Cymbidium sinense auxin conjugated CsGH3.1 gene and application thereof

Abstract

The invention discloses an auxin conjugated CsGH3.1 gene of cymbidium sinense 'Jianxiai' and application of the auxin conjugated CsGH3.1 gene. The CsGH3.1 gene provided by the invention belongs to an acyl amide synthetase family GH3 gene family, the CsGH3.1 is transformed into arabidopsis thaliana by constructing an expression vector, and the biological increment of callus induced by a transgenic plant explant in the dark is reduced, which shows that the CsGH3.1 is beneficial to culture of small plant type plants and improvement of division propagation efficiency, and the CsGH3.1 gene has a good application prospect. The stable industrial development is promoted; meanwhile, the blooming time of a transgenic plant overexpressing CsGH3.1 is delayed, and the blooming period becomes late, which indicates that the CsGH3.1 gene can change the blooming period and delay the blooming period of the plant, can be used for improving the blooming period of the plant and solves the problem of flowering asynchronism of the plant, and has important significance on breeding of the plant in the blooming period.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a 'Qijian Baimo' orchid auxin conjugated CsGH3.1 gene and its application. Background Art

[0002] Cymbidium sinense is an important orchid native to Guangdong Province and a material carrier of Chinese traditional culture. It has a variety of varieties, beautiful plant shape, elegant leaves, elegant flower colors, pleasant fragrance, high ornamental value, and is the largest-selling Chinese orchid with broad market prospects. However, due to the difficulty of germination of Cymbidium sinense seeds and the low efficiency of tissue culture rapid propagation, Cymbidium sinense still relies on division for propagation, which seriously hinders the industrialization of Cymbidium sinense. Carrying out genetic improvement of the characteristics of tissue culture rapid propagation of Cymbidium sinense and cultivating new varieties of Cymbidium sinense that are easy to industrialize are of extremely important practical significance for promoting the industrialization of Cymbidium sinense.

[0003] Transgenic and gene editing breeding are the development direction of Chinese orchid breeding. Obtaining breeding target trait genes is the basis for carrying out transgenic breeding and gene editing breeding. At present, some genes of Molan have been cloned, but there is no research report on cloning Molan GH3 gene.

[0004] The acylamide synthetase family GH3 (Gretchen Hagen 3, GH3) is a class of negative regulators that respond to auxin early. It maintains hormone homeostasis in plants by catalyzing the binding of amino acids to plant hormones such as IAA, JA and SA. GH3 genes usually have multiple family members in plants, and three subfamilies have been identified. Among them, the GH3 members in subfamily II have the function of auxin conjugation, which can catalyze the binding of IAA with multiple amino acids to form amino acidized IAA. The auxin conjugated GH3 gene affects the growth and development characteristics of plants and changes the resistance of plants to biotic and abiotic stresses by regulating the concentration of endogenous active auxin in plants. Therefore, cloning the auxin conjugated GH3 gene is of great significance to promote the molecular breeding and industrial development of orchids and further carry out orchid evolution research. Summary of the invention

[0005] The present invention provides a 'Qijian Baimo' black orchid auxin-conjugated CsGH3.1 gene and an application thereof.

[0006] The first object of the present invention is to provide a 'Qijian Baimo' orchid auxin conjugated CsGH3.1 gene.

[0007] The second object of the present invention is to provide a protein encoded by the 'Qijian Baimo' blue orchid auxin conjugated CsGH3.1 gene.

[0008] The third object of the present invention is to provide the application of 'Qijian Baimo' blue orchid auxin conjugated with CsGH3.1 gene.

[0009] The fourth object of the present invention is to provide a recombinant vector and a recombinant bacterium comprising the recombinant vector.

[0010] A fifth object of the present invention is to provide a preparation for improving the flowering period of plants.

