

(2) transferring the recombinant vector into Agrobacterium competent cells to construct a genetically engineered bacterium containing the recombinant vector described in step (1);

(3) Transforming the rice callus with the genetically engineered bacteria described in step (2) to obtain a transgenic positive rice plant.

Further, in step (1), the original expression vector of the recombinant vector is pSB130, and the recombinant vector also contains an insect-resistant gene.

Further, in step (2), the Agrobacterium is EHA105.

The present invention also provides the application of the rice gene OsISA1 as an inducible gene of rice borer in cultivating rice borer resistance, and the nucleotide sequence of the gene OsISA1 is shown in SEQ ID NO.3.

Further, the described application comprises the following steps:

(A) construct the recombinant vector containing rice gene OsISA1, the nucleotide sequence of described gene OsISA1 is as shown in SEQ ID NO.3;

(B) transferring the recombinant vector into Agrobacterium competent cells to construct a genetically engineered bacterium containing the recombinant vector described in step (A);

(C) transforming the rice callus with the genetically engineered bacteria described in step (B) to obtain a transgenic positive rice plant.

Further, in step (A), the original expression vector of the recombinant vector is pSB130, and the recombinant vector also includes the rice constitutive promoter pActin I.

Further, in step (B), the Agrobacterium is EHA105.

The present invention also provides a recombinant vector comprising the rice borer damage-inducible promoter pOsISA1; or, comprising the rice gene OsISA1 whose nucleotide sequence is shown in SEQ ID NO.3.

Further, the recombinant vector also includes an insect-resistant gene; further, the insect-resistant gene is a Bt gene.

The present invention also provides a transformant, which comprises the rice borer damage-inducible promoter pOsISA1; or the rice gene OsISA1 whose nucleotide sequence is shown in SEQ ID NO.3.

Further, the transformant also includes an insect-resistant gene; further, the insect-resistant gene is a Bt gene.

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

(1) The present invention finds a rice borer damage-inducible promoter pOsISA1 and gene OsISA1, discovers new uses of the promoter and gene in cultivating rice borer resistance, and is useful for cultivating intelligent rice borer resistance. Varieties provide important genetic resources.

(2) The present invention utilizes transgenic technology to provide a method for cultivating intelligent insect-resistant rice, and obtains an intelligent insect-resistant rice material.

Description of drawings

Figure 1 is a schematic diagram of the overall flow of the construction of intelligent insect-resistant rice according to the present invention.

Figure 2 shows the relative expression levels of the OsISA1 gene in each tissue in the japonica rice Nipponbare in Example 1, and the data comes from the Rice Gene Annotation Database (http://rice.plantbiology.msu.edu/).

FIG. 3 is the average relative expression level of transcripts of OsISA1 gene in Example 1 when it is damaged by Diploss spp.

Among them, HPL2 is a positive control, and its promoter is the damage-inducible type of C. chinensis. The abscissas 1-4 represent before treatment (0h), treatment 24h, 48h and 72h, respectively. The ordinate represents the relative expression of transcripts. A total of 4 biological replicates were set up.

FIG. 4 is a schematic diagram of the pSB130-rbcS-TP-Bt expression vector of Example 1. FIG.

FIG. 5 is a schematic diagram of the pOsISA1-Bt expression vector of Example 1. FIG.

Fig. 6 is the molecular detection of cry1Ab/cry1Ac target gene in the transgenic resistant callus of Example 1;

Among them, M: DL 2000 molecular weight marker; +: positive control; -: negative control; 1-33: callus after transformation; arrows indicate target bands.

Fig. 7 is the detection result of protein in the stem Cry1Ab/Cry1Ac of Example 2;

Among them, IC-3, IC-5, IC-8 are 3 independent transformants; ZY3 (TT51-derived line) is used as a positive control, NT is a non-transgenic negative control; IC3-T and other 3 are artificially treated with Dichilla After the results, and IC-3 and other 3 are the corresponding untreated control samples.

Fig. 8 is the detection result of Cry1Ab/Cry1Ac protein in the seed of embodiment 4;

Among them, IC-3, IC-5, IC-8 were 3 independent transformants; ZY3 (TT51-derived line) was used as a positive control; NT was a non-transgenic negative control.

FIG. 9 is a schematic diagram of the pOsISA1-OE expression vector in Example 5. FIG.

FIG. 10 shows the average relative expression levels of the transcripts of the OsISA1 gene in Example 5 when the OsISA1 gene is overexpressed.

