Molecular marker screening method of rice bakanae disease resistance gene

Abstract

The invention discloses a molecular marker screening method of a rice bakanae disease resistance gene, and relates to the field of biotechnology and plant genetic breeding. The method comprises the following components: S1, construction of a rice protein library and phage display, S2, specific binding screening with pathogenic bacteria of the rice bakanae disease, S3, identification of resistance proteins and corresponding genes, and S4, development of molecular markers. By constructing a rice protein library phage display system and utilizing a specific binding screening technology with rice bakanae disease pathogenic bacteria, the resistance-related protein and the corresponding gene can be efficiently screened from a large number of rice genes, and compared with a traditional method, the screening mode is more direct and efficient, and the screening cost is reduced. Errors caused by non-specific screening are reduced, and the efficiency and accuracy of resistance gene screening are greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and plant genetic breeding technology, specifically to a molecular marker screening method for rice bakanae disease resistance genes BACKGROUND

[0002] Rice is one of the most important food crops in the world, and its yield and quality are severely affected by various diseases, among which rice bakanae disease is a serious disease caused by pathogenic bacteria, which can cause rice plants to grow excessively, lodge and even die, seriously affecting the yield and quality of rice. In order to address this challenge, scientists have been working hard to find and identify resistance genes in rice in order to cultivate rice varieties with high resistance through genetic engineering.

[0003] In traditional technology, resistance gene screening usually relies on a large amount of phenotypic screening and genetic map construction work, which is not only time-consuming and laborious, but also often disturbed by environmental conditions and genetic background, resulting in low accuracy and reliability of screening results. In addition, traditional technology is difficult to directly identify specific genes related to resistance from complex genomes quickly and accurately, which limits the progress of disease resistance breeding work.

[0004] In view of the deficiencies of traditional technology in resistance gene screening, the present application provides a molecular marker screening method for rice bakanae disease resistance genes. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies of the prior art and provide a molecular marker screening method for rice bakanae disease resistance genes, which can efficiently screen proteins and corresponding genes related to resistance from a large number of rice genes by constructing a rice protein library phage display system and using specific binding screening technology with rice bakanae disease pathogen.

[0006] To solve the above technical problems, the present application provides the following technical solution: a molecular marker screening method for rice bakanae disease resistance genes, the specific steps of which are as follows: S1, construction of rice protein library and phage display Select different rice varieties, collect tissue samples at the critical period of rice growth, grind into powder, extract total RNA, reverse transcribe cDNA first strand, amplify rice protein coding gene fragments from cDNA using primers, insert the gene fragments into phage display vectors, introduce into E. coli competent cells and superinfect with helper phage, construct a rice protein library phage display system; S2, specific binding screening with rice bakanae disease pathogen The rice bacterial blight pathogen is separated, purified and cultured, the phage solution containing the rice protein library is incubated with the pathogen, the unbound phage is removed by washing, and the specific bound phage is eluted to obtain an enriched phage population; S3, identification of resistance proteins and corresponding genes The enriched phage population is used to infect fresh E. coli for amplification and culture, the DNA is extracted, the inserted rice protein coding gene fragment is amplified, sequenced and compared with the rice genome database to identify the resistance protein gene; S4, development of molecular markers According to the sequence of the resistance gene, primers are designed, and different rice variety genomic DNA is used as a template for PCR amplification, specific primer pairs are screened to obtain molecular markers, and a large-scale rice variety population or a genetic separation population is used to verify the correlation between the molecular markers and the resistance phenotype.

[0007] Further, in the step of selecting different rice varieties and collecting tissue samples at the key growth period of rice, the different rice varieties include rice varieties with high resistance, medium resistance and disease susceptibility, and the number of samples collected from each variety is not less than 3 biological replicates. The collected samples need to be surface sterilized before grinding, specifically soaked in 75% ethanol for 30 seconds and washed with sterile water for 3-5 times to remove possible bacterial contamination on the surface of the samples, ensuring the accuracy of the extracted RNA and gene fragments. During the grinding process, sufficient liquid nitrogen should be maintained to prevent sample melting and RNA degradation, which may affect the subsequent experimental results. By setting multiple biological replicates and strict sample processing procedures, the reliability and repeatability of the experiment can be improved, laying a foundation for subsequent accurate screening of resistance genes.

