Light-responsive gene AaHY5like9 from Actinidia arguta and its application

Abstract

This application belongs to the field of kiwifruit genetic engineering technology, specifically relating to a patent application for a light-responsive gene AaHY5like9 in hardy kiwifruit and its applications. The base sequence of the light-responsive gene AaHY5like9 in hardy kiwifruit is shown in SEQ ID No. 1. The protein encoded by this gene is a crop transcription factor related to light response. In this application, the inventors cloned a key light-responsive gene AaHY5like9 related to the fruit quality of hardy kiwifruit using whole-genome-level family identification, and confirmed that AaHY5like9 possesses light-responsive characteristics through the construction of relevant recombinant expression vectors and heterologous transformation verification. Based on this result, a certain genetic resource foundation can be laid for the regulation of hardy kiwifruit fruit quality and the breeding of high-quality hardy kiwifruit varieties.

Description

Technical Field

[0001] The present application belongs to the field of kiwifruit genetic engineering technology, and specifically relates to a light-responsive gene AaHY5like9 of Actinidia arguta and its application patent application. Background Art

[0002] The production efficiency of fruit trees depends largely on the quality of the fruit, and quality improvement is one of the important goals of fruit tree breeding. As an emerging fruit in recent years, soft-fleshed kiwifruit has the characteristics of special fruit shape, edible peel, no need for ripening, and can be eaten immediately after picking. In particular, the red soft-fleshed kiwifruit has a bright and beautiful appearance and is rich in anthocyanin antioxidants, which is loved by consumers. However, in actual cultivation, soft-fleshed kiwifruit generally has poor production quality problems such as poor coloring, poor taste, and fruit drop before harvest, which has greatly hindered the promotion of this fruit.

[0003] Genetic research shows that the manifestation of biological phenotypes depends on two important factors: genetic factors and environmental factors, namely genotype and environmental factors. Among them, genotype fundamentally determines whether the organism has the ability to express the trait, while environmental factors have a direct impact on the strength of the trait.

[0004] In recent years, with the rapid progress of genetic engineering technology and the development of kiwifruit genetic engineering, the discovery of genes related to kiwifruit fruit quality is of extremely important technical significance for improving fruit quality and cultivating new varieties. Summary of the invention

[0005] The purpose of this application is to provide a method for Actinidia arguta (Sieb. & Zucc) Planch. ex Miq), thus laying a certain technical foundation for related fruit quality regulation and the breeding of new varieties of Actinidia arguta.

[0006] The technical solution adopted in this application is described in detail as follows.

[0007] The base sequence of the light-responsive gene AaHY5like9 of Actinidia arguta is shown in SEQ ID No. 1, and the specific sequence is as follows:

[0008] ATGTCTAGTGAGAAAGAGAAAGAATCGGCCGAGGGCTCGTCGCGATCGACGGCGACCATAAGCCGGTATGAGTCGCAGAAGCGTCGAGACTGGAACACCTTTGGCCAGTATTTGAAAAATCACATGC CGCCTGTACCTCTGTCTCAGTGCACTTGCAACCATGTCCTCGAGTTCCTTCGTTATCTCGATCAGTTCGGGAAGACTAAGGTTCACTTACATGGGTGTGTGTTTTTCGGGCAGCCAGACCCACCTGCC CCGTGCACTTGCCCGCTGAGGCAAGCGTGGGGATCGCTGGATGCCCTGATCGGACGGCTGAGAGCCGCCTATGAGGAGCACGGCGGGTCCCCGGAGACGAACCCATTTGGGAACGGGGCGATACGTGT ATATTTACGGGAAGTTAAGGAGTGCCAAGCGAAGGCGCGAGGGATTCCGTACAAGAAGAAGAAGAAGAAGAAGAAGATGATCAAAGCAAACCATGAAGAACTCAAGTCTTCCAAACAGCCCACTTGA.

[0009] The light-responsive protein encoded by the light-responsive gene AaHY5like9 of Actinidia arguta has an amino acid sequence (169aa) as shown in SEQ ID No. 2, specifically:

[0010] MSSEKEKESAEGSSRSTATISRYESQKRRDWNTFGQYLKNHMPPVPLSQCTCNHVLEFLRYLDQFGKTKVHLHGCVFFGQPDPPAPCTCPLRQAWGSLDALIGRLRAAYEEHGGSPETNPFGNGAIRVYLREVKECQAKARGIPYKKKKKKKKMIKANHEELKSSKQPT.

