Photoinduced gene promoter, recombinant plasmid, recombinant bacterium and application thereof

Abstract

The invention discloses a light-induced gene promoter, a recombinant plasmid, a recombinant bacterium and an application of the light-induced gene promoter. The nucleotide sequence of the light-induced gene promoter is as shown in SEQ ID No: 1. The recombinant plasmid comprises an expression vector and the light-induced gene promoter. According to the invention, a light-induced gene promoter is used for replacing a constitutive gene promoter, and two recombinant vectors, namely, pbHLH93-LUC and pbHLH93-GUS, containing specific light-induced promoters are provided. The two constructed recombinant vectors have the advantages of being good in specificity, efficient in expression, easy to screen and the like, and the recombinant vector pbHLH93-GUS can completely rapidly obtain a transgenic material for photoinduced expression of a target gene on the basis of ensuring illumination, and is expected to play an important role in regulation and control of expression of the target gene.

Description

Technical Field

[0001] The present invention relates to the fields of plant biotechnology and genetic engineering, and in particular, to a light-inducible gene promoter, a recombinant plasmid, a recombinant bacterium, and their applications. Background Art

[0002] The growth, development, and growth cycle of plants are the results of differential gene expression in space and time. Transcriptional regulation is one of the main forms of plant gene expression regulation, which is achieved by the interaction of cis-acting elements and trans-acting factors. A promoter is one of the important cis-acting elements for plant gene transcriptional regulation. It is located upstream of the transcriptional start site at the 5' end of the structural gene and can activate RNA polymerase to accurately bind to the template DNA and have transcriptional initiation specificity. The promoter itself does not have the ability to control gene activity, and the trans-acting factor transcription factor can regulate the expression of the target gene by recognizing the specific DNA sequence on the promoter.

[0003] According to the usage characteristics of promoters, they can be divided into three categories: constitutive promoters, tissue-specific promoters, and inducible promoters. The regulation of constitutive promoters is not affected by external conditions, and the expression of the genes they initiate is persistent, but it will consume the substances and energy in the recipient cells excessively and cannot effectively control the spatio-temporality of gene expression. Therefore, there are certain defects in practical applications. Under the regulation of tissue-specific promoters, the expression of foreign genes generally occurs only in certain specific organs or tissue sites and often shows the characteristics of developmental regulation. It overcomes the waste caused by the non-specific, continuous, and high-efficiency expression of foreign genes initiated by constitutive promoters in recipient plants, and increases the effect of transgenes. Therefore, people pay more and more attention to the research and application of specific promoters (Song Yang et al., 2007). However, at present, the discovery of tissue-specific promoters in different species is mostly still in its infancy, and the specific mechanism is still unclear. Inducible promoters can rapidly induce the "on" and "off" of gene transcription under the stimulation of specific physical or chemical signals. Currently, light-inducible gene promoters, heat-inducible gene promoters, wound-inducible gene promoters, and fungal-inducible gene promoters have been isolated, etc.

[0004] Light is one of the important environmental factors during the growth of grapes. The absolute dependence on light during plant development has promoted the evolution of complex mechanisms, thereby enhancing the ability to perceive and transduce light signals (Zoratti et al., 2014). A large number of studies have shown that there are a series of endogenous genes in grapes that are induced by light. Appropriate enhancement of light can promote the expression of related genes, enhance the accumulation of condensed tannins in grapes, and ensure the quality of grape fruits.

[0005] Therefore, studying the promoters of these genes and developing a light-induced promoter-induced gene expression system have inestimable effects on grape genetic engineering research and application, and its application prospect is very broad. Summary of the Invention

[0006] In view of the above-mentioned prior art, the object of the present invention is to overcome the waste caused by the non-specific, continuous and high-efficiency expression of exogenous genes initiated by constitutive promoters in recipient plants, thereby providing a new light-induced gene promoter, recombinant plasmid and recombinant bacterium with advantages such as good specificity, high-efficiency expression and easy screening.

[0007] To achieve the above object, the present invention provides a light-induced gene promoter, and the nucleotide sequence of the light-induced gene promoter is as shown in SEQ ID No: 1.