[0008] Further, in the step of extracting total RNA from the sample by the improved Trizol method, after the Trizol reagent lyses the cells, chloroform is added before layering centrifugation. The sample is first placed at 4°C for 10-15 minutes to allow the cell fragments to fully precipitate. After centrifugation, the upper aqueous phase is aspirated, avoiding the protein precipitate in the middle layer to prevent protein contamination of the RNA. In the RNA precipitation process, pre-cooled isopropyl alcohol is used and precipitated at -20°C for 1-2 hours to improve the RNA precipitation efficiency. Then, the RNA precipitate is washed with 75% ethanol for 2-3 times to remove residual salt ions and organic solvents, ensuring the purity and quality of the extracted RNA, thereby providing a high-quality template for subsequent reverse transcription and gene amplification.

[0009] Further, in the step of amplifying the rice protein coding gene fragments from cDNA using primers, the primers are designed by using bioinformatics software to comprehensively analyze the rice genome database, specific primers are designed for different types of protein coding gene regions, the length of the primers is controlled to be 18-25 bases, the GC content is maintained to be 40%-60%, the annealing temperature is optimized by gradient PCR, a plurality of temperature gradients are set for pre-experiment, the temperature at which the amplification band is clear and specific is selected as the optimal annealing temperature, and enzyme cutting sites and protection bases are added to the 5' end and 3' end of the primers, respectively, so as to facilitate the subsequent connection of the gene fragments and the phage display vector and improve the connection efficiency and the success rate of the construction of the recombinant vector.

[0010] Further, in the step of incubating the phage solution containing the rice protein library with the pathogenic bacteria, the optimization of the incubation system includes: adjusting the incubation volume according to the concentrations of the phage and the pathogenic bacteria to make the ratio of the phage to the pathogenic bacteria in a suitable range, using gentle shaking to promote the full contact of the phage and the pathogenic bacteria and to avoid the destruction of the structure of the phage or the pathogenic bacteria caused by violent shaking, setting different incubation conditions for pathogenic bacteria at different growth stages, and appropriately prolonging the incubation time of mycelium to 6-12 hours and the incubation time of spores to 2-4 hours to ensure that the rice proteins displayed on the surface of the phage can fully specifically bind to the surface molecules of the pathogenic bacteria and improve the screening efficiency.

[0011] Further, in the step of washing the combined system multiple times using a washing buffer, the washing buffer contains 1×PBS, 0.1% Tween-20 and 0.05 mol / L NaCl to enhance the washing effect and remove non-specifically bound phage, the number of washing times is determined according to the pre-experiment results, the phage-pathogenic bacteria complex after different numbers of washing times is detected to analyze the retention of specifically bound phage and the removal effect of non-specific phage, and the number of washing times that can effectively remove non-specific phage and maximally retain specifically bound phage is selected, and in each washing process, the phage-pathogenic bacteria complex is precipitated by centrifugation to facilitate the replacement of the washing buffer and ensure the consistency of the washing effect.

[0012] Further, in the step of comparing the sequencing results with the rice genome database, high-throughput sequencing technology is used, and the Illumina sequencing platform is used for deep sequencing of the amplified gene fragments. The sequencing depth of each sample is not less than 1000x to ensure the accuracy and reliability of the sequencing results. In the comparison with the rice genome database, professional comparison software such as BLAST is used, and the comparison parameters are set. At the same time, a variety of bioinformatics analysis tools are used, HMMER is used for protein domain prediction, and InterProScan is used for function annotation. The resistance protein gene is comprehensively identified. For newly discovered genes, further homology analysis and phylogenetic tree construction methods are used to determine their potential role in rice resistance mechanism and evolutionary status.

