Apple phenylalanine ammonialyase and application thereof in improving resistance of apples to bactrocera dorsalis

Abstract

The invention provides apple phenylalanine ammonialyase and application thereof in improving the resistance of apples to bactrocera dorsalis, the amino acid sequence of the apple phenylalanine ammonialyase is SEQ ID NO: 1, and the nucleotide sequence of a coding gene of the apple phenylalanine ammonialyase is SEQ ID NO: 2. The invention further provides a method for improving the bactrocera dorsalis resistance of the apple strain, and according to the method, the content of the apple phenylalanine ammonialyase in the apple strain is increased. According to the apple phenylalanine ammonialyase PAL gene obtained through screening, the larva death rate of an overexpression test group is remarkably higher than that of a blank control group, and it is indicated that overexpression of the PAL gene can possibly affect survival of larvae.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological pesticides, and particularly relates to an apple phenylalanine ammonia-lyase and application thereof in improving the resistance of apples to Bactrocera dorsalis. BACKGROUND

[0002] Bactrocera dorsalis (Bactrocera dorsalis Bactrocera dorsalis belongs to the family Tephritidae of the order Diptera and is originally from southern India. In recent years, it has become a serious invasive pest of fruit trees in China. Adult females lay eggs in the fruit of host plants, and the larvae hatch and feed on the fruit, causing a decrease in fruit quality and possibly causing fruit drop and affecting yield, resulting in economic losses. The main hosts of B. dorsalis are citrus, apple, peach, plum, mango, guava, loquat, banana, tomato, and more than 250 other fruits and vegetables. Female adults lay eggs in the skin of mature or nearly mature fruits, and the larvae hatch and feed in the fruit, forming tunnels. The fruit around the tunnels rots into a jelly-like state. The larvae feeding on the fruit can cause the fruit to rot, drop, or turn color prematurely. The wounds caused by the larvae are easily infected by fungi and bacteria, further worsening the decay of the fruit. The damage caused by B. dorsalis can cause the affected fruit to be unsuitable for fresh consumption or processing, thereby losing its commercial value. When it occurs in large numbers, it can cause serious economic losses and is recognized internationally as a major fruit tree pest.

[0003] B. dorsalis has invaded many regions in Africa, Oceania, East Asia, and the Americas. It was first reported in China in 1937 and has since spread widely in southern China, mainly in Guangdong, Guangxi, Fujian, Hainan, Yunnan, Sichuan, and Taiwan. The core distribution area of B. dorsalis in China is the southern region south of the Yangtze River, where it occurs year-round and has overlapping generations. In recent years, due to climate change, the distribution of B. dorsalis has expanded northward. Prior to 2000, it was mainly limited to traditional tropical regions such as Guangdong, Guangxi, Fujian, and Hainan. Since 2010, it has been consistently present in Nujiang, Yunnan, and Panzhihua, Sichuan. In Ganzhou, Jiangxi, and Yongzhou, Hunan, the summer population density has increased significantly. Since 2020, overwintering populations have been detected in Wenzhou and Taizhou, Zhejiang. It has also been found sporadically in citrus orchards in Suzhou, Jiangsu, and Huangshan, Anhui. Currently, B. dorsalis is expanding its distribution from south to north and from east to west in China, with climate change and fruit trade being the main driving factors. It is predicted that the distribution of B. dorsalis will continue to expand in China in the coming years.

[0004] Phytophagous insects are one of the main enemy of plants, and the feeding behavior of phytophagous insects will cause many damages to plants. In the long-term co-evolution of plants and phytophagous insects, many plants have evolved certain resistance to insects, which is the result of the struggle between plants and phytophagous insects under the action of external environmental conditions. The resistance of plants to insects mainly includes antibiosis, tolerance and non-selectivity, among which antibiosis is one of the main mechanisms of plant resistance to insects, which shows that some secondary metabolites of plants have anti-insect effects, which can hinder the feeding, digestion and other behaviors of insects, or affect the physiological activities of insects such as molting and reproduction, and ultimately lead to the insects no longer feeding or growth inhibition, reduced reproductive capacity or even death. In this process, the genes related to plant secondary metabolism mainly play a role, which produce secondary metabolites with anti-insect effects by regulating the plant secondary metabolism pathway. The antibiosis of plants is an important mechanism for plants to resist insect damage, and if it can be utilized, it is possible to establish a pest defense system based on the antibiosis of the plant itself, to strengthen the plant's resistance to insects, and to reduce the use of chemical pesticides in traditional pest control to some extent, thereby reducing environmental pollution and killing natural enemies, reducing pesticide residues, and reducing control costs. SUMMARY