[0008] Preferably, the light-induced gene promoter is derived from Cabernet Sauvignon grapes.

[0009] The present invention also provides a recombinant plasmid, and the recombinant plasmid includes an expression vector and the light-induced gene promoter as described above.

[0010] Preferably, the expression vector is selected from the LUC vector;

[0011] and / or, the light-induced gene promoter is obtained by amplifying the genomic DNA of Cabernet Sauvignon grapes using the primer pairs shown in SEQ ID No: 4 and SEQ ID No: 5.

[0012] Preferably, the expression vector is selected from the GUS vector;

[0013] and / or, the light-induced gene promoter is obtained by amplifying the genomic DNA of Cabernet Sauvignon grapes using the primer pairs shown in SEQ ID No: 6 and SEQ ID No: 7.

[0014] Preferably, the recombinant plasmid is constructed by ligating the light-induced gene promoter to the recombinant plasmid after double digestion using a ligase.

[0015] Preferably, the double digestion process uses BsaI restriction endonuclease and Eco31I restriction endonuclease;

[0016] and / or, the ligase is selected from Biorun 2*EasyClone Mix ligase.

[0017] The present invention also provides a recombinant bacterium, and the recombinant bacterium is obtained by transforming Agrobacterium with the recombinant plasmid as described above.

[0018] The present invention also provides an application of the recombinant bacterium as described above in improving the light energy utilization efficiency of Arabidopsis thaliana, and the recombinant bacterium is obtained by transformation with a recombinant plasmid using a GUS vector as the expression vector.

[0019] Preferably, the application process includes: culturing Arabidopsis thaliana with a medium containing the recombinant bacterium based on the bud soaking method, and screening to obtain a homozygous mutant of Arabidopsis thaliana with stable inheritance.

[0020] The present invention uses a light-inducible gene promoter to replace the constitutive gene promoter, and provides two recombinant vectors containing specific light-inducible promoters, namely, using the pGreenII0800-LUC plasmid fused with the LUC reporter gene as the basic vector, and using the light-inducible gene promoter bHLH93 as the target sequence to form the recombinant vector pbHLH93-LUC. Using the pBWA(V)BII-GUS plasmid fused with the GUS reporter gene as the basic vector, and using the light-inducible gene promoter bHLH93 as the target sequence to form the recombinant vector pbHLH93-GUS. The two recombinant vectors constructed by the present invention have the advantages of good specificity, high-efficiency expression, easy screening, etc. Among them, the recombinant vector pbHLH93-GUS can completely obtain transgenic materials that can rapidly induce the expression of the target gene based on ensuring light, and is expected to play an important role in regulating the expression of the target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and are used to explain the present invention together with the following specific embodiments, but do not constitute a limitation to the present invention. In the drawings:

[0022] Figure 1 is the sequence analysis diagram of the bHLH93 promoter in Preparation Example 2;

[0023] Figure 2 is the structural diagram of the pbHLH93-LUC recombinant plasmid prepared in Preparation Example 3;

[0024] Figure 3 is the fluorescence intensity analysis diagram of tobacco leaves in the verification example;

[0025] Figure 4 is the structural diagram of the pbHLH93-GUS recombinant plasmid prepared in Preparation Example 4;

[0026] Figure 5 is the leaf picture of transgenic Arabidopsis thaliana cultured under different light conditions in Detection Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention.

[0028] The technical solutions of the present invention are described in detail below through specific examples.

[0029] Among them, Cabernet Sauvignon grapes were collected from the Shangzhuang Experimental Station of China Agricultural University; Arabidopsis thaliana of the Col-0 ecotype was provided by the Key Laboratory of Wine Processing of the Ministry of Agriculture and Rural Affairs;

[0030] The pMD19-T sequencing vector is a commercially available product with the brand number 3271Takara from Beijing Liuhetong Economic and Trade Co., Ltd.; the Escherichia coli competent DH5α is a commercially available product with the brand number CB101 from Beijing Tiangen Biotechnology Co., Ltd.; the pGreenII0800-LUC-ccdb plasmid vector is a preserved strain from Wuhan Boyuan Biotechnology Co., Ltd.; the pBWA(V)BII-GUS plasmid vector is a preserved strain from Wuhan Boyuan Biotechnology Co., Ltd.; the Agrobacterium tumefaciens GV3101 is a commercially available product with the brand number BC304-01 from Beijing Bomed Gene Technology Co., Ltd.