[0011] The sixth object of the present invention is to provide a method for constructing transgenic materials with delayed flowering period.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention cloned a gene with a cDNA full length of 2028bp in 'Qijian Baimo' Mo Lan for the first time. The gene belongs to the acylamide synthetase family GH3 gene family, which is the CsGH3.1 gene. Its cDNA sequence is shown in SEQ ID NO:1, wherein the 5'UTR is 57bp and the 3'UTR is 165bp; the CDS of CsGH3.1 is 1806bp, and its nucleotide sequence is shown in SEQ ID NO:2; the amino acid sequence of the encoded protein of the CsGH3.1 gene is 601aa in length, and its amino acid sequence is shown in SEQ ID NO:3.

[0014] The present invention clones an auxin conjugation gene GH3 from 'Qijian Baimo' Molan through RACE technology and PCR amplification method, constructs an expression vector, and transforms it into Arabidopsis thaliana. It is found that the biological increment of callus tissue induced by transgenic Arabidopsis root explants is reduced in the dark, indicating that CsGH3.1 can be used to change the proliferation of explants in the tissue culture process, that is, it can be used to reduce unnecessary tissue proliferation in tissue culture rapid propagation, which is beneficial to the cultivation of small plant type plants, improves the efficiency of ramet propagation, and promotes the stable development of the industry; at the same time, the stalking time of transgenic plants with overexpression of CsGH3.1 gene is delayed, and the flowering period becomes later, indicating that overexpression of Cs GH3.1 gene can change the flowering period, delay the flowering period of plants, can be used for plant flowering period improvement, and solve the problem of plant flowering period failure, which is of great significance for plant flowering period breeding, and lays a foundation for the application of 'Qijian Baimo' Molan Cs GH3.1 gene in molecular breeding.

[0015] Therefore, the present invention provides the use of blue orchid auxin conjugated CsGH3.1 gene in controlling the rapid propagation characteristics of plant tissue culture, regulating the flowering period of plants, improving breeding during the flowering period of plants, or preparing preparations for improving the flowering period of plants, or in constructing transgenic materials with delayed flowering period.

[0016] Furthermore, the control of the proliferation characteristics of plant tissue culture rapid propagation is to control the proliferation of unnecessary tissues in plant tissue culture rapid propagation, that is, to control the biological increment of callus tissue in plant tissue culture rapid propagation.

[0017] Preferably, the controlling of the biological increment of callus in plant tissue culture rapid propagation is to reduce the biological increment of callus. Reducing the biological increment is conducive to solving the problems of difficulty in plant differentiation and growth of tissue culture seedlings due to excessively fast proliferation.

[0018] Furthermore, the regulating the flowering period of plants is to delay the flowering period of plants.

[0019] Preferably, the plant is Cymbidium orchid.

[0020] The invention provides a recombinant vector comprising a Cymbidium orchid auxin conjugated to a CsGH3.1 gene.

[0021] The present invention provides a recombinant bacterium, comprising the above-mentioned recombinant vector.

[0022] The invention also provides a preparation for improving the flowering period of plants, comprising a reagent for overexpressing the CsGH3.1 gene.

[0023] The present invention also provides a method for constructing a transgenic material with delayed flowering period, wherein the Cymbidium orchid auxin conjugated CsGH3.1 gene is transformed into a plant material through a plant expression vector, thereby obtaining a transgenic material containing the Cymbidium orchid auxin conjugated CsGH3.1 gene.

[0024] Preferably, the plant expression vector is constructed first, then the plant expression vector is transformed into Agrobacterium, and then the plant is transformed by the Agrobacterium inflorescence infection method.

[0025] More preferably, the plant expression vector is formed by connecting the pCAMBIA1301 vector and the CsGH3.1 gene to form pCAMBIA1301-CsGH3.1, and the method for constructing the plant expression vector is: first synthesizing the cDNA sequence of the CsGH3.1 gene, then amplifying the coding region sequence to obtain the target sequence; finally connecting the target sequence to the vector to obtain the plant expression vector.

[0026] As a preferred embodiment of the above technical solution, the primers used to amplify the coding region sequence are:

[0027] CsGH3.1-OE-F (SEQ ID NO.12): 3'-acgggggactctagaggatccATGGCTGCGGGGAATTCG-5';

[0028] CsGH3.1-OE-R (SEQ ID NO. 13): 3'-cgatcggggaaattcgagctcCTAATCAAACCGGCTCGGACT-5'.

