IbGH3.4b gene for coding sweet potato auxin amide synthetase and application of IbGH3.4b gene

Abstract

The invention discloses an IbGH3.4b gene for encoding sweet potato auxin amide synthetase and application of the IbGH3.4b gene. The nucleotide sequence of the gene is shown as SEQ ID NO.1; the amino acid sequence of the protein is as shown in SEQ ID NO. 2; the invention also provides a related recombinant vector and a host cell. The invention further discloses application of any one of the IbGH3.4b gene for coding the sweet potato auxin amide synthetase, the protein coded by the IbGH3.4b gene, the recombinant vector and the host cell in regulation and control of plant growth and development and / or regulation and control of auxin anabolism. The acquisition of the gene for coding the sweet potato auxin amide synthetase IbGH3.4b provides a precious gene resource for molecular plant breeding, and meanwhile, the gene has important significance for analyzing the molecular mechanism of plant growth and development and auxin synthesis and metabolism. The gene can be applied to plant genetic improvement.

Description

Technical Field

[0001] The invention relates to an IbGH3.4b gene encoding sweet potato auxinamide synthase and application thereof, belonging to the field of genetic engineering. Background Art

[0002] Auxin is a hormone that plays a key role in the entire growth and development of plants. It participates in physiological processes such as apical dominance, root formation, stress adaptation and aging by inducing rapid and high expression of early response genes. Auxin early response genes refer to some genes that can be rapidly activated for transcription after auxin treatment and do not require new protein synthesis. They generally include three categories: Aux / IAAs, SAURs (Small auxin up RNAs) and GH3s (Gretchen Hagen 3) gene families, which play a very important role in the growth and development of plants. GH3s genes, encoding auxin amide synthase, are widely distributed in the plant kingdom and consist of three groups of adenylases: aryl and acyl CoA synthetases, firefly luciferase and the amino acid activation adenylation domain of non-ribosomal peptide synthetase (NRPS). GH3 is a cytoplasmic acid amide synthase of firefly luciferase, which can couple auxin, jasmonate and benzoic acid derivatives with various amino acids, thus playing an important role in plant growth and development and response to adversity. GH3 can regulate the homeostasis of auxin in plants. When the concentration of IAA is high, the free IAA growth hormone is linked to amino acids under the catalysis of auxin amide synthase to form IAA amino acid products, thereby reducing the concentration of free IAA; when the concentration of IAA is low, the amino acid products can be hydrolyzed by proteolytic enzymes to become active free IAA, but when IAA is combined with aspartic acid or glutamic acid, the bound IAA will be further oxidatively metabolized and no longer hydrolyzed to free IAA.

[0003] Sweet potato is not only the bottom line crop for national food security, but also an emerging nutritional health food in modern life. New functional sweet potato varieties with health-care effects have greatly changed the concept of traditional edible sweet potatoes, and have made a qualitative leap in the development and application of sweet potato functional foods. Sweet potatoes are widely grown in more than 100 countries in the world. my country is the world's largest producer, and sweet potato production accounts for about 80% of the world's sweet potatoes. Clarifying the growth and development regulation mechanism of sweet potatoes can better guide the cultivation of sweet potatoes. Cloning auxin synthesis and regulatory genes in sweet potatoes and studying their basic biological characteristics and functions can provide a theoretical basis for the entire plant growth and development gene regulatory network, and provide a certain material basis for improving crop stress resistance. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides an IbGH3.4b gene encoding sweet potato auxin amide synthase and its application. The heterologous expression of this gene can effectively affect the plant leaf morphology, plant type and plant auxin synthesis and metabolism, which is of great significance to molecular plant breeding.

[0005] In order to achieve the above object, the present invention adopts a gene IbGH3.4b encoding sweet potato auxinamide synthase, and its nucleotide sequence is shown in SEQ ID NO.1.

[0006] A protein, the protein is encoded by the IbGH3.4b gene encoding sweet potato auxinamide synthase, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0007] A recombinant vector comprises the IbGH3.4b gene encoding sweet potato auxinamide synthase.