[0031] Other reagents used in the present invention are conventional reagents that can be understood and used by those skilled in the art, and will not be elaborated here.

[0032] Preparation Example 1. Cloning of a light-induced gene promoter (denoted as the bHLH93 promoter)

[0033] According to the promoter region approximately 1 Kb upstream of the published Pinot noir bHLH93 (bHLH93: XM_002264371.4) gene sequence, a first primer pair as shown in SEQ ID NO: 2 (upstream primer, denoted as pbHLH93-F: 5'-GCTTTCCAGTAGAAATTTGGCAC-3') and SEQ ID NO: 3 (downstream primer, denoted as pbHLH93-R: 5'-CTCCCTTTTCTCTTTTCTCTCTCC-3') was designed.

[0034] Using the genomic DNA of Cabernet Sauvignon grapes as a template, PCR amplification was carried out with the first primer pair. The obtained PCR product was the light-induced gene promoter bHLH93, and the target fragment of 1023 bp was recovered by agarose gel electrophoresis. The PCR reaction system is shown in Table 1 (total system of 40 μL). The PCR reaction conditions were: Heat Lid was maintained at 105 °C until the end of the program; 98 °C, 30 s; (98 °C, 10 s; 55 °C, 30 s; 72 °C, 40 s), 35 cycles; 72 °C, 10 min; 4 °C, ∞. The amplification product was obtained.

[0035] Table 1 PCR reaction system

[0036] 2×High-Fidelity PCR Master Mix 25 μL <![CDATA[Upstream primer shown in SEQ ID NO:2 (10 μM, diluted with ddH2O)]]> 1 μL <![CDATA[Downstream primer shown in SEQ ID NO:3 (10 μM, diluted with ddH2O)]]> 1 μL DNA template 1 μL <![CDATA[ddH2O]]> 12 μL

[0037] Since the 3' end of the DNA fragment amplified by 2× High-Fidelity PCR Master Mix does not contain base A, the above amplification product needs to be subjected to TA cloning. The TA cloning reaction system is shown in Table 2 (50 μL total system). The TA cloning reaction conditions are as follows: React at 72 °C for 20 min and then immediately place on ice for 2 min. The amplified product after adding A (i.e., the bHLH93 promoter) is obtained.

[0038] Table 2 TA cloning reaction system

[0039] 10×A-Tailing Buffer 5 μL dNTP Mixture 4 μL A-Tailing Enzyme 0.5 μL Blunt-ended DNA fragment (i.e., the aforementioned amplification product) 0.5 - 5 μg <![CDATA[ddH2O]]> Make up to 50 μL

[0040] The obtained bHLH93 promoter and the pMD19-T sequencing vector are ligated at 16 °C for 1 h according to the reaction system shown in Table 3 to obtain the ligation product pMD19-T-pbHLH93.

[0041] Table 3 Ligation reaction system

[0042] bHLH93 promoter 1 μL pMD19-T sequencing vector 1 μL Solution I 5 μL <![CDATA[ddH2O]]> 3 μL

[0043] The obtained ligation product pMD19-T-pbHLH93 is transformed into Escherichia coli: Add 50 μL of Escherichia coli competent DH5α and 10 μL of the ligation product pMD19-T-pbHLH93 to a 1.5 mL centrifuge tube, gently flick to mix, and then incubate on ice for 30 min; then transfer the centrifuge tube to a 42 °C water bath, heat shock for 90 s, and then quickly place on ice for 2 min; add 900 μL of LB liquid medium to the centrifuge tube, mix well, culture at 37 °C and 180 rpm for 45 min, then centrifuge at 8000 rpm for 1 min, discard 700 μL of the supernatant, use a pipette to blow and mix the cell pellet and the remaining liquid, and then take 100 μL and spread it on the LB solid medium with the corresponding resistance. Seal it and invert it in a 37 °C constant temperature incubator for 16 h. Pick the cultured colonies into 1 mL of LB liquid medium (containing kanamycin), and culture with shaking at 37 °C for 2 h. Perform colony PCR identification, and send the positive bacterial liquid to the company for sequencing to obtain the correctly sequenced bHLH93 promoter sequence.