Among them, NT is the negative control. The abscissa represents 4 independent transformants. The ordinate represents the relative expression of transcripts. A total of 3 biological replicates were set up.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, the following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

Example 1

1. Excavation of the damage-inducible genes and promoters of Diploss spp.

The transcriptome sequencing data of rice stems at tillering stage before (0h) and after (24h, 48h, and 72h) damage by S. chinensis were taken, and bioinformatics technology was used to screen for rapid response to S. The target gene that is almost not expressed when the borer is infested.

The characteristics of the above-mentioned genes are as follows: before the treatment of S. chinensis, the gene transcription level is low, and the limit is FPKM ≤ 20; In the rice gene annotation database (http://rice.plantbiology.msu.edu/), the FPKM value of each tissue and part is extremely low, especially the detection value in the endosperm is required to be 0.

According to the above requirements, a cytochrome P450-like gene (LOC_Os04g08824) was screened and named OsISA1 (Induced by Striped stem borers Attacked 1) (Fig. 2). However, the above-mentioned transcriptome sequencing data of S. japonica showed that the expression of OsISA1 was sharply up-regulated after being damaged by Diploss spp. (Fig. 3). Therefore, its promoter is predicted to have the function of rapidly and efficiently responding to the damage of Dilophos chinensis. According to the Nipponbare sequencing sequence published in the Rice Gene Annotation Database (http://rice.plantbiology.msu.edu/), the size of about 2Kb before ATG was used as the predicted promoter region. In this patent, 2360bp before the start codon ATG of the OsISA1 gene is selected as the promoter and named as pOsISA1.

2. Construction of intelligent anti-insect expression module

According to the sequence information provided by the rice gene annotation website (http://rice.plantbiology.msu.edu/) and the sequence information of the expression vector pSB130-rbcS-TP-Bt (Fig. 4), refer to the one-step rapid cloning kit Hieff

Plus One Step Cloning Kit instructions for designing cloning primers containing kozak sequences (pOsISA1-F: 5'-TAAAACGACGGCCAGTGCCGCTTGTGAAATTTACTCTGTAA-3', pOsISA1-R: 5'-GCAGTTGTTGTCCAT GGTGGC GCTAGATAACTGATCAGGTGGC-3').

Using the high-quality DNA of Nipponbare wild-type rice as a template, a total of 2400bp DNA fragments of pOsISA1 sequence of 2360bp, Kozak sequence (GCCACC ATGG , excluding underlined partial bases) and homologous sequences on both sides of the vector backbone were amplified by KOD FX high-fidelity enzyme. , connected to the vector backbone pSB130-Em-Bt after the rbcS-TP fragment was excised from pSB130-rbcS-TP-Bt, and the HindIII and SalI restriction sites at the homologous junction were not retained.

The specific experimental methods are as follows:

2.1 Target fragment cloning

KOD FX reaction system (40μL):

The reaction program was 94 °C for 3 min, 98 °C for 10 s, 68 °C for 3 min for 15 sec, amplification for 32 cycles, and a final extension at 68 °C for 7 min. The above products were separated by 1% agarose gel electrophoresis.

2.2 Recovery of target fragments

Use Zymoclean Gel DNA Recovery Kit to recover the above-mentioned target fragment gel. The specific steps are as follows: cut off the target band on the agarose gel with a blade and put it into a 1.5mL centrifuge tube; add 3 times the volume of ADB to the centrifuge tube buffer, that is, 100μL (mg) gel plus 300μL ADB buffer, and then placed in a metal bath at 55°C for 5-10 minutes to completely melt the gel; transfer the agarose solution to the adsorption column set on the collection tube, and centrifuge at 12000rpm for 30sec , discard the effluent; add 200 μL of Wash Buffer to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the effluent; repeat the washing twice, then centrifuge the empty tube for 2 min; cover the adsorption column on a new 1.5 mL centrifuge tube, add 6-20 μL Elution Buffer, stand for 1 min and centrifuge for 1 min to elute the DNA.

2.3 Vector linearization

The pSB130-rbcS-TP-Bt vector was double digested with HindIII and SalI from TaKaRa company. The specific steps are as follows:

After incubation at 37°C for 1 hour, the vector backbone pSB130-Em-Bt with a larger fragment was recovered by electrophoresis.