[0005] The purpose of the present application is to provide an apple phenylalanine ammonia-lyase and its application in improving the resistance of apples to Bactrocera dorsalis, that is, a gene associated with the resistance of apples to Bactrocera dorsalis is screened, and a method for improving the resistance of apples to Bactrocera dorsalis is provided.

[0006] The present application first provides an apple phenylalanine ammonia-lyase, the amino acid sequence of which is SEQ ID NO: 1; MEAETITQNGKKGHHQNGAVESPLCIKKDPLNWGLAADSLKGSHLDEVKRMVAEYRKPVVKLGGESLTISQVAAIATHDTGVKVELSESARAGVKASSDWVMDSMGKGTDSYGVTTGFGATSHRRTKQGAALQKELIRFLNAGVFGSATESGHTLPHQATRAAMLVRINTLLQGYSGIRFEILEAITKFLNSNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPNGQTLNASEAFELVGIDSGFFELQPKEGLALVNGTAVGSGLASTVLFETNILALLAEILSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAAKKLHEQDPLQKPKQDRYALRTSPQWLGPQIEVIRYSTKSIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNTRLAIASIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTAEAVDILKLMSSTFLVALCQSVDLRHLEENLRNTVKNTVSQVAKRTLTTGVNGELHPSRFCEKDLLKVVDREYVFAYIDDPCSATYPLMQKLRQVLVEHALTNGESEKNASTSIFQKIGAFEEELKALLPKEVESARSAIESGNAAVPNRIAECRSYPLYKFVREELGGEYLTGEKVRSPGEECDKVFQAICQGKIIDPILGCLEGWNGAPLPIC (SEQ ID NO: 1); The phenylalanine ammonia-lyase of the apple, a nucleotide sequence of an encoding gene is SEQ ID NO: 2;

[0007] The present application also provides a Gateway recombination expression vector, wherein the nucleotide sequence of the apple phenylalanine ammonia-lyase gene is inserted. The present application further provides an Agrobacterium, wherein the Gateway recombination expression vector is carried.

[0008] The present application further provides a use of the apple phenylalanine ammonia-lyase, which is used for improving the resistance of apple strains to Bactrocera dorsalis. The present application further provides a method for improving the resistance of apple strains to Bactrocera dorsalis, which comprises increasing the content of the apple phenylalanine ammonia-lyase in the apple strains. Further, the method comprises using the Agrobacterium to infect the apple strains.

[0009] The apple phenylalanine ammonia-lyase screened in the present application PAL The mortality of the larvae in the overexpression test group is significantly higher than that in the blank control group, which indicates that PAL The overexpression of the gene can affect the survival of the larvae. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 : The figure of the amplified bands of the target gene; wherein M represents a DNA Marker; lanes 1 and 2 are the amplification results of the PAL gene (2194bp). PAL gene

[0011] Figure 2 : The figure of the amplified bands of the attB-PCR; wherein M represents a DNA Marker; lanes 1 and 2 are the amplification results of the PAL gene (2194bp) in the Red Fuji and Xiaoguoguang strains. Figure 3 : The figure of the amplified bands of the LR reaction; wherein M represents a DNA Marker; lanes 1-3 are the amplification results of the PAL gene (2194bp).

[0012] Figure 4 : The figure of the colonies after being transferred into the Agrobacterium.