[0044] Preparation Example 2, Sequence analysis of the bHLH93 promoter

[0045] Import the original files of paired-end sequencing into SeqMan software for splicing. During splicing, discard the bases with poor paired-end peak shapes, and keep the other parameters default. After splicing is completed, export the result. Import the spliced sequence and the template into CLC Genomic Workbench software for alignment. The strain corresponding to the correctly aligned sequence is used for subsequent experiments. Analyze and predict the cis-acting elements of the correctly aligned bHLH93 promoter sequence through the online website of PlantCARE and the PLACE database. Predict the transcription start site of the promoter using the online website of Softberry TSSP. The bHLH93 promoter sequence (as Figure 1 shown) contains not only the transcriptional core elements TATA-box and CAAT-box, but also some cis-acting elements that respond to environmental stress and plant hormones, as well as DNA binding sites for MYB, bHLH, and WRKY transcription factors to bind. These results indicate that the bHLH93 promoter may be involved in the defense processes of many biotic and abiotic stresses in grapes.

[0046] Among the numerous cis-acting elements, the bHLH93 promoter has a large number of light-responsive elements (LREs) and multiple types. They are I box core, AE-box, Box 4, GT1-motif, Gap-box, TCCC-motif, and TCT-motif respectively. These 7 types of LREs appear a total of 10 times in the bHLH93 promoter region, and are mostly distributed between -500 - 1000 bp. The above results indicate that the bHLH93 promoter may be a light-inducible promoter.

[0047] Preparation Example 3: Preparation of the first recombinant bacterium using the LUC plasmid as a vector

[0048] Using the ligation product pMD19-T-pbHLH93 containing the bHLH93 promoter sequence with correct sequencing in Preparation Example 1 as a template, perform PCR amplification according to the amplification system and amplification conditions in Preparation Example 1 using the second primer pair shown in SEQ ID NO:4 (upstream primer, denoted as pbHLH93-LUC-F: 5’-CGAGGTCGACGGTATCGATAgctttccagtagaaatttggcacata aacatttttagaag-3’) and SEQ ID NO:5 (downstream primer, denoted as pbHLH93-LUC-R: 5’-GCGTCTTCCATGGTCCCCCGctcccttttctcttttctctctccctc-3’). The obtained PCR product is the first light-inducible gene promoter bHLH93 with infusion sites at both ends, and perform agarose gel electrophoresis to recover the target fragment.

[0049] The pGreenII 0800-LUC-ccdb plasmid vector fused with the LUC reporter gene was digested with BsaI and Eco31I, and the digested products were recovered and purified by agarose gel electrophoresis to obtain the digested pGreenII 0800-LUC-ccdb plasmid vector. Among them, the double digestion reaction system is shown in Table 4 (the total system is 20 μL). The reaction conditions for the double digestion process were reaction at 37 °C for 1 h.

[0050] Table 4 Double digestion reaction system

[0051] Nuclease-free Water 13 μL 10*Buffer 2 μL BsaI / Eco3II 1 μL pGreenII 0800-LUC-ccdb 4 μL

[0052] The first light-inducible gene promoter bHLH93 fragment was ligated into the digested pGreenII 0800-LUC-ccdb plasmid vector to construct the recombinant plasmid as shown in Figure 2 . This recombinant plasmid was designated as pbHLH93-LUC. The ligation system is shown in Table 5 (the total system is 20 μL). The reaction conditions for the ligation process were reaction at 37 °C for 30 h.