[0029] The present invention has the following beneficial effects:

[0030] The present invention clones an auxin conjugation gene GH3 from 'Qijian Baimo' Molan through RACE technology and PCR amplification method, transforms CsGH3.1 into Arabidopsis thaliana by constructing an expression vector, and finds that the biological increment of callus induced by transgenic Arabidopsis root explants is reduced in the dark, indicating that CsGH 3.1 can be used to change the proliferation of explants in the tissue culture process, that is, it can be used to reduce unnecessary tissue proliferation in tissue culture rapid propagation, which is beneficial to the cultivation of small plants, improves the efficiency of ramet propagation, and promotes the stable development of the industry; it shows that the 'Qijian Baimo' Molan CsGH3.1 gene can inhibit callus proliferation in the proliferation stage of tissue culture, which has broad application prospects in changing the proliferation characteristics in plant tissue culture and changing the growth trend of plants.

[0031] At the same time, the bolting time of transgenic plants overexpressing the CsGH3.1 gene was delayed and the flowering period became later, indicating that overexpressing the CsGH3.1 gene would change the flowering period and delay the flowering period of the plant. It can be used to improve the flowering period of plants and solve the problem of inconsistent flowering period of plants. It is of great significance for plant flowering period breeding and lays the foundation for the application of the CsGH3.1 gene in molecular breeding of 'Qijian Baimo' Molan. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 1% agarose gel electrophoresis diagram of the cDNA fragment of the CsGH3.1 gene of the present invention (A is the electrophoresis diagram of the 5'-RACE amplification product; B is the electrophoresis diagram of the 3'-RACE amplification product);

[0033] Figure 2 The 1% agarose gel electrophoresis diagram of the CsGH3.1 target gene amplification product containing the restriction site and homology arms of the pCAMBIA1301 vector of the present invention;

[0034] Figure 3 This is the vector map of the overexpression vector pCAMBIA1301 whose promoter of the target gene is 35SN;

[0035] Figure 4 This is a PCR positive verification image of Arabidopsis thaliana transfected with CsGH3.1 gene of the present invention;

[0036] Figure 5 The quantitative results of RT-qPCR of CsGH3.1 gene of Arabidopsis thaliana PCR positive plants of the present invention;

[0037] Figure 6 This is a comparison of callus proliferation of T3 generation Arabidopsis thaliana transgenic with CsGH3.1 gene and wild-type Arabidopsis thaliana root explants when they were propagated on hormone-free MS medium in the dark for 10 days;

[0038] Figure 7The figure is a comparison of the callus proliferation (A) and proliferation rate (B) of the T3 generation Arabidopsis thaliana transgenic with CsGH3.1 gene and wild-type Arabidopsis root explants induced callus on CIM medium for 5 days and transferred to MS medium without hormones in the dark for 10 days;

[0039] Figure 8 This is a comparison chart of the growth conditions of T3 generation Arabidopsis thaliana transgenic with CsGH3.1 gene of the present invention and wild-type Arabidopsis thaliana plants. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] The plant materials 'Qijian Baimo' Molan used in the following examples were all stored in the flower breeding laboratory of South China Agricultural University in the form of sterile tissue culture materials, the test tube seedlings were transplanted in the flower greenhouse of the teaching experimental base on the campus of South China Agricultural University, and the Arabidopsis thaliana was cultivated in the greenhouse on the campus of South China Agricultural University and managed daily in a conventional cultivation manner.

[0043] Example 1 Mining and cloning of CsGH3.1 gene

[0044] 1. Sequence mining and alignment

[0045] Three periods of rhizome proliferation of Cymbidium 'Xiaofeng' and Cymbidium 'Qijian Baimo' were selected to extract RNA and construct transcriptome sequencing data of adventitious root cDNA library. Through bioinformatics methods, a unigene sequence (Unigene0063066) that was differentially expressed in the proliferation periods of the two materials was found. This unigene sequence was searched in the NCBI database Blast, and it was found that it had the highest similarity with the GH3 genes of other species. It was speculated that it might be the GH3 gene of Cymbidium 'Qijian Baimo', and it was named CsGH3.1 gene.