2.4 Purpose vector construction

Use the one-step rapid cloning kit Hieff to use the recovered PCR product and linearized vector

The Plus One Step Cloning Kit is used to construct the expression vector. The system is as follows:

After mixing and centrifuging, place it in a PCR instrument for 20 minutes at 50°C, and cool it on ice for 5 minutes to transform E. coli DH5α. The specific steps are as follows:

Take out the DH5α competent cells from the -80°C refrigerator, thaw on ice, add the above ligation product, and mix gently; ice bath for 30 minutes, heat shock in a 42°C water bath for 45s, and cool on ice for 2 minutes; add 500 μL LB The liquid medium was placed on a shaker at 37°C, and recovered at 200 rpm for 1 h; centrifuged at 5000 rpm for 3 min to precipitate the bacteria, aspirated 400 μL of the supernatant, and mixed the remaining supernatant with the bacteria by pipetting. On the LB solid medium of 37°C, invert overnight (12-16h) in a constant temperature incubator at 37°C; select 3 single clones, inoculate them into LB liquid medium containing karamycin, and place them on a shaker at 37°C and 250rpm for culture. When the solution was turbid, positive clones were detected by bacterial liquid PCR and sent for sequencing identification. Samples with correct sequencing are stored in bacterial solution and plasmid for future use.

The correctly sequenced expression vector was named pOsISA1-Bt. The expression vector map is shown in Figure 5. So far, the intelligent anti-insect expression module has been successfully constructed.

3. Agrobacterium-mediated genetic transformation of rice

3.1 Expression vector transformation of Agrobacterium EHA105

The transformation of Agrobacterium by pOsISA1-Bt vector was carried out by electroporation, and the Agrobacterium strain used was EHA105. The specific procedure is as follows: add 0.5 μL of plasmid to a 1.5 mL centrifuge tube containing 50 μL of Agrobacterium EHA105 electroshock competent cells, mix it with a pipette, and then transfer it to the electrode cup; after electric shock, quickly add 1 mL of LB liquid medium , pipette and mix evenly, transfer it to the previous 1.5mL centrifuge tube, and shake it on a constant temperature shaker at 28°C for 1h; L kanamycin, 25mg/L rifampicin) surface, the petri dish was inverted and placed in a 28°C incubator for 2 days; after the positive clones were verified by colony PCR, the positive clones were shaken to preserve the bacterial solution (1mL of the bacterial solution was added 250 μL of 80% sterile glycerol). Store at -80°C for later use.

3.2 Agrobacterium-mediated genetic transformation of rice

Rice transformation was carried out according to the method and steps reported by Chen Hao (Chen Hao. Development of "gene switch" system and its application in breeding "green" insect-resistant rice with zero endosperm expression [D]. Zhejiang: Zhejiang University, 2016.) .

The specific procedures are as follows:

Take out the Agrobacterium liquid stored at -80°C, draw 200μL from it, and spread it evenly on the surface of LB solid medium containing 25mg/L rifampicin and 50mg/L kanamycin, and culture at 28°C overnight; The single colony was expanded and cultivated, and the liquid medium used was the same as described above; after that, 200-300 μL of fresh bacterial liquid was drawn from it and inserted into 20 mL of LB liquid medium containing 25 mg/L rifampicin and 50 mg/L kanamycin. , 28 ℃ shaking (220rpm) cultured for 16-18h. Take a sufficient amount of bacterial liquid and centrifuge it at 4000rpm for 15min, discard the LB medium supernatant; add 20mL of 0.1M _MgSO4 solution to resuspend Agrobacterium (resuspend gently by pipetting), and centrifuge at 4000rpm for 10- 15min, discard the MgSO ₄ supernatant containing antibiotics; add 5 mL of AA-AS infection medium containing 200 μM acetosyringone (AS) to resuspend Agrobacterium, and then add an appropriate amount of AA-AS infection medium to adjust the bacteria The OD600 value of the solution was finally adjusted between 0.2-0.8; the bacterial solution was dispensed into a sterile 50mL centrifuge tube, 20-25mL/tube, for use.