[0053] Table 5 Ligation reaction system

[0054] Biorun 2*EasyClone Mix 10 μL The first bHLH93 digested fragment 5 μL Digested pGreenI1 0800-LUC-ccdb 5 μL

[0055] The obtained recombinant plasmid pbHLH93-LUC was transformed into Agrobacterium tumefaciens GV3101 (i.e., Agrobacterium competent cells). The specific transformation process was as follows: 1 μg of the recombinant plasmid pbHLH93-LUC was added to 100 μL of Agrobacterium competent cells, gently flicked and mixed evenly, then ice-bathed for 5 min, immediately frozen in liquid nitrogen for 5 min, transferred to a 37 °C water bath for heat shock for 5 min, and then ice-bathed for 5 min; then 800 μL of LB liquid medium was added, and cultured at 28 °C and 220 rpm for 2 - 3 h; after the culture was completed, centrifuged at 8000 rpm for 1 min, about 700 μL of the supernatant was discarded, the cell pellet and the remaining liquid were pipetted and mixed evenly, and 100 μL was taken and spread on the corresponding resistant (20 mg / L Rif, 50 mg / L Kan) LB solid medium, sealed and inverted in a 28 °C constant temperature incubator for 48 - 72 h; after single colonies grew out, they were identified by colony PCR, the positive bacteria with correct identification were preserved, stored at -80 °C for later use, and the first recombinant bacteria were obtained.

[0056] Verification example

[0057] The first recombinant bacteria obtained above were transformed into tobacco leaves. The specific operation process includes: taking 100 μL of the agrobacterium liquid (i.e., the first recombinant bacteria) with the recombinant plasmid pbHLH93-LUC prepared above and adding it to 4 mL of LB (20 mg / L Rif, 50 mg / L Kan) liquid medium, culturing at 28 °C and 220 rpm for about 20 h; sucking an appropriate amount of the cultured bacteria liquid into 50 mL of LB (20 mg / L Rif, 50 mg / L Kan) liquid medium to make the initial OD600 0.19 - 0.2, and culturing at 28 °C and 220 rpm until the OD600 reaches 1.0 - 1.2; transferring all the bacteria liquid after the enlarged culture to a sterilized 50 mL centrifuge tube, centrifuging at 4 °C and 5000 rpm for 5 min, and then discarding the supernatant; resuspending the thallus with 30 mL of tobacco injection buffer (prepared in the conventional manner in the art, for example, it can contain 50 mM sucrose, 10 mM MES, 0.03% (v / v) Silwet-77, 100 μM acetosyringone, 10 mM MgCl2, 2 mM ascorbic acid. Of course, the present invention is not limited to this specific composition and dosage, and any conventional tobacco injection buffer formula that can be understood and used by those skilled in the art can be used here), centrifuging at 4 °C and 5000 rpm for 5 min, and then discarding the supernatant, repeating this step twice; resuspending the thallus with an appropriate amount of tobacco injection buffer to make the OD600 of the bacteria liquid between 0.6 and 0.8; allowing the prepared agrobacterium liquid (i.e., the bacteria liquid with OD600 between 0.6 and 0.8 prepared as described above) to stand at room temperature for 2 - 3 h, using the agrobacterium liquid containing the empty vector as a negative control; injecting the bacteria liquid into the back of the Nicotiana benthamiana leaves about seven weeks old with a syringe without a needle. When injecting, first gently scratch a small opening with the tip of the needle, hold it with your finger, and inject the bacteria liquid with a syringe without a needle (do not cross the leaf veins). After avoiding light for 12 h, place it in the greenhouse for normal culture for 3 days; apply a layer of D-luciferin solution on the back of the leaves injected with agrobacterium, take pictures with a fluorescence imaging system, observe and save the experimental results. And analyze and measure the fluorescence intensity with ImageJ software. As Figure 3 shown, it can be seen that the bHLH93 promoter has activity.

[0058] Preparation Example 4: Preparation of the second recombinant bacteria with the GUS plasmid as the vector

[0059] Using the ligated product pMD19-T-pbHLH93 containing the bHLH93 promoter sequence with correct sequencing in Preparation Example 1 as a template, PCR amplification was carried out according to the amplification system and amplification conditions in Preparation Example 1 using the third primer pair shown in SEQ ID NO:6 (upstream primer, denoted as pbHLH93-GUS: 5’-CGCCTGCAGGTCTAGATAGAgctttccagtagaaatttggcacataaa catttttagaag-3’) and SEQ ID NO:7 (downstream primer, denoted as pbHLH93-GUS-R: 5’-CCCTCAGATCTACCATCGCActcccttttctcttttctctctccctc-3’). The resulting PCR product was the second light-inducible gene promoter bHLH93 with ends for infusion, and the target fragment was recovered by agarose gel electrophoresis.