[0013] Further, in the step of verifying the screened molecular marker, the verification process includes indoor seedling inoculation identification and field natural infection identification. In indoor seedling inoculation identification, more than 100 different rice seedlings are selected, and the rice bacterial leaf blight pathogen is inoculated on the seedlings by needle inoculation or root immersion inoculation. After 2-3 weeks of cultivation under suitable temperature and humidity conditions, the incidence of seedlings is observed and recorded, and the molecular marker is detected. In field natural infection identification, more than 200 rice varieties are planted in a rice bacterial leaf blight high-incidence area. During the growth of rice, the incidence is investigated regularly, and the yield and other agronomic traits are counted after harvesting. The molecular marker is detected. Through the combination of indoor and field identification methods, the correlation between the molecular marker and the rice bacterial leaf blight resistance phenotype is comprehensively evaluated to ensure that the screened molecular marker has high application value in actual breeding.

[0014] Further, in the step of inserting the amplified rice protein coding gene fragment into the phage display vector, different types of phage display systems are considered when selecting the vector. The selection is based on the size of the gene fragment and the subsequent experimental requirements. In the ligation process, T4 DNA ligase is used for ligation reaction. The molar ratio of gene fragment to vector in the ligation system is optimized through pre-experiment and controlled between 3:1-5:1. The ligation reaction is carried out at 16℃ overnight to improve the ligation efficiency. When transforming the ligation product into E. coli competent cells, the electric transformation or chemical transformation method is used. In electric transformation, appropriate voltage, capacitance and resistance parameters are set. In chemical transformation, competent cells are prepared by the CaCl2 method, and the temperature and time conditions in the transformation process are optimized to ensure that the recombinant vector is efficiently introduced into E. coli cells, and the rice protein library phage display system is successfully constructed.

[0015] Compared with the prior art, the rice bacterial leaf blight resistance gene molecular marker screening method has the following beneficial effects: ​I. This method constructs a rice protein library phage display system and utilizes a screening technique based on specific binding with the rice bakanae disease pathogen to efficiently screen for resistance-related proteins and corresponding genes from a large number of rice genes. Compared with traditional methods, this screening method is more direct and efficient, reduces errors caused by non-specific screening, and greatly improves the efficiency and accuracy of resistance gene screening.

[0016] Second, this method not only screened out resistance proteins and corresponding genes, but also further developed molecular markers closely linked to these resistance genes. These molecular markers can rapidly and accurately detect resistance genes in different rice varieties through PCR amplification technology, providing a powerful tool for rice disease resistance breeding. By combining indoor seedling inoculation identification and field natural disease identification, the correlation between these molecular markers and resistance phenotypes was verified, ensuring the application value of molecular markers in actual breeding and helping to accelerate the breeding process of disease-resistant rice varieties.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 Flowchart of molecular marker screening method for rice bakanae disease resistance genes. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below. Example 1

[0021] Molecular marker screening for bakanae disease resistance in rice varieties from a major rice-producing area was conducted. Rice varieties exhibiting high, moderate, and susceptible resistance levels were selected. Leaf tissue samples were collected at the tillering stage, with three biological replicates for each variety. After surface sterilization, the samples were ground into powder under sufficient liquid nitrogen. Total RNA was extracted using a modified Trizol method. During extraction, cells were lysed with Trizol reagent and then allowed to stand at 4°C for a period to allow cell debris to precipitate. After centrifugation, the supernatant was collected to avoid protein contamination. RNA was precipitated using pre-cooled isopropanol at -20°C, followed by washing with 75% ethanol. The extracted RNA was reverse transcribed into cDNA first strand. The final RNA was then analyzed using biological markers. Informatics software was used to analyze the rice genome database. Specific primers were designed for different protein-coding gene regions, with primer lengths controlled within a reasonable range and GC content meeting requirements. The annealing temperature was optimized using gradient PCR. Enzyme restriction sites and protective bases were added to both ends of the primers. Rice protein-coding gene fragments were amplified from cDNA using the primers. A suitable phage display vector was selected based on the gene fragment size and experimental requirements. The gene fragment was ligated to the vector using T4 DNA ligase. After optimizing the molar ratio, the reaction was carried out overnight at 16°C. The ligation product was introduced into competent E. coli cells via electroporation or chemical transformation. After helper phage superinfection, a rice protein library phage display system was constructed.