[0046] 2. Total RNA extraction from rhizomes of 'Qijian Baimo' Cymbidium orchid during proliferation stage

[0047] Take 1 g of fresh rhizomes of 'Qijian Baimo' orchid in the proliferation stage, quickly grind them into powder in liquid nitrogen, use TakaRaMiniBEST Plant RNA Extraction Kit (TaKaRa Biotechnology Co., Ltd) kit according to the instructions to extract total RNA of 'Qijian Baimo' orchid, dissolve it in sterile ultrapure water, detect it by 1% agarose gel electrophoresis, and store it in a -80℃ ultra-low temperature refrigerator until use.

[0048] 3. Full-length cloning of CsGH3.1 gene

[0049] The amplification of the full length of the CsGH3.1 gene includes the amplification of the gene core sequence, the amplification of the 3' end sequence, and the amplification of the 5' end sequence. Among them, we designed the gene core primers and 3'RACE and 5'RACE primers based on the unigene conserved sequence. The primers are:

[0050] Primers for amplification of the gene core sequence:

[0051] CsGH3.1-F (SEQ ID NO.4): 3'-CATAACAGGCATAAGGAGACT-5';

[0052] CsGH3.1-R (SEQ ID NO.5): 3'-TGCTGCTTGGAGATGGAGGAGG-5';

[0053] 5' RACE primers:

[0054] CsGH3.1-G1 (SEQ ID NO.6): 3'-GATTACGCCAAGCTTGACACCAGCCGTCGTCTCAGCCAAC-5';

[0055] CsGH3.1-G2 (SEQ ID NO.7): 3'-GATTACGCCAAGCTTAAGGGGGGC ACATTGGCTTCAGATT-5';

[0056] 3' RACE primers:

[0057] CsGH3.1-G3 (SEQ ID NO.8): 3'-GATTACGCCAAGCTTCCCCCCTTC CGAAATCTCCTACACC-5';

[0058] CsGH3.1-G4 (SEQ ID NO.9): 3'-GATTACGCCAAGCTTCGGTTTACAGGCAGAGTAGGGGTGGCG-5';

[0059] The specific operation steps are as follows: The PCR products were subjected to agarose electrophoresis and recovered using a gel recovery kit. The recovered products were purified by pMD TM 18-T Vector was used for TA cloning, and the competent E. coli DH5α was transformed. The positive single clones were picked and sent to Guangzhou Qingke Biotechnology Co., Ltd. for Sanger double-end sequencing.

[0060] The results of agarose electrophoresis of PCR products were as follows Figure 1 As shown, in the 5'-RACE amplification product ( Figure 1 A) and 3'-RACE amplification products ( Figure 1 B) shows that the final CsGH3.1 gene core sequence was 1147 bp, 3' RACE sequencing obtained a sequence of 416 bp, and 5' RACE sequencing obtained a sequence of 465 bp.

[0061] The three partial sequences amplified were aligned and spliced ​​using BioEdit software, and finally a cDNA of the CsGH3.1 gene with a full length of 2028 bp was obtained, the specific sequence of which is shown in SEQ ID NO.1, wherein the 5'UTR is 57 bp and the 3'UTR is 165 bp; the CDS of its coding region is 1806 bp, the specific sequence of which is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is 601 aa in length, the specific sequence of which is shown in SEQ ID NO.3.