[0060] The plasmid vector pBWA(V)BII-GUS fused with the GUS reporter gene and the PCR product light-inducible gene promoter bHLH93 prepared in step 3) were double digested with BsaI and Eco31I, and the digested products were recovered and purified by agarose gel electrophoresis to obtain the digested plasmid vector pBWA(V)BII-GUS. Among them, the double digestion reaction system is shown in Table 6 (the total system is 20 μL). The reaction conditions for the double digestion process were to react at 37 °C for 1 h.

[0061] Table 6 Double digestion reaction system

[0062] Nuclease-free Water 13 μL 10*Buffer 2 μL BsaI / Eco3II 1 μL pBWA(V)BII-GUS 4 μL

[0063] The promoter bHLH93 (i.e., the second light-inducible gene promoter bHLH93) obtained by amplification with the third primer pair was ligated into the digested plasmid vector pBWA(V)BII-GUS to construct a recombinant plasmid as shown in Figure 4 The recombinant plasmid was denoted as pbHLH93-GUS. The ligation system is shown in Table 7 (the total system is 20 μL). The reaction conditions for the ligation process were to react at 37 °C for 30 h.

[0064] Table 7 Ligation reaction system

[0065] Biorun 2*EasyClone Mix 10 μL The second bHLH93 digested fragment 5 μL Digested pBWA(V)BII-GUS 5 μL

[0066] The obtained recombinant plasmid pbHLH93-GUS was transformed into Escherichia coli competent DH5α. The specific transformation process was as follows: Add 50 μL of Escherichia coli competent DH5α and 10 μL of the ligation product (i.e., recombinant plasmid pbHLH93-GUS) into a 1.5 mL centrifuge tube. After gently flicking to mix, place it on ice for 30 min; transfer the centrifuge tube to a 42 °C water bath, heat shock for 90 s, and then quickly place it on ice for 2 min; add 900 μL of LB liquid medium, mix well, and culture at 37 °C and 180 rpm for 45 min; after centrifuging at 8000 rpm for 1 min, discard 700 μL of the supernatant, use a pipette to blow and mix the cell pellet and the remaining liquid, and then take 100 μL to coat on the LB solid medium with the corresponding resistance, seal it, and invert it and culture it in a 37 °C constant temperature incubator for 16 h. Pick colonies into 1 mL of LB liquid medium (containing kanamycin), and culture it with shaking at 37 °C for 2 h. Perform colony PCR identification, and send the positive bacterial solution to the company for sequencing, so as to obtain the recombinant plasmid pbHLH93-GUS with correct sequencing.

[0067] The recombinant plasmid pbHLH93-GUS with correct sequencing was transformed into Agrobacterium tumefaciens GV3101. The specific transformation process was as follows: Add the recombinant plasmid pbHLH93-GUS with correct sequencing into 100 μL of Agrobacterium tumefaciens competent cells. After gently flicking to mix, place it on ice for 5 min, immediately place it in liquid nitrogen for quick freezing for 5 min, then transfer it to a 37 °C water bath for heat shock for 5 min, and then place it on ice for 5 min; add 800 μL of LB liquid medium, culture at 28 °C and 220 rpm for 2 - 3 h; centrifuge at 8000 rpm for 1 min, discard about 700 μL of the supernatant, use a pipette to blow and mix the cell pellet and the remaining liquid, and then take 100 μL to coat on the LB solid medium with the corresponding resistance (20 mg / L Rif, 50 mg / L Kan), seal it, and invert it and culture it in a 28 °C constant temperature incubator for 48 - 72 h; after single colonies grow out, perform identification by colony PCR, preserve the correctly identified positive bacteria, and store them at -80 °C for later use, that is, the second recombinant bacterium was obtained.