Transfer Xiushui 134 embryogenic callus pre-cultured for about 7 days from the subculture dish to an empty petri dish covered with sterile filter paper, and air-dry it on the ultra-clean workbench for about 10-15min. Slowly roll the callus with a stainless steel spoon to make it fully dry; after it is dry, transfer it into a 50mL centrifuge tube containing bacterial liquid, shake it gently (not too vigorously) at room temperature for about 40min, and put the centrifuge tube in an ultra-clean work. Let stand on the table for 10 min; discard the bacterial liquid, and place the embryogenic callus on sterile filter paper to dry for about 15 min; then, transfer the infected callus to a sterile filter paper-covered surface containing AS (200 μM). On the co-cultivation medium (Table 1), cultivate in the dark for 50-55h at 28°C; select the embryogenic callus with no massive growth of Agrobacterium or contamination on the surface, and move it to the antibacterial culture containing 500mg/L cephalosporin On the base (Table 2), the antibacterial culture was carried out in a dark room at 28°C for 3-4 days; the callus after the antibacterial culture was then transferred to the screening medium containing 500 mg/L cephalosporin and 75 mg/L hygromycin, and 28 ℃ darkroom culture; in the first week, the Agrobacterium contamination should be checked every day. If the contamination cannot be controlled, the screening medium should be replaced in time, and the callus with good growth status should be selected every half month and subculture on the fresh screening medium. , and adjust the concentration of cephalosporin in the medium according to the degree of Agrobacterium self-contamination. In general, it can be considered to halve the concentration of cephalosporin in the third or fourth round of sub-screening.

A total of 33 independent transformants were obtained according to the above procedure.

Table 1 AA medium formula

Table 2. CC Medium Recipe

3.3 PCR identification of transgenic resistant callus

3.3.1 Extraction of genomic DNA from resistant callus

Weigh 0.1 g of transgenic resistant calli from each independent transformant subcultured and place it in a sterilized 1.5 mL centrifuge tube filled with grinding beads; add 500 μL of 1.5×CTAB extract to the sample grinder Grind to homogenate, place in 56°C water bath for 20-30min (repeatedly invert and mix 2 times during the water bath); then add 500μL of chloroform, invert up and down several times and mix thoroughly, then centrifuge at room temperature for 10min (8000rpm); pipette 400μL Clear to a new centrifuge tube, add 800 μL of absolute ethanol, mix well and place at -30°C for about 30 minutes; centrifuge at 12,000 rpm and room temperature for 5 minutes and discard the supernatant; wash the DNA pellet with 75% ethanol and discard Ethanol and dried at room temperature; add 100 μL of sterile water to dissolve overnight for use.

3.3.2 Molecular detection of cry1Ab/cry1Ac in transgenic resistant calli

Molecular detection of cry1Ab/cry1Ac in transgenic resistant calli was performed using conventional PCR techniques. The detection primers used are: Bt-F: 5'-TGGTTCTGCCCAAGGTATCG-3' and Bt-R: 5'-AACGGTTCCGCTCTTTCTGT-3', the size of the amplified target fragment is about 369 bp, and the PCR reaction system is the same as the conventional system. The PCR reaction program was as follows: denaturation at 94°C for 5 min; denaturation at 95°C for 15sec, annealing at 55°C for 30sec and extension at 72°C for 30sec (32 cycles); extension at 72°C for 10min and storage at 12°C at low temperature. The PCR amplification products obtained were separated and identified by 1% agarose gel electrophoresis.

PCR detection results showed that cry1Ab/cry1Ac genes could be detected in 18 of the 33 screened transgenic resistant calli in a ratio of about 54.5% (Fig. 6). It indicated that the target gene had been integrated into the recipient plant, and the negative single plant indicated that the resistant callus might have only integrated the Hpt gene and could grow on the selective medium without integrating the target gene.

Therefore, the negative material was discarded, and only the positive material was regenerated for redifferentiation.

3.3.3 Differentiation of cry1Ab/cry1Ac positive callus

Differentiation of cry1Ab/cry1Ac positive callus according to Chen Hao (Chen Hao. Development of "gene switch" system and its application in breeding "green" insect-resistant rice with zero endosperm expression [D]. Zhejiang: Zhejiang University, 2016. ) reported by the method. The specific experimental procedure is as follows: transfer the resistant callus positive for the target gene cry1Ab/cry1Ac to N6 differentiation medium (N6 basic medium + 2 mg/L Kinetin + 1 mg/L NAA + 4% Gelrite), in a dark room at 28 °C Medium pre-differentiation for 7-9 days, then transferred to fresh differentiation medium, and differentiated green shoots in a light room at 25°C (usually, green dots can be seen after 7-14 days, and green shoots can be differentiated after 3 weeks) . The obtained green seedlings, after washing the medium adhered to the root system, directly (root and shoot differentiation type) or after rooting in the rooting medium (bud first differentiation type) are transferred into Yoshida culture medium for transitional culture, and wait for their growth. After the condition is good and stable, it is transplanted to the greenhouse until maturity.