[0011] The primer pair used for PCR amplification of the light-responsive gene AaHY5like9 of Actinidia arguta was specifically designed as follows:

[0012] F: ATGTCTAGTGAGAAAGAGAAAGAATCG;

[0013] R:TCAAGTGGGCTGTTTGGAAGA.

[0014] The kiwifruit light response gene AaHY5like9 is used in kiwifruit, and the protein encoded by the kiwifruit light response gene AaHY5like9 serves as a transcription factor and is related to light response.

[0015] The recombinant overexpression vector containing the light-responsive gene AaHY5like9 of Actinidia arguta is based on the pBI121 plasmid and recombines the AaHY5like9 gene.

[0016] The method for breeding a new variety of Arabidopsis thaliana using the Actinidia arguta light-responsive gene AaHY5like9 is used to regulate the hypocotyl length of Arabidopsis thaliana seeds after germination by overexpressing the Actinidia arguta light-responsive gene AaHY5like9.

[0017] After years of investigation and research, the inventor believes that in the cultivation of soft-fleshed kiwifruit, insufficient or uneven light exposure to the fruit caused by poor lighting (shaded orchard) is the main reason for the poor fruit quality. The technical principle is: on the one hand, light provides energy for the normal photosynthesis of the plant and ensures the growth and development of the fruit. On the other hand, light participates in the morphological construction of the fruit as an environmental signal. Therefore, receiving sufficient light during the growth and development of the fruit is a prerequisite for the formation of high-quality fruit. In view of the decisive role of genes in fruit quality, it is of great technical significance to improve fruit quality by exploring key light-responsive genes.

[0018] In this application, the inventors used family identification at the whole genome level to clone a key light-responsive gene AaHY5like9 related to the fruit quality of Actinidia arguta, and combined with the construction of relevant recombinant expression vectors and heterologous transformation verification, confirmed that AaHY5like9 has light-responsive characteristics. Based on this result, a certain genetic resource foundation can be laid for the quality control of Actinidia arguta fruit and the cultivation of high-quality new varieties of Actinidia arguta. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the chromosome location distribution of 15 HY5like genes in Actinidia chinensis;

[0020] Figure 2 This is the structural analysis of 15 HY5like genes in Actinidia chinensis;

[0021] Figure 3 Phylogenetic analysis of 15 HY5like members in Actinidia chinensis and HY5 genes in 32 other species;

[0022] Figure 4 The homologous cloning of AaHY5like gene in Actinidia arguta and the expression pattern analysis of candidate genes were carried out;

[0023] a is the homologous cloning result of HY5like genes from 15 Actinidia chinensis in Actinidia arguta;

[0024] b is the expression level analysis of AaHY5like2 in Actinidia arguta;

[0025] c is the expression level analysis of AaHY5like9 in Actinidia arguta;

[0026] d is the expression level analysis of AaHY5like11 in Actinidia arguta;

[0027] e is the cluster analysis of the expression patterns of the three genes AaHY5like2, AaHY5like9, and AaHY5like11 in Actinidia arguta;

[0028] Figure 5 The results of subcellular localization of AaHY5like9 in Actinidia arguta.

[0029] a is a schematic diagram of the structure of the constructed recombinant plasmid vector pBI121::AaHY5like9, which carries a 35S promoter and a GFP tag;

[0030] b is the observation result of green fluorescence signal, which shows that the fluorescence of empty vector pBI121 carrying only GFP tag is located in the nucleus and cytoplasm, while AaHY5like9-GFP is localized in the nucleus;

[0031] Figure 6 The results of PCR positive detection of Arabidopsis hy5 mutant genetic transformation; among them: the first 16 lanes are positive plants, the fragment size is 670 bp, and the last lane is the negative control (NC);

[0032] Figure 7 This is the complementation result of Arabidopsis thaliana hy5 mutant; among them:

[0033] a shows the hypocotyl phenotypes of wild type, hy5 mutant, and AaHY5like9 transformed hy5 mutant;

[0034] b is the statistical result of hypocotyl length. DETAILED DESCRIPTION

[0035] The present application is further explained below with reference to the embodiments.