[0022] The pathogen of rice bakanae disease in this production area was isolated, purified, and cultured. The incubation volume was adjusted according to the concentration of bacteriophage and pathogen to ensure that the ratio of the two was within an appropriate range. The bacteriophage solution containing a rice protein library was incubated with the pathogen under gentle shaking conditions. Different incubation times were set for different growth stages of the pathogen, such as appropriately extending the incubation time for mycelium and relatively shortening the incubation time for spores. After incubation, the system was washed multiple times with a washing buffer containing 1×PBS, 0.1% Tween-20, and 0.05 mol / L NaCl. The number of washes that could effectively remove non-specific bacteriophages while retaining specific bacteriophages to the greatest extent was determined by detection and analysis. Each wash was centrifuged to precipitate the bacteriophage-pathogen complex. After washing, the specific bacteriophages were eluted to obtain an enriched bacteriophage population.

[0023] Enriched phage populations were infected with fresh E. coli for amplification and culture. DNA was extracted and amplified to insert rice protein-coding gene fragments. High-throughput sequencing technology was used to perform deep sequencing of the gene fragments on the Illumina sequencing platform, ensuring that the sequencing depth of each sample met the requirements. The sequencing results were compared with the rice genome database using BLAST software. Simultaneously, HMMER was used for protein domain prediction, and InterProScan was used for functional annotation to comprehensively identify resistance protein genes. For newly discovered genes, homology analysis and phylogenetic tree construction were used to clarify their potential roles and evolutionary positions in the rice resistance mechanism.

[0024] Primers were designed based on the identified resistance gene sequences. PCR amplification was performed using genomic DNA from different rice varieties in the production area as templates. Specific primer pairs were screened to obtain molecular markers. The verification process included indoor seedling inoculation identification and field natural disease identification. In the indoor process, more than 100 seedlings of different varieties were selected, and the pathogen was inoculated onto the seedlings by needle prick or root soaking. The seedlings were cultured under suitable temperature and humidity conditions for 2-3 weeks, and the disease incidence was observed and recorded, and molecular markers were detected. In the field, more than 200 varieties were planted in areas with high incidence of bakanae disease, and the disease incidence was investigated regularly. After harvest, agronomic traits such as yield were statistically analyzed, and molecular markers were detected. The correlation between molecular markers and resistance phenotypes was evaluated by combining the two identification methods. Example 2

[0025] In hybrid rice breeding, molecular marker screening for bakanae disease resistance genes was conducted. Parental varieties with different resistance levels (highly resistant, moderately resistant, susceptible) were selected. Stem tissue samples were collected during the booting stage, with at least three biological replicates for each variety. After surface sterilization, the samples were ground into powder under sufficient liquid nitrogen. Total RNA was extracted using a modified Trizol method. Cells were lysed with Trizol reagent and incubated at 4°C for 10-15 minutes. After centrifugation, the upper aqueous phase was carefully aspirated, avoiding the intermediate protein precipitation layer. RNA precipitation was performed using pre-cooled isopropanol at -20°C for 1-2 hours, followed by washing 2-3 times with 75% ethanol. The RNA was then reverse transcribed into cDNA first strand. Bioinformatics software was used to analyze the rice genome database. Specific primers were designed for different types of protein-coding gene regions, with primer length and GC content controlled within specified ranges. The annealing temperature was optimized using gradient PCR. Enzyme sites and protective bases were added to the 5' and 3' ends of the primers to amplify rice protein-coding gene fragments from cDNA. A suitable phage display vector was selected based on the gene fragment size and subsequent experimental requirements. The gene fragment and vector were ligated using T4 DNA ligase, with the optimal molar ratio between the two being 3:1-5:1. Ligation was performed overnight at 16°C. The ligation product was transformed into competent E. coli cells (electroplation or chemical transformation). A rice protein library phage display system was constructed through helper phage superinfection.

[0026] The main pathogens of rice bakanae disease in the local area were isolated, purified, and cultured. The incubation volume was adjusted according to the concentration of bacteriophage and pathogen to ensure an appropriate ratio. The bacteriophage solution containing a rice protein library was incubated with the pathogen under gentle shaking. Different incubation times were set according to the growth stage of the pathogen (mycelium or spores). After incubation, the system was washed multiple times with a washing buffer containing 1×PBS, 0.1% Tween-20, and 0.05 mol / L NaCl. The optimal number of washing times was determined by detection. After each washing, centrifugation was performed to precipitate the bacteriophage-pathogen complex. After washing, the specifically bound bacteriophages were eluted to obtain an enriched bacteriophage population.