Example 2 Qualitative detection of insect-resistant proteins in transformants such as IC-3 of positive transgenic plants

The Cry1Ab/Cry1Ac protein in the positive transgenic plants IC-3 and other three transformants was detected and analyzed with the Cry1Ab/Cry1Ac test strips from Shanghai Youlong Company. Protein extraction and test paper detection in rice stems at heading stage were carried out according to the steps described in the product instructions with slight modifications. The stalks damaged by S. chinensis (artificially inoculated for 48 hours) and the stems not damaged by S. spp. were selected for comparison experiments, and the above three transformants were detected to verify the function of the intelligent anti-insect module.

The specific steps are as follows: respectively take transgenic rice stalks of about 2-3 cm in length, place them in a mortar, and add liquid nitrogen to fully grind them. Take about 0.2 g of powder and add it to a 1.5 mL centrifuge tube pre-installed with 0.5 mL of the protein extraction Buffer that comes with the kit, vortex and mix, and then centrifuge at 12,000 rpm for 30 sec. Transfer 0.3 mL of the supernatant to another sterile 1.5 mL centrifuge tube. Then put in the test strip, and observe the color development of the test strip for about 3 minutes.

The results showed that the protein samples of the positive transgenic plants could detect the target band after being damaged by Diloquat, but could not be detected in the corresponding controls (Fig. 7). It is not expressed when it is not harmed by Dilophos, but only after it is harmed. Based on this, it is inferred that the intelligent anti-insect module can work normally according to the expected design.

Example 3 Identification of Insect Resistance of To Generation Individual Plants of Transformants such as IC-3

The above three independent transformants including IC-3 were selected for artificial inoculation identification, and the inoculation identification was carried out by artificial in vitro stalk method.

The specific steps of the in vitro stalk method are as follows: take 2 rice seedlings of the main ear at the heading stage, wipe the rice seedlings dry, cut the 2 rice seedlings into 2 5cm stalks including nodes and leaf sheaths, and then place them on the stalks. A small filter paper sheet impregnated with 0.1 g/L benzodiazepine fresh-keeping solution was pressed on the end. Then each stalk was connected to 10 ant borers (Ceratophobia), and the 2 stalks were transferred to the inner diameter of a small flat-bottom glass tube (9.5cm×1.5cm), and the mouth of the tube was plugged with absorbent cotton. The mortality of larvae and the weight of live worms were examined or determined on the 7th day after the start of infestation. Meanwhile, on the 3rd day, small filter paper sheets impregnated with 0.1 g/L benzopyrazole fresh-keeping liquid were added at both ends of the stems to ensure that the stems were kept moisturizing. On the 7th day, the rice plants were stripped and inspected, the number of larvae survived was recorded, and the mass of live worms in each glass tube was weighed if necessary.

The test results confirmed that the three independent transformants such as IC-3 showed high resistance to the artificially inoculated C. chinensis. As shown in Supplementary Table 4, the mortality rate of the inoculated C. chinensis on the control was 10%, which was significantly lower than that of the three transformants, among which the mortality rate in IC-8 was as high as 90%. It was indicated that the above three transformants could intelligently respond to the damage of Diplostis chinensis and express anti-insect protein in a targeted manner to achieve the effect of anti-insect. Finally, according to the transgenic new material and the parental inoculation, the difference in the mortality rate of Diplostis chinensis is extremely significant.

Example 4 Qualitative detection of Cry1Ab/Cry1Ac protein in To generation seeds of transformants such as IC-3

The Cry1Ab/Cry1Ac protein in the seeds of the T0 generation of the positive transgenic plants IC-3 and other three transformants was detected and analyzed by the Cry1Ab/Cry1Ac test strips from Shanghai Youlong Company.

Protein extraction and test strip detection in rice seeds were performed according to the procedures described in the product instructions with slight modifications. About 50 seeds with glumes were selected for testing.

The specific steps are as follows: about 50 transgenic rice seeds are respectively taken and placed in a grinding sample box equipped with steel balls with a diameter of 1.5 cm, and the grinding sample box is placed on a sample grinding machine to grind the seeds into powder. Take about 0.2 g of powder and add it to a 1.5 mL centrifuge tube pre-installed with 0.5 mL of the protein extraction Buffer that comes with the kit, vortex and mix, and then centrifuge at 12,000 rpm for 30 sec. Transfer 0.3 mL of the supernatant to another sterile 1.5 mL centrifuge tube. Then put in the test strip, and observe the color development of the test strip for about 3 minutes.