[0036] Example 1

[0037] In view of the important influence of light on the fruit quality of fruit trees, combined with the needs of kiwifruit cultivation and fruit quality improvement, the inventors believe that it is necessary to further explore and analyze the light response-related genes in kiwifruit. Therefore, this embodiment first briefly introduces the mining process of related genes as follows.

[0038] (I) Identification of the HY5 gene family at the whole genome level in kiwifruit

[0039] First, the inventor used Actinidia chinensis ( Actinidia chinensis Planch.'Red-5' genome was used as the reference genome, and SHORT HYPOCOTYL was used to search for candidate genes in the genome annotation file. The results showed that there were 15 results annotated as Protein LIGHT-DEPENDENT SHORT HYPOCOTYLS like.

[0040] Furthermore, according to the annotation information, these 15 genes were named HY5like1-HY5like15 according to the order of chromosome arrangement. At the same time, these 15 HY5like genes were irregularly distributed on 10 chromosomes (Chr.3, Chr.12, Chr.13, Chr.15, Chr.17, Chr.18, Chr.22, Chr.23, Chr.26, and Chr.28) (see Figure 1 ).

[0041] Analysis of the gene structure of these 15 HY5like members showed that they all contained basic gene structures such as UTR, CDS, Intron, and Motifs: Motif1, Motif2, and Motif3. Therefore, based on the similarities and differences in structure, these 15 members can be divided into two subfamilies, A and B. Subfamily A includes: HY5like1 / 4 / 5 / 6 / 10 / 12 / 13 / 14 / 15, and subfamily B includes: HYlike2 / 3 / 7 / 8 / 9 / 11 (classification results are shown in Figure 2 as shown).

[0042] In order to determine the evolutionary characteristics of these 15 members, phylogenetic analysis was performed on the HY5 members reported in 32 other species and the 15 HY5 members in kiwifruit. It was found that the 15 HY5 members in kiwifruit were clustered into a single group and had no cluster overlap with the HY5 members of other species (the results are shown in Figure 3 This result indicates that the 15 HY5 genes in kiwifruit have independent evolutionary patterns.

[0043] (II) Cloning of the HY5 gene family in Actinidia arguta

[0044] Based on the research needs of Actinidia arguta, in order to determine whether these 15 HY5like genes also exist in Actinidia arguta, based on the aforementioned series of HY5like gene sequences in Actinidia chinensis, the inventors designed relevant primers and conducted trial cloning of relevant gene sequences. The specific process is briefly described as follows.

[0045] First, based on the HY5like genes in Actinidia chinensis, the inventors designed a series of primer sequences for PCR amplification as shown in Table 1 below:

[0046] Table 1. Primer sequences designed for PCR amplification based on HY5like gene of Actinidia chinensis

[0047] .

[0048] Subsequently, the fruits of Actinidia arguta cultivar ‘Ruby Star’ (fruits were collected from the National Horticultural Germplasm Resource Bank-Kiwifruit Branch, Zhengzhou, Henan) were used as experimental samples, and total RNA was extracted from the fruits (polysaccharide and polyphenol plant RNA extraction kit, Huayueyang Biotechnology Co., Ltd., Beijing), and reverse transcribed into cDNA (high-efficiency reverse transcription kit FsQ-101, Toyobo Co., Ltd., Japan) for use;

[0049] Then, the cDNA prepared above was used as a template, and Phanta® Max Super-Fidelity DNA Polymerase (P505, Novozymes Biotech Co., Ltd., Nanjing) was used to perform PCR amplification with the primers designed above. The 20µL amplification system was designed as follows:

[0050] Mix, 10 µL;

[0051] cDNA template, 1 µL;

[0052] Upstream and downstream primers, 1 µL each;

[0053] ddH2O, 7 µL;

[0054] The amplification program was as follows: preheating at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 90 s, 33 cycles; termination extension at 72°C for 10 min;

[0055] Finally, the PCR amplification products were detected by 1% agarose gel electrophoresis.