[0062] Example 2 Construction of Plant Expression Vector of CsGH3.1 Gene of 'Qijian Baimo' Orchid

[0063] 1. Synthesis of the first-strand cDNA

[0064] Total RNA was extracted from the rhizomes of the proliferating stage of 'Qijian Baimo' Molan, and the extraction method was the same as step 2 of Example 1; the synthesis of the first-strand cDNA of 'Qijian Baimo' Molan was carried out with the help of the FastKing RT Kit (With gDNase) (TIANGEN Biotech Co., Ltd) reverse transcription kit, and the reaction system was: Total R NA, 1 μg; 5×gDNA Buffer, 2.0 μL; RNase-Free ddH2O, Up to 10 μL. The reaction conditions were: incubate at 42°C for 3 minutes, and then place on ice. Then the following reaction solutions were added: 10×King RT Buffer, 2.0 μL; FastKing RT Enzyme Mix, 1.0 μL; FQ-RT Primer Mix, 2.0 μL; RNase-Free ddH2O, Up to 10 μL. After thorough mixing, the reaction was carried out according to the following procedure: incubate at 42°C for 15 min, incubate at 95°C for 3 min, and place on ice to obtain cDNA, which was stored at -80°C.

[0065] 2. Amplification of CsGH3.1 gene CDS sequence

[0066] Using the cDNA obtained from the rhizomes of 'Qijian Baimo' Molan as a template, specific primers were designed at the 5'UTR and 3'UTR of the CsGH3.1 gene for PCR amplification. The reaction conditions were: 95℃ 5min; 95℃ 30s, 58℃ 30s, 72℃ 1min, 35cycles; 72℃ 10min. The 1858bp target fragment containing the ORF of the CsGH3.1 gene of 'Qijian Baimo' Molan was obtained. TA cloning was performed to connect the target gene into pMD TM 18-T Vector, stored in E. coli as a plasmid.

[0067] The primer sequences are:

[0068] CsGH3.1-1F (SEQ ID NO.10): 3'-TTCCATTCCTCCCAAACTAATCCA CAG-5';

[0069] CsGH3.1-1R (SEQ ID NO.11): 3'-CCACTCCAACCCGGTCTAATCAA AC-5';

[0070] Design primers containing the coding region sequence and introduce restriction sites and protective bases at both ends of the primers. Perform PCR amplification from the plasmid containing the target gene. The test results are as follows: Figure 2As shown, the CsGH3.1 target gene amplification product containing the restriction site and homology arm of the pCAMBIA1301 vector is displayed; primers are then designed for vector connection, and the specific primers are as follows:

[0071] CsGH3.1-OE-F (SEQ ID NO.12): 3'-acgggggactctagaggatccATGGCTGCGGGGAATTCG-5';

[0072] CsGH3.1-OE-R (SEQ ID NO. 13): 3'-cgatcggggaaattcgagctcCTAATCAAACCGGCTCGGACT-5'.

[0073] 3. Connect the target gene to the vector

[0074] Select the target gene promoter as 35SN overexpression vector pCAMBIA1301 vector (such as Figure 3 As shown), and the vector was subjected to restriction digestion reaction. The reaction system was: 4 μL of pCAMBIA1301 vector, 2 μL of 10× buffer, 1 μL each of KpnI and SacI, 13 μL of ddH2O, a total of 20 μL. The reaction conditions were: 37°C, restriction digestion for 1 hour.

[0075] The target gene PCR product containing KpnI and SacI restriction sites was connected to the pCAMBIA1301 vector by homologous recombination using the One Step Cloning Kit from Vazyme. The reaction system was: 2 μL of pCAMBIA1301 vector, 2 μL of insert fragment, 5 μL of 2×ClonExpress Mix, 1 μL of ddH2O, a total of 10 μL. The reaction conditions were: 37°C for 30 min, and then immediately placed on ice for cooling.

[0076] 4. Transform the recombinant product into competent cells

[0077] The above recombinant product was transferred into the competent E. coli DH5α by heat shock method. The specific steps are as follows: 10 μL of the recombinant product was added to 100 μL of competent cells and placed on ice for 30 minutes. After heat shock in a 42°C water bath for 45 seconds, it was immediately placed on ice for 2-3 minutes; 900 μL of LB liquid culture medium was added, and the bacteria were shaken at 200 rpm and 37°C for 1 hour; the bacteria were collected by centrifugation at 5,000 rpm for 5 minutes, and 900 μL of supernatant was discarded. 100 μL of resuspended bacteria were gently spread on the plate containing Kan resistance; inverted and cultured overnight in a 37°C incubator.