[0013] Figure 5 : The figure of the PCR identification bands of the Agrobacterium GV3101 colonies; wherein M represents a DNA Marker; lanes 1 and 2 are the identification results of the PAL gene (2194bp). PAL gene Figure 6

[0014] Figure 7 : The schematic diagram of the injection hole; PAL : Red Fuji (left) and Xiaoguoguang (right)​​Figure 8 Gene expression level diagram after gene overexpression; PAL : Red Fuji PAL Effect diagram of gene overexpression for 6 days on the mortality (left) and average weight (right) of B. cucurbitae larvae; Figure 9: Xiaoguoguang Figure 10 Effect diagram of gene overexpression for 6 days on the mortality (left), average weight (right) of B. cucurbitae larvae; PAL : Red Fuji Figure 11 Effect diagram of gene overexpression for 9 days on the mortality of B. cucurbitae larvae; PAL : Xiaoguoguang Figure 12 Effect diagram of gene overexpression for 9 days on the mortality (left), average weight (right) of B. cucurbitae larvae; Figure 13 : Diagram of part of the dissected larvae; PAL : Photo diagram of part of the larvae; DETAILED DESCRIPTION

[0015] The fruit tree pest research group of Qingdao Agricultural University found that some apple varieties such as Xiaoguoguang and Red Fuji have relatively high resistance to B. cucurbitae, and after allowing B. cucurbitae to cause damage to apples and then determining the transcriptome, it was found that the expression amount of phenylalanine ammonia-lyase gene (PAL) of the apple varieties subjected to B. cucurbitae damage changed. Reagent name

[0016] The present application uses Gateway cloning technology to construct a gene overexpression vector, and through artificial intervention of the expression degree of the target gene, the target gene of the test group is overexpressed. And by comparing the growth and development of B. cucurbitae larvae under different expression degrees of the target gene, the resistance of the two target genes to B. cucurbitae is verified.

[0017] The test fruits in the embodiments of the present application are Xiaoguoguang and Red Fuji apple early development stage fruits collected in Laizhou, Shandong in September 2024 under the unified management mode and consistent development stage.

[0018] ​E. coli DH5α competent cells, E. coli OmniMAX 2-T1 competent cells from Bomeida Biological Company; E. coli vector pMD19-T from TaKaRa Company; pDONR221 vector (BP reaction donor vector), pH7FWG2-RR-293 vector (overexpression vector) were presented by Professor Hu Yuzhen of Beijing Agricultural University. Agrobacterium GV3101 competent cells were from Shanghai Generay Biotech Co., Ltd. Primers were synthesized by Shanghai Generay Biotech Co., Ltd. Test insects were from the South China strain of B. cucurbitae which were reared in the laboratory using artificial feed and had been reproducted for many generations, and the insect source was stable.

[0019] The present application will be described in detail below in conjunction with examples and drawings.

[0020] Example 1, gene cloning 1. Primer design Primers were designed according to the cds sequences of the two genes using Primer Primier 5 software (Table 1). Primers were synthesized by Shanghai Generay Biotech Co., Ltd.

[0021] Table 1: List of primer sequences required for the test

[0022] 2. Extraction of total RNA from plants and synthesis of cDNA RNA was extracted from Xiaoguoguang and Red Fuji fruits using the Nuoyuan FastPure Universal Plant Total RNA Isolation Kit (RC411-01) kit, and the specific steps were referred to the instruction manual. The RNA product was determined for concentration and stored at -80°C.

[0023] The extracted RNA was subjected to cDNA synthesis using the TaKaRa kit PrimeScript® RT reagent Kit with gDNA Eraser, and the specific steps were referred to the instruction manual.

[0024] 3. Amplification of target genes The target genes were subjected to PCR amplification using the primers in Table 1 with cDNA as the template, and the amplification reaction system and reaction conditions are shown in Table 2 and Table 3.

[0025] Table 2: PCR amplification reaction system Amount used Xiaoguoguang cDNA / Red Fuji cDNA 2 × Taq Plus Master Mix II (Dye Plus) (P213-01) 1µl PAL_OE_F 12.5µl PAL_OE_R 0.5µl PAL 0.5µl dd H2O 10.5µl Table 3: PAL gene PCR amplification reaction conditions

[0026] PCR products were detected by agarose gel electrophoresis and the fragment size was observed in the gel imaging system, Figure 1 The length of the target gene fragment was 2194 bp, and the band size was consistent with the expected value, indicating that the target gene fragment was successfully amplified. PAL The gel corresponding to the length of the fragment was cut under ultraviolet light, and the FastPure Gel DNA Extraction Mini Kit from Novizen was used for gel purification and recovery.