[0056] It should be noted that the related operations refer to the existing technology or the kit instructions, and conventional operations can be performed and will not be described in detail.

[0057] The electrophoresis results are as follows Figure 4 As shown ( Figure 4a). It can be seen that only AaHY5like2, AaHY5like9 and AaHY5like11 can normally amplify specific single bands, that is, in Actinidia arguta, only three homologous genes of the HY5like family, AaHY5like2, AaHY5like9 and AaHY5like11, exist, and these three genes are single homologous and can be used as candidate genes for subsequent research.

[0058] The three homologous genes were further sequenced and analyzed, and the sequence of AaHY5like9 (510 bp) is shown in SEQ ID No. 1, as follows:

[0059] ATGTCTAGTGAGAAAGAGAAAGAATCGGCCGAGGGCTCGTCGCGATCGACGGCGACCATAAGCCGGTATGAGTCGCAGAAGCGTCGAGACTGGAACACCTTTGGCCAGTATTTGAAAAATCACATGC CGCCTGTACCTCTGTCTCAGTGCACTTGCAACCATGTCCTCGAGTTCCTTCGTTATCTCGATCAGTTCGGGAAGACTAAGGTTCACTTACATGGGTGTGTGTTTTTCGGGCAGCCAGACCCACCTGCC CCGTGCACTTGCCCGCTGAGGCAAGCGTGGGGATCGCTGGATGCCCTGATCGGACGGCTGAGAGCCGCCTATGAGGAGCACGGCGGGTCCCCGGAGACGAACCCATTTGGGAACGGGGCGATACGTGT ATATTTACGGGAAGTTAAGGAGTGCCAAGCGAAGGCGCGAGGGATTCCGTACAAGAAGAAGAAGAAGAAGAAGAAGATGATCAAAGCAAACCATGAAGAACTCAAGTCTTCCAAACAGCCCACTTGA.

[0060] After further analysis, the sequence of the protein (169aa) encoded by AaHY5like9 is shown in SEQ ID No. 2, as follows:

[0061] MSSEKEKESAEGSSRSTATISRYESQKRRDWNTFGQYLKNHMPPVPLSQCTCNHVLEFLRYLDQFGKTKVHLHGCVFFGQPDPPAPCTCPLRQAWGSLDALIGRLRAAYEEHGGSPETNPFGNGAIRVYLREVKECQAKARGIPYKKKKKKKKMIKANHEELKSSKQPT.

[0062] 3. Further determination of research subjects

[0063] In the previous research work, the inventors found that bagging can inhibit the fruit coloring of Actinidia arguta. In combination with the research purpose of this application, in order to screen the key HY5like genes that respond to light, the inventors selected samples of Actinidia arguta fruit with different treatments and different periods, and analyzed the expression levels of the three candidate HY5like genes mentioned above. The specific samples (samples were collected from the National Horticultural Germplasm Resource Bank-Kiwifruit Branch, the variety is 'Ruby Star', planted in Zhengzhou Fruit Research Institute, Zhengzhou, Henan) include: UF70 (fruit 70 days after flowering without bagging, the fruit is green at this time), BF70 (fruit 70 days after flowering with bagging, the fruit is green at this time), UF110 (fruit 110 days after flowering without bagging, the fruit is red at this time), BF110 (fruit 110 days after flowering with bagging, the fruit is green at this time). The specific process is briefly described as follows.

[0064] First, the PCR amplification primer sequences for expression detection were designed as follows:

[0065] Targeting the AaHY5like2 gene:

[0066] AaHY5like2-F: TAGCAGGTACGAGTCACAGAAGC,

[0067] AaHY5like2-R:TGATCGAGGTAACGAAGGAAAT;

[0068] Targeting the AaHY5like9 gene:

[0069] AaHY5like9-F: TGCCGCCTGTACCTCTGTCT,

[0070] AaHY5like9-R:TCCCAAATGGGTTCGTCTCC;

[0071] Targeting the AaHY5like11 gene:

[0072] AaHY5like11-F:TCCTTCGTTACCTCGATCAGTT,

[0073] AaHY5like11-R:GCCTTTGCTTGGCAGTCCTTA;

[0074] Subsequently, RNA was extracted and cDNA template was prepared according to the above operation.