[0027] Enriched phage populations were infected with fresh E. coli for amplification and culture. DNA was extracted and amplified to insert rice protein-coding gene fragments. The gene fragments were then deep sequenced using the Illumina high-throughput sequencing platform, with a sequencing depth of no less than 1000× for each sample. The sequencing results were compared with the rice genome database using BLAST software. HMMER was used for protein domain prediction, and InterProScan was used for functional annotation to identify resistance protein genes. For new genes, homology analysis and phylogenetic tree construction were used to clarify their roles and evolutionary positions in the resistance mechanism.

[0028] Primers were designed based on resistance gene sequences. PCR amplification was performed using genomic DNA from different parents and progeny materials in hybrid rice breeding as templates. Specific primer pairs were screened to obtain molecular markers. For verification, seedlings of more than 100 different materials were selected indoors and inoculated with pathogens by needle prick or root soaking. They were cultured at suitable temperature and humidity for 2-3 weeks, and the disease incidence was observed and molecular markers were detected. In the field, more than 200 materials were planted in areas with high incidence of bakanae disease, and the disease incidence was investigated regularly. After harvest, agronomic traits were statistically analyzed and molecular markers were detected. The correlation between molecular markers and resistance phenotypes was evaluated through indoor and field identification, providing a basis for molecular marker-assisted selection in hybrid rice breeding.

[0029] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for screening a molecular marker of a rice blast resistance gene, characterized in that, The specific steps of the method are: S1, construction of a rice protein library and phage display Different rice varieties are selected, and tissue samples are collected at critical growth stages of the rice, ground into powder, and then total RNA is extracted, cDNA first strand is synthesized by reverse transcription, and rice protein coding gene fragments are amplified from cDNA using primers, and the gene fragments are inserted into a phage display vector, introduced into E. coli competent cells and superinfected with helper phage, and a rice protein library phage display system is constructed; S2, specific binding screening with rice bacterial leaf blight pathogen The rice bacterial leaf blight pathogen is isolated and purified and cultured, the phage solution containing the rice protein library is incubated with the pathogen, and the unbound phage is removed by washing, and the specific binding phage is eluted to obtain an enriched phage population; S3, identification of resistance proteins and corresponding genes The enriched phage population is used to infect fresh E. coli for amplification and culture, and the DNA is extracted, and the inserted rice protein coding gene fragments are amplified, sequenced and compared with the rice genome database to identify the resistance protein genes; S4, development of molecular markers According to the sequence of the resistance gene, primers are designed, and different rice variety genomic DNA is used as a template for PCR amplification, specific primer pairs are screened to obtain molecular markers, and the correlation between the molecular markers and the resistance phenotype is verified using a large population of rice varieties or a genetic separation population.

2. The method of claim 1, wherein the molecular marker is selected from the group consisting of SEQ ID NOs: 1 to 6. In the step of selecting different rice varieties and collecting tissue samples at critical growth stages of the rice, different rice varieties include rice varieties with high, medium and susceptible resistance levels, and the number of samples collected from each variety is not less than 3 biological replicates. The samples collected need to be surface sterilized before grinding, and sufficient liquid nitrogen should be maintained during the grinding process.

3. The molecular marker screening method for rice bakanae disease resistance genes according to claim 1, characterized in that, In the step of extracting total RNA from the sample using the improved Trizol method, after the Trizol reagent lyses the cells, chloroform is added before layering and centrifuging. First, the sample is allowed to stand at 4°C for 10-15 minutes to allow the cell debris to fully precipitate. After centrifugation, avoid sucking the protein precipitate in the middle layer to prevent protein contamination of the RNA. In the RNA precipitation process, pre-cooled isopropyl alcohol is used, and the RNA precipitate is precipitated at -20°C for 1-2 hours, and then the RNA precipitate is washed with 75% ethanol for 2-3 times.