[0027] The screened PAL The nucleotide sequence of the gene is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1.

[0028] Example 2: Construction of Gateway Recombinant Expression Vector 1. Primer design Using Primer Primier 5 software, primers were designed according to the cds sequence of the gene as shown in Table 4. Reagent name

[0029] Table 4: List of primer sequences required for the test

[0030] 2. Ligation of the target fragment into pMD19-T vector According to the pMD19-T vector instructions of TaKaRa company, the purified target fragment was ligated into pMD19-T vector, and the ligation system is shown in Table 5.

[0031] Table 5: Ligation system table of pMD19-T vector Amount used pMD19-T Vector*1 Gel recovery product 1µl Solution I 4µl Reagent name 5µl Ligation reaction conditions: 4°C overnight (12-20 hours), 16°C for one hour. The ligation product was transformed into DH5α E. coli.

[0032] 1) Add 10 μl of ligation reaction solution to 50 μl of DH5α competent cells, mix gently, and incubate on ice for 30 min.

[0033] 2) Use a water bath to heat shock at 42°C for 90s, and quickly ice bath for 2 min.

[0034] 3) Add 500 μl of LB liquid medium without antibiotics, shake culture at 37°C, 200 r / min for 2 h.

[0035] 4) Centrifuge at 7500 rpm for 5 min, remove the supernatant, and add 200 μl of LB liquid medium.

[0036] ​5) Spread 100µl of bacterial culture onto an LB agar plate containing ampicillin, place it upright for 30 min, then invert it and incubate at 37°C for 12 h. Pick a single white colony and shake it to incubate.

[0037] The ligated colonies were identified by culture PCR and sent to Qingke Biotechnology for sequencing. The received results were compared with the original sequence to determine whether the target gene was successfully constructed into the pMD19-T vector. The successfully aligned bacterial cultures were then propagated. After propagation, plasmids were extracted using the plasmid mini-prep kit (DP103) from Tiangen Biotech Co., Ltd., following the manufacturer's instructions.

[0038] 3. attB-PCR AttB insertion sites were constructed on the plasmid using attB-PCR to produce PCR products containing the attB insertion, which were then used for subsequent Gateway cloning reactions. Using the extracted plasmid as a template, attB-PCR amplification was performed using the primers listed in Table 1. The amplification reaction system and conditions are shown in Tables 6 and 7 below: Table 6: attB-PCR Reaction System Amount used Plasmid 2 × Taq Plus Master Mix II (Dye Plus) (P213-01) 1µl PAL_OE_F 12.5µl PAL_OE_R 0.5µl Figure 2 0.5µl dd H2O 10.5µl Table 7: PAL gene attB-PCR amplification reaction conditions

[0039] As a result, the attB-PCR products were detected by agarose gel electrophoresis and the fragment size was observed using a gel imaging system. Reagent name The band size matches the target gene fragment size, indicating that the plasmid has successfully contained the attB insertion and is ready for Gateway cloning.

[0040] 4. BP reaction Prepare the reaction solution according to the system in Table 7, and perform the BP reaction using the Thermo Fisher Scientific Gateway® BP Clonase™ II Enzyme Mix kit. The steps are shown in Table 8 below: Table 8: BP Reaction System Amount used attB-PCR product pDONR221 vector (150 ng / µl) 1-7µl TE buffer (pH 8.0) 1µl To 8 µl Reagent name 1) Add each ingredient from Table 8 to a 1.5 ml test tube and mix at room temperature.

[0041] 2) Melt the Invitrogen BP Clonase II enzyme mixture on ice for about 2 minutes. Briefly vortex the BP Clonase II enzyme twice (2 seconds each time).

[0042] 3) Add 2 μΐ of BP Clonase II enzyme mix to each sample (step 1 above) and mix thoroughly by vortexing briefly twice. Briefly centrifuge.

[0043] 4) Store the BP Clonase II enzyme mix at -20°C or -80°C.