[0075] Finally, PCR amplification and detection analysis were performed; in the fluorescence quantitative PCR, kiwifruit β-actin was used as the internal reference gene;

[0076] The 20µl reaction system is designed as follows:

[0077] qPCR mix, 10 µL;

[0078] Upstream and downstream primers, 1 µL each;

[0079] cDNA template, 3 µL;

[0080] ddH2O, 5 µL;

[0081] The program of the fluorescence quantitative PCR system (Roche 480) was: 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 20 s, 45 cycles, and 65°C for 1 min.

[0082] The results are as follows Figure 4 As shown ( Figure 4 b. Figure 4 c. Figure 4 d). The analysis showed that the three genes had different expression patterns, but the expression of AaHY5like9 was closely related to fruit color (appearance quality), that is, it showed significant high and low expression levels in UF110 and BF110 fruit samples, respectively.

[0083] Further cluster analysis results showed that ( Figure 4 e): AaHY5like9 was clustered into a separate group, which was significantly separated from AaHY5like2 and AaHY5like11. This result further indicated that AaHY5like9 had an independent expression pattern. Based on this, AaHY5like9 was selected as a candidate light-responsive gene for further study.

[0084] Example 2

[0085] On the basis of the above-mentioned Example 1, in order to further clarify and confirm the light response characteristics of the AaHY5like9 gene, the inventors constructed an overexpression vector containing the AaHY5like9 gene, and experimentally verified it by heterologous transformation of the Arabidopsis thaliana hy5 mutant. The specific experimental process and results are briefly described as follows.

[0086] 1. Construction of recombinant overexpression vector

[0087] Using pBI121 plasmid as a vector, a recombinant plasmid vector pBI121:: AaHY5like9 , the specific process is as follows.

[0088] (1) Referring to the above operation, PCR amplification was performed to prepare the AaHY5like9 gene, and the PCR amplification product was detected by 1% agarose gel electrophoresis, and the target fragment was recovered by gel excision (EasyPure® Quick Gel Extraction Kit, Quanshijin Biotechnology Co., Ltd., Beijing).

[0089] (2) Connect the recovered AaHY5like9 gene fragment to the T vector; the connection system is:

[0090] Gel recovery product, 4 µL;

[0091] 5×TA / Blunt-Zero Cloning Mixa, 1 µL;

[0092] Incubate in PCR instrument at 37°C for 5 min.

[0093] (3) The above ligation products were transformed into DH5α Escherichia coli competent cells and plated for culture. Positive clones were selected for PCR verification, and the plasmids extracted from the positive bacterial solution were further sequenced for verification.

[0094] (4) Design primer sequences with restriction enzyme cutting sites (Xba Ⅰ, Bam HI) as follows:

[0095] Upstream primer: gagaacacgggggactctagaATGTCTAGTGAGAAAGAGAAAGAATCG,

[0096] Downstream primer: gcccttgctcaccatggatccAGTGGGCTGTTTGGAAGACTTG;

[0097] The above-mentioned plasmid with correct sequencing was used as a template and PCR amplification was performed using the above primers (to obtain the plasmid without the stop codon). AaHY5like9 Gene), and perform electrophoresis detection and gel excision recovery;

[0098] The recovered product was further connected to the T vector and transformed into DH5α Escherichia coli competent cells, and plate culture, bacterial liquid PCR verification, and sequencing verification were performed.

[0099] (5) Perform double restriction digestion on the previously sequenced plasmid and pBI121. The 40 µL restriction digestion system is designed as follows:

[0100] Plasmid (or pBI121 vector), 15 µL;

[0101] 10 × FastDigest Buffer, 4 µL;

[0102] XB Ⅰ Restriction enzyme, 2 µL;

[0103] Bam HI restriction enzyme, 2 µL;

[0104] ddH2O, 17 µL;

[0105] 37℃ digestion for 15 min;

[0106] The digested products were detected by agarose gel electrophoresis, and the small plasmid fragments and large vector fragments were recovered, and further ligated using T4 ligase. The ligation system was:

[0107] AaHY5like9 , 10 µL;

[0108] pBI121 vector, 1.5 µL;

[0109] T4 DNA ligase, 1 µL;

[0110] Ligation Buffer, 2.5 µL;

[0111] ddH2O, 10 µL;

[0112] Incubate at 16°C for 12 h.