4. The molecular marker screening method for rice bakanae disease resistance genes according to claim 1, characterized in that, In the step of amplifying rice protein coding gene fragments from cDNA using primers, bioinformatics software is used to analyze the rice genome database when designing primers. Specific primers are designed for different types of protein coding gene regions. The length of the primers is controlled at 18-25 bases, the GC content is maintained at 40%-60%, the annealing temperature is optimized by gradient PCR, multiple temperature gradients are set for pre-experiments, and the temperature with clear and specific amplification bands is selected as the optimal annealing temperature. In addition, enzyme cutting sites and protection bases are added to the 5' and 3' ends of the primers.

5. The molecular marker screening method for rice bakanae disease resistance genes according to claim 1, characterized in that, In the step of incubating the phage solution containing the rice protein library with the pathogenic bacteria, the optimization of the incubation system includes: adjusting the incubation volume according to the concentrations of the phage and the pathogenic bacteria, so that the ratio of the phage to the pathogenic bacteria is in a suitable range; in the incubation process, a gentle shaking mode is adopted to promote the sufficient contact between the phage and the pathogenic bacteria, while avoiding the structural damage of the phage or the pathogenic bacteria caused by violent shaking; different incubation conditions are set for pathogenic bacteria in different growth stages, and the incubation time of mycelium can be extended to 6-12 hours, and the incubation time of spores is 2-4 hours.

6. The molecular marker screening method for rice bakanae disease resistance genes according to claim 1, characterized in that, In the step of washing the combined system multiple times with a washing buffer, the washing buffer contains 1×PBS, 0.1% Tween-20 and 0.05 mol / L NaCl; the retention of specific binding phage and the removal effect of non-specific phage are analyzed by detecting the phage-pathogenic bacteria complex after different washing times; the washing times that can effectively remove non-specific phage and maximize the retention of specific binding phage are selected; and in each washing process, the phage-pathogenic bacteria complex is precipitated by centrifugation.

7. The molecular marker screening method for rice bakanae disease resistance genes according to claim 1, characterized in that, In the step of sequencing the amplified gene fragments and comparing the sequencing results with the rice genome database, high-throughput sequencing technology is used, and the Illumina sequencing platform is used for deep sequencing of the amplified gene fragments; the sequencing depth of each sample is not less than 1000x; when compared with the rice genome database, professional alignment software such as BLAST is used, the alignment parameters are set, and various bioinformatics analysis tools are used for protein domain prediction, function annotation, comprehensive identification of resistance protein genes; for newly discovered genes, further homology analysis and phylogenetic tree construction methods are used to determine their potential role in rice resistance mechanism and evolutionary status.

8. The molecular marker screening method for rice bakanae disease resistance genes according to claim 1, characterized in that, In the step of verifying the screened molecular markers, the verification process includes indoor seedling inoculation identification and field natural infection identification; in indoor seedling inoculation identification, more than 100 seedlings of different rice varieties are selected, the rice bakanae pathogen is inoculated on the seedlings by needle inoculation or root immersion inoculation, and the seedlings are cultured for 2-3 weeks under suitable temperature and humidity conditions; the incidence of seedlings is observed and recorded, and the molecular markers are detected; in field natural infection identification, more than 200 rice varieties are planted in a rice bakanae high-incidence area; the incidence is investigated regularly during the growth of rice; the yield and other agronomic traits are counted after harvesting; and the molecular markers are detected; the correlation between the molecular markers and the rice bakanae resistance phenotype is comprehensively evaluated by combining indoor and field identification methods.

9. The molecular marker screening method for rice bakanae disease resistance genes according to claim 1, characterized in that, In the step of inserting the amplified rice protein-coding gene fragment into the phage display vector, different types of phage display systems are considered when selecting the vector, based on the size of the gene fragment and subsequent experimental requirements. During the ligation process, T4 DNA ligase is used for the ligation reaction. The molar ratio of the gene fragment to the vector in the ligation system is optimized through preliminary experiments and controlled between 3:1 and 5:

1. The ligation reaction is carried out overnight at 16°C. When transforming the ligation product into competent E. coli cells, electroporation or chemical transformation methods are used. For electroporation, voltage, capacitance, and resistance parameters are set, while for chemical transformation... We prepared competent cells and optimized the temperature and time conditions during the transformation process.

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