[0044] 5) Incubate at 25°C for 1 hour.

[0045] 6) Add 1 μΐ of Proteinase K solution to each sample to stop the reaction. Vortex briefly. Incubate at 37°C for 10 minutes.

[0046] 7) Transform 1 μΐ of each BP reaction into 50 μΐ of Invitrogen One Shot OmniMAX 2 T1 phage resistance cells (Cat# C8540-03).

[0047] 8) Incubate on ice for 30 minutes.

[0048] 9) Heat shock the cells at 42°C for 30 seconds.

[0049] 10) Add 250 μΐ of S.O.C. medium and incubate at 37°C with shaking for 1 hour.

[0050] 11) Plate 100 μΐ of each transformation onto selective plates containing kanamycin sulfate and incubate at 37°C for 12 hours. Pick single white colonies and grow in liquid culture. Perform colony PCR on the ligated colonies and send for sequencing. Align the results received with the original sequence to determine if the gene of interest was successfully constructed into the pDONR221 vector. Expand the liquid culture of the colonies that aligned successfully and extract the plasmid using the Plasmid Miniprep Kit (DP103) from Tiangen Biochemical (Teiangen) according to the manufacturer's instructions.

[0051] 5. LR reaction The plasmid with the pDONR221 vector ligated extracted in the previous step was used as the entry clone for the LR reaction. The overexpression treatment group was ligated into the pH7FWG2-RR-293 vector (overexpression vector). The reaction solution was prepared according to the system shown in Table 8, and the LR reaction was performed using the Thermo Fisher Scientific Gateway® LR Clonase™ II Enzyme Mix kit, as shown in Table 9.

[0052] Table 9: LR reaction system table Amount used Gateway entry clone (50-150 ng) pH7FWG2-RR-293 vector (150 ng / µl) 1-7µl TE buffer (pH 8.0) 1µl To 8 µl PAL 1) Add each component in Table 9 to a 1.5 ml tube at room temperature and mix.

[0053] 2) Thaw the Invitrogen LR Clonase II enzyme mix on ice for about 2 minutes. Vortex the LR Clonase II enzyme mix briefly twice (2 seconds each).

[0054] 3) Add 2 μΐ of the LR Clonase II enzyme mix to each sample (from step 1 above) and mix thoroughly by vortexing briefly twice. Centrifuge briefly.

[0055] 4) Store the LR Clonase II enzyme mix at -20°C or -80°C.

[0056] 5) Incubate at 25°C for 1 hour.

[0057] 6) Add 1 μΐ of the Proteinase K solution to each sample to stop the reaction. Vortex briefly. Incubate at 37°C for 10 minutes.

[0058] 7) Transform 1 μΐ of each BP reaction into 50 μΐ of Invitrogen One Shot OmniMAX 2 T1 phage resistance cells (Cat# C8540-03).

[0059] 8) Incubate on ice for 30 minutes.

[0060] 9) Heat shock the cells at 42°C for 30 seconds.

[0061] 10) Add 250 μΐ of S.O.C. medium and incubate at 37°C with shaking for 1 hour.

[0062] 11) Plate 100 μΐ of each transformation onto selective plates containing spectinomycin and incubate at 37°C for 12 hours. Pick single white colonies and grow in liquid culture. Perform colony PCR using primers shown in Table 4 to identify the correct size band. If the correct size band is observed, the Gateway cloning reaction is complete and the overexpression vector is successfully constructed. Extract the plasmid using the Plasmid Miniprep Kit (DP103) from Tiangen Biotech Co., Ltd.

[0063] Perform colony PCR on the colonies transformed after the LR reaction. The electrophoresis gel shows the correct size band for the desired fragment (2194 bp). Figure 3 The Gateway cloning reaction is complete and the overexpression vector is successfully constructed. Figure 4).

[0064] Example 3: Apple fruit transient transformation and biological parameter determination 1. Vector transformation to Agrobacterium 1) Transform the Gateway cloning constructed plasmid into GV3101 Agrobacterium competent, and transform the empty pH7FWG2-RR-293 vector which has not been Gateway cloned into another part of Agrobacterium as a blank control group. Take the Agrobacterium competent stored at -80°C, and insert it into ice when it is in an ice water mixed state after being partially thawed at room temperature or in the palm of the hand for a few moments.