The results showed that the target protein could not be detected in the protein samples of the seeds of the transformants such as IC-3, which was consistent with the wild-type control (Fig. 8). The results indicated that the Cry1Ab/Cry1Ac protein was not expressed in the seeds. So far, new germplasms of intelligent insect-resistant rice, represented by transformants such as IC-3, have been successfully created.

Example 5 Acquisition and functional verification of OsISA1 gene overexpression material

In the phytozome rice genome database (https://phytozome.jgi.doe.gov/pz/portal.html#!search show=KEYWORD&method=Org_Osativa), use keywords to search for the OsISA1 gene (LOC_Os04g08824), and obtain its genome sequence (including Exon, intron and 5 ' and 3 ' UTR sequence) (SEQ ID No.3), adopt the experimental method in embodiment 1 to clone OsISA1 gene, and construct in the expression vector pLB (Chen Hao. Development of "gene switch" system and its application in breeding "green" insect-resistant rice with zero endosperm expression [D]. Zhejiang: Zhejiang University, 2016.) Excision on the backbone of LNL-Bt fragment.

The cloning primer of this gene is (ISA1-OE-F: 5′-GCTTTTTGTAGGTAGACTGCAGTATCGTCTCTGCACTCACACTG-3′, ISA1-OE-R: 5′-CGATCGGGGAAATTCGTCGACAATAAAAATGGTTTTATTTGTA-3′), and the expression vector backbone was linearized with Pst I and Sal I enzymes. The construction method was the same as that in Example 1, and the constructed expression vector was named pOsISA1-OE (Fig. 9).

Similarly, it was transformed into a rice variety Xiushui 134 by Agrobacterium infection, and the expression level of OsISA1 gene in the T0 generation positive seedlings was detected. Compared with the non-transgenic control, the transcription level of this gene was increased by about 61.36- to 377.18-fold in 4 independent transformants, respectively (Fig. 10). According to the characteristic of the sharp up-regulation of OsISA1 expression after the nematode A. chinensis, it is inferred that when the gene is overexpressed, it may enhance the resistance to S. chinensis to a certain extent. Therefore, this genetic resource can be used to breed new insect-resistant rice materials.

Finally, it should also be noted that the above list is only a few specific embodiments of the present invention. Objectively, the present invention is not only limited to the above embodiments, and can also have a considerable part of variations. Therefore, all modifications that those of ordinary skill in the art can directly derive or associate from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims

A rice borer damage-inducible promoter pOsISA1, characterized in that it at least comprises the nucleotide sequence shown in SEQ ID NO.1.
The rice borer damage-inducible promoter pOsISA1 according to claim 1 is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.2.
Application of the rice borer damage-inducible promoter pOsISA1 according to any one of claims 1 or 2 in cultivating intelligent borer-resistant rice.
application as claimed in claim 3, is characterized in that, comprises the following steps:

(1) construct a recombinant vector containing the rice borer damage-inducible promoter pOsISA1 described in any one of claims 1 or 2;

(2) transferring the recombinant vector into Agrobacterium competent cells to construct a genetically engineered bacterium containing the recombinant vector described in step (1);

(3) Transforming the rice callus with the genetically engineered bacteria described in step (2) to obtain a transgenic positive rice plant.
The application of the rice gene OsISA1 as an inducible gene of rice borer damage in cultivating intelligent rice borer resistance is characterized in that the nucleotide sequence of the gene OsISA1 is as shown in SEQ ID NO.3.
application as claimed in claim 5, is characterized in that, comprises the following steps:

(A) construct the recombinant vector containing rice gene OsISA1, the nucleotide sequence of described gene OsISA1 is as shown in SEQ ID NO.3;

(B) transferring the recombinant vector into Agrobacterium competent cells to construct a genetically engineered bacterium containing the recombinant vector described in step (A);

(C) transforming the rice callus with the genetically engineered bacteria described in step (B) to obtain a transgenic positive rice plant.
A kind of recombinant vector, it is characterized in that, comprise the rice borer damage inducible promoter pOsISA1 as described in any one of claim 1 or 2; Or, comprise the rice with nucleotide sequence as shown in SEQ ID NO.3 Gene OsISA1.
The recombinant vector of claim 7, further comprising an insect resistance gene.
A transformant, comprising the rice borer damage-inducible promoter pOsISA1 according to any one of claims 1 or 2; or comprising the rice gene OsISA1 whose nucleotide sequence is shown in SEQ ID NO.3.
The transformant of claim 9, further comprising an insect resistance gene.