[0078] 5. Positive identification of recombinant plasmid

[0079] Pick up single colonies grown on 5 plates, place them in 3-5 mL of liquid culture medium containing the corresponding antibiotics, and shake at 37°C and 220 rpm for 12 hours; extract E. coli plasmid DNA, and use it as a template for PCR amplification using primers containing the exogenous DNA insert. The detection primer sequences are:

[0080] P130135SN-F (SEQ ID NO.14): 3'-ACGGGGGACTCTAGAGGAT-5';

[0081] P130135SN-R (SEQ ID NO.15): 3'-CAGGAAACAGCTATGACCATGA-5';

[0082] The fragment size was then detected by gel electrophoresis, and the correctly detected plasmid vector was sent to the company for sequencing; the sequencing results were compared using Snap Gene software to check whether the target gene was inserted correctly. The correct band was 2127bp. 500μL of the successfully constructed pCAMBIA1301-CsGH3.1 expression vector was taken, 500μL of the bacterial solution was added and 500μL of sterile glycerol (concentration of 50%) was mixed and stored at -80℃.

[0083] Example 3 Agrobacterium-mediated transformation of Arabidopsis thaliana with the CsGH3.1 gene of 'Qijian Baimo' Molan and its identification

[0084] 1. Transformation of competent cells of Agrobacterium EHA105

[0085] Take 2.5 μL pCAMBIA1301-CsGH3.1 recombinant plasmid vector plasmid and add it to 100 μL Agrobacterium competent cells, place it on ice for 30 minutes, freeze it in liquid nitrogen for 5 minutes, and place it in a 37°C water bath for 5 minutes; add 300 μL liquid YEP medium, shake the bacteria at 220 rpm and 28°C for 4 hours; take 150 μL bacterial solution and evenly spread it on a plate containing Kan and Rif resistance, and invert and culture at 28°C for 2 days; pick a single colony and after the test is correct, culture it in a liquid medium containing Kan and Rif resistance at 28°C and 220 rpm until OD 600 The value is about 0.8, which is used for subsequent Agrobacterium infection, and 400 μL of bacterial solution is mixed with 50% sterile glycerol in equal volumes in a sterile centrifuge tube and stored at -80° C. Detection verification is performed, and the detection primers are the same as step 6 of Example 2.

[0086] 2. Arabidopsis planting

[0087] Wild-type Arabidopsis thaliana seeds (Columbia ), after disinfection, sow on MS plates, vernalize in the dark at 4℃ for 3 days, transfer to 20℃, 16h light conditions, culture for 10 days, transplant to the moist substrate (peat soil: vermiculite = 3:1) that has been watered in advance, cover with a layer of plastic wrap to keep it moist for 2-3 days, and inflorescence can be seen about 3-4 weeks after planting.

[0088] 3. Arabidopsis thaliana inflorescence dip genetic transformation

[0089] The inflorescence of the blooming Arabidopsis thaliana was infected with the Agrobacterium suspension solution detected correctly for 2-3 seconds, and then covered with plastic wrap, sealed and kept away from light for 24 hours. This operation was repeated 3 times in total, with an interval of 7 days each time. After infection, the Arabidopsis thaliana T0 generation seeds were harvested after culturing under normal conditions until maturity.

[0090] 4. Identification of Arabidopsis plants overexpressing CsGH3.1 in 'Qijian Baimo'

[0091] 4.1 Screening of resistant plants

[0092] After disinfection, Arabidopsis T0 seeds were sown on 1 / 2MS medium containing hygromycin resistance. After vernalization in a 4°C refrigerator for 3 days, they were moved to a light incubator at 22°C and 16 h of light conditions for culture. After about 2 weeks, the surviving Arabidopsis were selected and transplanted into a mixed nutrient soil of substrate and vermiculite for normal culture.