[0113] (6) The above ligation products were transformed into DH5α E. coli competent cells, and further plated for screening. Positive clones were selected for bacterial liquid PCR verification and sequencing verification to ensure that the recombinant plasmid was constructed correctly. Finally, the recombinant plasmid vector pBI121:: AaHY5like9 (Schematic diagram of the recombinant plasmid structure is shown in Figure 5 a).

[0114] Furthermore, to facilitate subsequent transformation applications, the correct recombinant plasmid vector pBI121:: AaHY5like9 Transform Agrobacterium GV3101 competent cells, screen and identify to obtain transformed bacteria for infection. At the same time, transform Agrobacterium GV3101 competent cells with pBI121 empty vector to prepare control infection bacteria.

[0115] 2. Infection of Nicotiana benthamiana leaves

[0116] The prepared transformed bacteria for infection (or control bacteria for infection (or invasion) were streaked onto LB solid medium containing antibiotics and cultured upside down for 72 h;

[0117] The cells were collected by centrifugation at 4000 r / min and resuspended in infection solution (10 mM MES, 10 mM MgCl2 and 200 µM AS) to an OD of 600 = about 0.8, and after standing at room temperature for two hours, it is used as the bacterial solution for infection.

[0118] Select Nicotiana benthamiana leaves of about 6 weeks old, and use a 1 mL syringe without a needle to gently inject the infection solution from the back of the leaves (the infection solution can be seen gradually penetrating the leaves with the naked eye). At the same time, the infection solution prepared with an empty vector is injected in the same way as a control.

[0119] The tobacco plants after infection and injection were shaded for 24 h, and then cultured in a normal light incubator with alternating light and dark for 48 h, and the GFP fluorescence signal was observed under a laser confocal scanner.

[0120] The results are as follows Figure 5 ( Figure 5 b). Analysis shows that the green fluorescence signal of the empty vector pBI121 carrying only the GFP tag is located in the cell nucleus and evenly distributed in the cytoplasm; while in the leaf samples injected with the recombinant plasmid vector AaHY5like9-GFP, the green fluorescence signal was only observed in the cell nucleus. Analysis suggests that this is because transcription factors usually participate in regulation by activating downstream gene expression, and all need to play a role in the cell nucleus. In other words, the subcellular localization results show that the AaHY5like9 gene encodes a transcription factor with the general characteristics of transcription factors. It belongs to the transcription factor protein and can play a transcriptional regulatory function in the cell nucleus.

[0121] (3) Arabidopsis thaliana hy5 Mutants

[0122] Arabidopsis hy5 The mutant (a T-DNA insertion mutant, the research material can be publicly obtained from the relevant seed resource library, and the materials used in this embodiment are donated by the applicant's cooperative unit), as a HY5 gene function loss mutant, its phenotype is: under normal light conditions, the long hypocotyl is significantly longer, and the length of the hypocotyl is about 1.8 cm after germination and cultivation of seeds for 7 days (while the normal wild type is only about 0.2 cm). Based on this mutant, the inventor transformed it and performed heterologous expression of the AaHY5like9 gene to investigate whether the mutant missing gene can be synonymously compensated. The specific experimental process is briefly described as follows.

[0123] First, Arabidopsis hy5 mutant seeds were sterilized and cultured in a greenhouse until they sprouted and flowered (growth conditions: relative humidity 60%, temperature 22°C, 16 h light / 8 h dark);

[0124] Then, refer to the above operation, activate the Agrobacterium containing the recombinant plasmid vector, and resuspend the bacteria in transformation buffer (OD600 = about 1.0)

[0125] Conversion Buffer: Each liter contains 25 mL of MS macromolecules, 0.5 mL of MS micromolecules, 5 mL of MS organic, 2.5 mL of MS iron salt, 50 mL of sucrose, 10 µL of 1 mg / mL 6-BA, 400 µL of SILWET-77, pH=5.8;

[0126] During transformation, the immersion method (infection method) was used: all inflorescences of Arabidopsis thaliana that were flowering were immersed in the transformation buffer solution for 30 seconds, and the transformation was repeated once after 7 days;

[0127] After the infection is completed, Arabidopsis thaliana is continued to be cultured. After 3 weeks of culture, the number of watering is reduced to accelerate plant senescence, and finally the seeds are harvested and recorded as T0 generation seeds.