[0068] Application example: Application of the bHLH93 promoter in the preparation of transgenic Arabidopsis thaliana

[0069] Arabidopsis thaliana of the Col-0 ecotype was stably transformed using the flower bud soaking method, and the specific process is as follows: All the already-opened flowers and the developed siliques were removed in advance, leaving only the inflorescences; 100 μL of the Agrobacterium (i.e., the second recombinant bacterium) solution carrying the expression recombinant vector obtained in Preparation Example 4 was taken and placed in 4 mL of LB (20 mg / L Rif, 50 mg / L Kan) liquid medium, and cultured at 28 °C and 220 rpm for about 20 h; An appropriate amount of the bacterial solution was pipetted into 50 mL of LB (20 mg / L Rif, 50 mg / L Kan) liquid medium to make the initial OD600 0.19 - 0.2, and cultured at 28 °C and 220 rpm until the OD600 reached 0.6 - 1.0; The bacterial solution was all transferred to a sterilized 50 mL centrifuge tube, centrifuged at 4 °C and 8000 rpm for 8 min, and then the supernatant was discarded; The cells were resuspended with an appropriate amount of transformation solution to make the OD600 of the bacterial solution about 0.8, and then the corresponding amount of Silwet-77 (final concentration 200 μL / L) was added to improve the transformation efficiency; The prepared Agrobacterium bacterial solution was left standing at room temperature for 2 - 3 h, with the Agrobacterium bacterial solution containing the empty vector as the negative control; The bacterial solution was poured into a petri dish, and the inflorescences of Arabidopsis thaliana were immersed in the bacterial solution for 30 s. If there were too many inflorescences, it could be carried out in batches. After completion, it was bagged and cultured in the dark for 12 - 16 h, then the bag was removed and placed in the greenhouse for normal cultivation. The harvested seeds were T1 generation seeds, and the seeds were stored in a centrifuge tube and stored at room temperature with a desiccant added.

[0070] The T1 generation seeds were cultured on 1 / 2 MS solid medium containing hygromycin, vernalized and then cultured under light. The T1 generation positive plants growing normally in the transferred medium were transferred to nutrient soil and grown at 22 °C in the greenhouse. After the T1 generation positive plants grew to full maturity, the T2 generation seeds were harvested.

[0071] The T2 generation seeds were normally planted, and the T2 generation plants with good growth vigor were subjected to genotype identification, the genotypes of the T2 generation were analyzed, and the stably inherited homozygous mutants of the T3 generation were screened.

[0072] The screened homozygous mutants of the T3 generation were cultured according to the following method:

[0073] Washing of seeds: An appropriate number of T3 generation seeds were taken and placed in a 1.5 mL centrifuge tube, 1 mL of 75% ethanol was added, sterilized for 10 min, the 75% ethanol was aspirated out in a laminar flow hood, and immediately resuspended with 1 mL of absolute ethanol. The seeds were aspirated out with a 1 mL pipette tip and placed on qualitative filter paper until completely dry.

[0074] Spot sowing (for observing the phenotype of young roots): The disinfected seeds were spot-sown onto a square phenotype medium containing 1 / 2 MS (antibiotics or hormones could be added according to needs) with a toothpick. Try to keep all the seeds on the same straight line for easy observation.

[0075] Arabidopsis thaliana culture method (for observing root phenotypes): After vernalizing the sown seeds for three days, place them vertically in a light incubator, ensuring that the roots do not enter the petri dish to affect later observations.

[0076] Detection example, histochemical analysis of GUS in transgenic Arabidopsis thaliana leaves under dark culture and light induction

[0077] Dark treatment: Place the transgenic Arabidopsis thaliana homozygous plants carrying the pbHLH93-GUS recombinant vector (i.e., the homozygous mutant T3 generation plants obtained in the application example) that have grown for about 4 weeks in a climate chamber at a temperature of 22°C and a light intensity set to 0 LUX for 8 hours. After 8 hours, take one leaf from each Arabidopsis thaliana plant and immerse it in an appropriate amount of GUS staining solution (1.0 mmol / L X-gluc, 50 mmol / L PBS (pH 7.0), 2 mmol / L EDTA, 0.12% Triton X-100, 20% methanol, 0.4 mmol / L potassium ferrocyanide, 0.4 mmol / L potassium ferricyanide), and place it at 37°C overnight until color development; at room temperature, decolorize with 75% alcohol and then observe and take pictures under a stereomicroscope.