[0093] 4.2 Genomic PCR identification

[0094] Young leaves of resistant Arabidopsis plants were selected, DNA was extracted using the CTAB method, PCR was performed using P130135SN-F and P130135SN-R as primers, and PCR products were detected using 1.5% agarose. A total of 10 transgenic Arabidopsis plants were tested, and the results were as follows: Figure 4 As shown, the transgenic plants amplified a product fragment of the target length, the correct band was 2127 bp, and the positive rate detected was 100%.

[0095] 4.3 RT-qPCR identification

[0096] The above transgenic Arabidopsis thaliana with CsGH3.1 gene identified by genomic PCR was planted to T3 generation, and the leaf RNA of the transgenic Arabidopsis thaliana plants with positive PCR identification was extracted for RT-qPCR identification. The cDNA obtained by reverse transcription of the total RNA of the three transgenic Arabidopsis thaliana OE-1, OE-2, and OE-3 with CsGH3.1 gene was used as a template, and the primers CsGH3.1-qPCR-F (SEQ ID NO.16): 3'-GGCGGCGGAAG GAAGAGG-5'; and CsGH3.1-qPCR-R (SEQ ID NO.17): 3'-CGTATCTCAAGCGGTCCAATCG-5'; were used to detect the expression of the CsGH3.1 gene. The reaction conditions were: 95℃ 2min; 95℃ 20s, 60℃ 20s, 72℃ 20s, 35cycles; 72℃ 5min.

[0097] Actin(UBQ10)-F (SEQ ID NO.18): 3'-GGCCTTGTATAATCCC TGATGAA-5'; and Actin(UBQ10)-R (SEQ ID NO.19): 3'-AGAAGTTCG ACTTGTCATTAGAAAGAAA-5'; were used as internal reference genes to calculate the relative expression level of CsGH3.1.

[0098] The relative expression results of CsGH3.1 are shown in Figure 5 As shown, the expression levels of CsGH 3.1 in OE-1, OE-2, and OE-3 were significantly increased, indicating that CsGH3.1 was expressed to varying degrees in transgenic Arabidopsis.

[0099] Example 4 Phenotypic observation of transgenic Arabidopsis thaliana carrying the CsGH3.1 gene of 'Qijian Baimo' Molan

[0100] 1. Genetically modified materials

[0101] The Arabidopsis transgenic materials are positive T3 generation transgenic plants of CsGH3.1 Arabidopsis OE-1, positive T3 generation transgenic plants of CsGH3.1 Arabidopsis OE-2, and positive T3 generation transgenic plants of CsGH3.1 Arabidopsis OE-3.

[0102] 2. Investigation of proliferation capacity in tissue culture

[0103] Seeds of wild-type Arabidopsis and three T3 generation CsGH3.1 gene overexpression lines were sterilized and sown on 1 / 2MS medium, placed in a 4°C refrigerator for 2 days of vernalization, and then placed vertically on a culture dish on a culture rack and cultured at 26°C, 16h light / 8h dark. After 10 days of growth, roots of wild-type Arabidopsis and transgenic Arabidopsis seedlings with consistent growth were selected as explants, cut into about 1 cm, and placed on CIM medium (Gamborg's B-5 medium, 20.0g / L sucrose, 0.5g / L MES, 0.5mg / L 2,4-D, 0.05mg / L kinetin, 5g / L agar, PH 5.8), and cultured at 26°C in the dark for 5 days to induce callus. The induced callus was transferred to ordinary hormone-free 1 / 2MS culture medium plates. Each plate was divided into two areas, and 12 wild-type and transgenic Arabidopsis callus were inoculated respectively. Three replicates were inoculated for each strain, and five culture dishes were inoculated for each replicate. The culture was continued at 26°C in the dark for 10 days. The differences in the proliferation characteristics of transgenic Arabidopsis and wild-type Arabidopsis callus were observed, and the proliferation rate was calculated.

[0104] The callus proliferation of transgenic Arabidopsis and wild-type Arabidopsis Figure 6 As shown, after callus was induced from Arabidopsis root explants and transferred to hormone-free medium, the biomass increment of transgenic Arabidopsis callus was reduced compared with that of wild-type Arabidopsis.