[0128] Referring to the above operation and the conventional technology, the T0 generation seeds were sterilized and then planted on a 1 / 2 MS screening medium plate containing kanamycin (100 µg / mL) to screen positive plants. Referring to the above operation, the positive plants obtained by screening were cultured until the seeds were harvested, which were recorded as T1 generation seeds. The same screening and culture operation was performed to obtain T2 generation seeds.

[0129] During the screening process, when further identifying the positive plants obtained by screening with kanamycin-containing medium, plant leaves were used as samples (genomic DNA was extracted as a template), primers were designed based on the kanamycin resistance gene NPT, and PCR identification was performed (the target fragment size of the positive plant was 670 bp); the specific design of the primer sequence is as follows:

[0130] F:AAGATGGATTGCACGCAGGT,

[0131] R:TCACGGGTAGCCAACGCT.

[0132] Finally, 16 positive strains were identified (partial PCR identification results are shown in Figure 6 as shown).

[0133] For further phenotypic evaluation, the T2 generation seeds (i.e., AaHY5like9 transformed mutants) were sterilized and then planted on 1 / 2MS medium and placed vertically in a culture room for 7 days to observe the phenotypic changes of the hypocotyls (at the same time, wild-type Arabidopsis, hy5 mutants as controls).

[0134] The results are as follows Figure 7 As shown. It can be seen that the wild type presents a short hypocotyl phenotype. hy5 The mutant showed a long hypocotyl phenotype, while the transformed AaHY5like9 gene hy5 The hypocotyl phenotype of the mutant transgenic Arabidopsis is between the two ( Figure 7 a).

[0135] Further measurement of hypocotyl length showed that: hy5 The hypocotyl length of the mutant was significantly different from that of the wild type and the transgenic ( Figure 7 b).

[0136] The results of hypocotyl measurement of some samples are as follows (unit: cm)

[0137] .

[0138] The above results show that the stable genetic transformation of AaHY5like9 gene can significantly compensate for hy5 The mutant has a long hypocotyl phenotype, which indicates that AaHY5like9 can act as a light-responsive gene and participate in the occurrence and regulation of related biological traits.

[0139] It needs to be explained that in actual research, based on intuitiveness and measurability, the light response characteristics are usually indirectly reflected by changes in hypocotyl length. Taking Arabidopsis as an example, its main technical theory is: for wild-type plants, under normal light conditions, the plants have short hypocotyls, and under dark conditions, they have long hypocotyls. But for hy5 mutant plants, they have long hypocotyls regardless of normal light or dark conditions. Therefore, hy5 in Arabidopsis is usually identified as a key factor (gene) for light response. In the above transformation results, the mutant transformed with AaHY5like9 can restore the long hypocotyl phenotype, so it can be determined that AaHY5like9 has the function of light response and is a light response gene.

Claims

1. The light-responsive gene AaHY5like9 of Actinidia arguta is characterized by: Its base sequence is shown in SEQ ID No.

1.

2. The protein encoded by the light-responsive gene AaHY5like9 of Actinidia arguta according to claim 1 is characterized in that: Its amino acid sequence is shown in SEQ ID No.

2.

3. A primer pair for PCR amplification of the light-responsive gene AaHY5like9 of Actinidia arguta described in claim 1, characterized in that: The specific design is as follows: F: ATGTCTAGTGAGAAAGAGAAAGAATCG; R:TCAAGTGGGCTGTTTGGAAGA.

4. An overexpression vector prepared using the light-responsive gene AaHY5like9 of Actinidia arguta L. according to claim 1, characterized in that: The pBI121 plasmid is used as a vector, and the light-responsive gene AaHY5like9 of the Actinidia arguta mentioned in claim 1 is recombined.

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