[0078] Low light treatment: Place the transgenic Arabidopsis thaliana homozygous plants carrying the pbHLH93-GUS recombinant vector that have grown for about 4 weeks in a climate chamber at a temperature of 22°C and a light intensity set to 4000 LUX for 8 hours. After 8 hours, take one leaf from each Arabidopsis thaliana plant and immerse it in an appropriate amount of GUS staining solution (1.0 mmol / L X-gluc, 50 mmol / L PBS (pH 7.0), 2 mmol / L EDTA, 0.12% Triton X-100, 20% methanol, 0.4 mmol / L potassium ferrocyanide, 0.4 mmol / L potassium ferricyanide), and place it at 37°C overnight until color development; at room temperature, decolorize with 75% alcohol and then observe and take pictures under a stereomicroscope.

[0079] High light treatment: Place the transgenic Arabidopsis thaliana homozygous plants carrying the pbHLH93-GUS recombinant vector that have grown for about 4 weeks in a climate chamber at a temperature of 22°C and a light intensity set to 8000 LUX for 8 hours. After 8 hours, take one leaf from each Arabidopsis thaliana plant and immerse it in an appropriate amount of GUS staining solution (1.0 mmol / L X-gluc, 50 mmol / L PBS (pH 7.0), 2 mmol / L EDTA, 0.12% Triton X-100, 20% methanol, 0.4 mmol / L potassium ferrocyanide, 0.4 mmol / L potassium ferricyanide), and place it at 37°C overnight until color development; at room temperature, decolorize with 75% alcohol and then observe and take pictures under a stereomicroscope.

[0080] The results obtained are as Figure 5as shown

[0081] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0082] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

[0083] In addition, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A light-induced gene promoter, characterized in that, The nucleotide sequence of the light-inducible gene promoter is shown in SEQ ID No:

1.

2. The light-inducible gene promoter according to claim 1, wherein The light-inducible gene promoter is derived from Cabernet Sauvignon grapes.

3. A recombinant plasmid, characterized in that, The recombinant plasmid comprises an expression vector and the light-inducible gene promoter as claimed in claim 1 or 2.

4. The recombinant plasmid according to claim 3, characterized in that, The expression vector is selected from the LUC vector; and / or, the light-inducible gene promoter is obtained by amplifying the genomic DNA of Cabernet Sauvignon grapes using the primer pair shown in SEQ ID No: 4 and SEQ ID No:

5.

5. The recombinant plasmid according to claim 3, characterized in that, The expression vector is selected from the GUS vector; and / or, the light-inducible gene promoter is obtained by amplifying the genomic DNA of Cabernet Sauvignon grapes using the primer pair shown in SEQ ID No: 6 and SEQ ID No:

7.

6. The recombinant plasmid according to any one of claims 3-5, characterized in that, The recombinant plasmid is constructed by ligating the light-inducible gene promoter to the recombinant plasmid after double digestion with a ligase.

7. The recombinant plasmid according to claim 6, characterized in that, The double digestion process uses BsaI restriction endonuclease and Eco31I restriction endonuclease; and / or, the ligase is selected from Biorun 2*EasyClone Mix ligase.

8. A recombinant bacterium, characterized in that, The recombinant bacterium is obtained by transforming Agrobacterium with the recombinant plasmid as claimed in any one of claims 3-7.

9. Use of a recombinant bacterium as described in claim 8 in the response of Arabidopsis thaliana to light, characterized in that, The recombinant bacterium is obtained by transforming with a recombinant plasmid having an expression vector of GUS vector.

10. The application according to claim 9, wherein the application process comprises: Based on the floral dip method, Arabidopsis thaliana is cultured in a medium containing the recombinant bacterium, and a stably inherited Arabidopsis thaliana homozygous mutant is screened.

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