[0105] The results are as follows Figure 7 As shown in A, the induction and proliferation of transgenic Arabidopsis callus were significantly inhibited compared with wild-type Arabidopsis callus. Figure 7 As shown in B, it shows that the biological increment of transgenic Arabidopsis callus is reduced. This trait plays an important role in changing the proliferation characteristics during tissue culture. Reducing the biological increment can prevent the difficulty of plant differentiation in Mo Lan tissue culture due to excessive proliferation rate, which is not conducive to the growth of tissue culture seedlings; the Mo Lan CsGH3.1 gene of 'Qijian Baimo' Mo Lan can effectively reduce unnecessary tissue proliferation in tissue culture rapid propagation, and plays an important role in cultivating small plants and promoting the stable development of the industry.

[0106] 3. Growth survey

[0107] Seeds of wild-type Arabidopsis and three T3 generation CsGH3.1 gene overexpression strains were disinfected and evenly sown on 1 / 2MS culture medium plates. Each plate was divided into two areas, and 70 wild-type and transgenic Arabidopsis seeds were sown respectively. Three replicates were sown for each strain, and five culture dishes were sown for each replicate. After vernalization in a 4°C refrigerator for 2 days, they were cultured at 26°C, 16 h light / 8 h dark conditions. After growing for 2 days, wild-type Arabidopsis and transgenic Arabidopsis seedlings with consistent growth were selected and transferred to ordinary 1 / 2MS culture medium. After the culture dishes were sealed with sealing film, the culture dishes were placed vertically on the culture rack and continued to be cultured at 26°C, 16 h light conditions for 10 days. The transgenic Arabidopsis and wild-type Arabidopsis grown on the culture medium for 10 days were transplanted to a mixed nutrient soil of substrate and vermiculite for normal culture, and the difference in growth between transgenic Arabidopsis and wild-type Arabidopsis plants was observed.

[0108] The results are as follows Figure 8 As shown, compared with the wild-type Arabidopsis, the transgenic Arabidopsis plants have delayed bolting time and later flowering period, indicating that the CsGH3.1 gene of 'Qijian Baimo' Molan can delay the flowering period of the plant and change the flowering period, which can be used to solve the problem of the flowering period of Molan. This trait is of great significance for plant type and flowering period breeding.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A 'Qijian Baimo' orchid auxin conjugated CsGH3.1 gene, characterized in that: The full-length cDNA sequence of the gene is shown in SEQ ID NO: 1, and the coding sequence of the gene is shown in SEQ ID NO:

2.

2. The protein encoded by the 'Qijian Baimo' blue orchid auxin conjugated CsGH3.1 gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

3.

3. Use of the Cymbidium orchidii auxin conjugated to the CsGH3.1 gene according to claim 1 in controlling the rapid propagation characteristics of plant tissue culture.

4. Use of the Cymbidium orchidii auxin conjugated to the CsGH3.1 gene according to claim 1 in regulating the flowering period of plants.

5. Use of the Cymbidium orchidii auxin conjugated to the CsGH3.1 gene according to claim 1 in improving plant flowering period breeding or in preparing a preparation for improving plant flowering period.

6. Use of the Cymbidium orchidii auxin conjugated to the CsGH3.1 gene according to claim 1 in constructing transgenic materials with delayed flowering period.

7. A recombinant vector, characterized in that The invention comprises the Cymbidium orchid auxin conjugated CsGH3.1 gene as described in claim 1.

8. A recombinant bacterium, characterized in that Comprising the recombinant vector according to claim 7.

9. A preparation for improving the flowering period of plants, characterized in that Contains a reagent for overexpressing the CsGH3.1 gene.

10. A method for constructing a transgenic material with delayed flowering, characterized in that: The Cymbidium orchid auxin conjugated CsGH3.1 gene described in claim 1 is transformed into plant materials through a plant expression vector, thereby obtaining a transgenic material containing the Cymbidium orchid auxin conjugated CsGH3.1 gene.

Images