[0065] 2) Add 0.01-1 μg of plasmid DNA to each 100 μl of competent (the amount added depends on the concentration of the plasmid obtained in the previous step, when the plasmid concentration is 400 ng / μl, add 2 μl), mix well by hand, and then sequentially stand on ice for 5 minutes, liquid nitrogen bath for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes.

[0066] 3) Add 700 μl of LB liquid medium without antibiotics, and shake culture at 28°C for 2-3 hours. Centrifuge at 6000 rpm for 1 minute to collect the bacteria, resuspend the bacterial pellet by gently blowing and mixing about 100 μl of supernatant, and spread on LB plates containing the corresponding antibiotics, and incubate at 28°C for 2-3 days. Pick single white colonies and shake culture, and perform colony PCR identification (see Figure 5 ), and observe whether the band size is consistent with the size of the target gene fragment. Take the bacterial solution with the correct band size for expansion culture. The identification results are shown in Figure 6 .

[0067] 2. Apple transient infection and larval inoculation Shake culture the bacterial solution that has been tested and found to be correct, and control the OD600 to be around 1.0, and prepare the infection solution according to the ratio of 200 μM AS + 10 μM MES + 10 μM MgCl2. Collect the bacterial bodies by centrifugation at 5000 rpm for 10 minutes using a high-speed centrifuge, add the infection solution to dilute the bacterial solution, and control the OD600 to be around 1.0, which can be used to infect apples.

[0068] Take fresh Xiaoguoguang and Red Fuji apple fruits that are picked at the same time, have the same size, and have similar shapes, and use a fine awl with a diameter of about 1 mm to pierce holes on the apple surface near the calyx. There are three groups of holes, each with three holes, and the depth of each hole is about 3 cm. The holes are spaced 8-10 mm apart, and the three groups of holes are evenly distributed on the apple surface. Reagent .

[0069] The transformed Agrobacterium infection solution was injected into the apple fruit using a 1 ml sterile syringe, 80 μl was injected into the two side holes, and 60 μl was injected into the middle hole, and the transient transformation of the apple fruit was completed. About half an hour to one hour after the injection was completed, 1 orange fruit fly newly hatched larva was inoculated into the middle injection hole of each group using a sterilized brush, and 3 larvae were inoculated into each apple. A total of 16 apples were treated, of which 12 apples were inoculated with larvae, and 4 apples were not inoculated with larvae. The apple fruit after transient infection was placed in a breathable container and placed in a temperature (24±1) ℃, relative humidity (75±5)% environment. After 72 h of treatment, the flesh of the 4 apples without larvae was sampled about 1 cm around the injection hole and about 3 cm deep, and the sample was quickly placed in liquid nitrogen and stored at -80°C for real-time fluorescent quantitative PCR detection. The larvae were dissected from the inoculated apples after 6 d and 9 d of treatment, and the mortality and body weight of the larvae were recorded.

[0070] 3. Real-time fluorescent quantitative PCR detection of gene expression 1) Reaction system The sample was subjected to RNA extraction using the Tiangen RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (DP441), and the specific steps were referred to the instruction manual. The RNA product was measured for concentration and stored at -80°C. The extracted RNA was subjected to cDNA synthesis using the TaKaRa kit PrimeScript® RT reagent Kit with gDNA Eraser, and the specific steps were referred to the instruction manual.

[0071] The fluorescent quantitative PCR reaction system is shown in Table 10 below: Table 10: Fluorescent quantitative PCR reaction system Amount used (µL) 2×RealStar Green Fast Mixture Forward Primer 5.0 Reverse Primer 0.25 cDNA 0.25 dd H2O 1.0 Up to 10.0 Figure 7 The reaction condition was 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing and extension for 30 s, 40 cycles; the reaction was warmed up at a speed of 0.6°C / s.

[0072] The gene expression level after overexpression was identified as PAL , and the qRT-PCR detection showed that the overexpression of Red Fuji RF-PAL in the test group PAL compared with the blank control group (CK-293), Ralls-PAL the expression level of the gene was significantly increased, and the PAL gene was effectively overexpressed; there was no significant difference between the test group of Xiaoguoguang PAL Figure 8 and the blank control group. The results showed that the treatment effect of PAL gene overexpression in the Xiaoguoguang group was not significant, while the treatment effect in the Red Fuji group was significant.

[0073] 2) Effects of overexpressing the target gene on oriental fruit fly larvae 6 days later from Figure 9 The results showed that the mortality rate of larvae in the PAL gene overexpression test group of Red Fuji was higher than that in the blank control group (293), and the average weight of larvae was slightly lower than that in the blank control group (293), but there were no significant differences.

[0074] from Figure 10 The results showed that the mortality rate of larvae in the PAL gene overexpression test group was higher than that in the blank control group (293), and the average weight of larvae was lower than that in the blank control group (293), but there were no significant differences.

[0075] 3) Effects of overexpressing the target gene on oriental fruit fly larvae 9 days later from PAL As a result, Red Fuji Figure 11 The mortality rate of larvae in the overexpression group was significantly higher than that in the blank control group (293), and no larvae survived in the experimental group. Combining bioassay results and qRT-PCR data, the expression level of the PAL gene in the Red Fuji apples was significantly increased in the experimental group. After overexpression of the PAL gene in apples using molecular biology techniques, the larvae of the oriental fruit fly exhibited a high mortality rate on day 9, indicating that the PAL gene has an impact on the growth of oriental fruit fly larvae.

[0076] from Figure 12 The results showed that the mortality rate of larvae in the PAL gene overexpression experimental group was significantly higher than that in the blank control group (293), and the average weight of larvae in the experimental group after PAL gene overexpression was lower than that in the blank control group. These results indicate that PAL gene overexpression may affect larval growth.

[0077] In the Red Fuji PAL gene overexpression experimental group, qRT-PCR detection showed that the expression level of the gene increased significantly, indicating that the target gene overexpression treatment was effective; Figure 13 The results showed that the larvae of the oriental fruit fly caused damage to apples at both 6 and 9 days. Dissection results indicated that at 6 days, there was no significant difference in larval mortality and average weight between the experimental group and the blank control group (293). At 9 days, the larval mortality rate in the experimental group was significantly higher than that in the control group, and the average weight of surviving larvae was lower in the experimental group. PAL It can be seen that the larvae in the overexpression experimental group were generally smaller in size than those in the blank control group.

[0078] The above results indicate that the 9-day treatment had a stronger effect on the growth and development of *Bacteroides citrinum* larvae than the 6-day treatment. Combined with bioassay and qRT-PCR results, it was confirmed that the PAL gene can affect the growth and development of *Bacteroides citrinum* larvae, thus verifying its resistance effect against *Bacteroides citrinum*.

Claims

1. An apple phenylalanine ammonia-lyase, characterized in that, The apple phenylalanine ammonia-lyase comprises: 1) a protease with an amino acid sequence of SEQ ID NO: 1; 2) a protease derived from 1) by substituting, deleting or adding one or several amino acids on the protease of 1).

2. A gene, characterized in that, The gene encodes the apple phenylalanine ammonia-lyase of claim 1.

3. The gene of claim 2, wherein The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A Gateway recombination expression vector, characterized in that, The recombinant expression vector has inserted therein a nucleotide sequence encoding the apple phenylalanine ammonia-lyase of claim 1.

5. The Gateway recombination expression vector of claim 4, wherein, The nucleotide sequence has the sequence of SEQ ID NO:

2.

6. An Agrobacterium characterized in that, The Agrobacterium carries the Gateway recombinant expression vector of claim 4.

7. The apple phenylalanine ammonia-lyase of claim 1 is used to improve the resistance of an apple strain to Bactrocera dorsalis.

8. A method of increasing resistance of an apple cultivar to B. cucurbitae, comprising, The method is used to increase the content of the apple phenylalanine ammonia-lyase of claim 1 in an apple strain.

9. The method of claim 8, wherein, The method is used to use the Agrobacterium of claim 6 to dip the apple strain.

Images