[0008] A host cell comprises the IbGH3.4b gene encoding sweet potato auxinamide synthase or the recombinant vector.

[0009] Another object of the present invention is to disclose the use of any one of the IbGH3.4b gene encoding sweet potato auxinamide synthase, the protein encoded by it, the recombinant vector, and the host cell in regulating plant growth and development and / or regulating auxin anabolism;

[0010] Specifically, heterologous expression of the IbGH3.4b gene can affect plant growth and development; and / or the synthesis and metabolism of plant auxins.

[0011] Among them, the impact on plant growth and development involves plant leaf morphology and plant type, which is specifically manifested as: heterologous expression of the IbGH3.4b gene reduces plant leaf area and causes leaf curling; the impact on plant auxin synthesis and metabolism is specifically manifested as: heterologous expression of the IbGH3.4b gene reduces the content of free auxin in plants and increases the content of bound auxin.

[0012] Specifically, the TCP-type transcription factors that regulate the expression of the IbGH3.4b gene include IbTCP11 and IbTCP17.

[0013] Finally, the present invention also provides a plant cultivation method for changing leaf morphology, plant type and endogenous free auxin content, comprising: constructing a plant expression vector containing the IbGH3.4b gene; transforming plant cells with the constructed plant expression vector; and cultivating the transformed plant cells into transgenic plants.

[0014] The present invention isolates a complete cDNA of a GH3-type auxin early response gene IbGH3.4b from sweet potato, connects it to a plant expression vector, transforms Arabidopsis thaliana by Agrobacterium infection, and obtains transgenic plants; plant phenotypic results show that Arabidopsis thaliana transformed with a pCAMBIA1301-35S-IbGH3.4b vector has curled leaves, a significantly reduced leaf area compared with a control group (WT), and an increased content of bound auxin; and at the same time, the expression of the IbGH3.4b gene is regulated by TCP transcription factors IbTCP11 and IbTCP17.

[0015] The acquisition of the sweet potato auxinamide synthase IbGH3.4b gene of the present invention provides a precious gene resource for molecular plant breeding, and is of great significance for analyzing the molecular mechanism of plant growth and development and auxin synthesis and metabolism. This gene can be applied to plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a sequence alignment analysis diagram of IbGH3.4b gene;

[0017] Figure 2 The tissue expression pattern analysis diagram of the IbGH3.4b gene in Example 1; B is Bud; YL is youngleaf; L1 is the first mature leaf; ML is the mature leaf; ST is the stem; FR is the fiber root; PR is the pencil root; SR is the storage root;

[0018] Figure 3 The figure is a subcellular localization diagram of the IbGH3.4b gene in Example 2; in the figure, GFP driven by the 35S promoter is used as a control, and the left, left middle, right middle and right represent photos taken under dark, DAPI, bright and merged views, respectively; the fluorescence signal of the fusion of IbGH3.4b and GFP protein is located in the cell nucleus, which is consistent with the DAPI staining result, and there is also a fluorescence signal in the cytoplasm; indicating that IbGH3.4b is localized in the cell nucleus and cytoplasm;

[0019] Figure 4 Schematic diagram of the IbGH3.4b overexpression vector pCAMBIA1301-35S-IbGH3.4b in Example 3; in the figure, LB and RB are the left and right boundaries of T-DNA, Hyg is the hygromycin resistance gene, 35S-P is the 35S promoter, and NOS-T is the NOS terminator;

[0020] Figure 5This is a diagram for screening and identifying positive strains of Arabidopsis thaliana heterologously expressing IbGH3.4b in Example 3; in the figure, A is an agarose gel electrophoresis of PCR amplification products of Arabidopsis thaliana transgenic plants heterologously expressing IbGH3.4b; B is an analysis of the expression level of IbGH3.4b in Arabidopsis thaliana transgenic plants heterologously expressing IbGH3.4b;

[0021] Figure 6 It is a phenotypic observation diagram of the Arabidopsis positive strain heterologously expressing IbGH3.4b in Example 4; in the figure, A is a phenotypic photo of the Arabidopsis transgenic positive strain heterologously expressing IbGH3.4b; B is a phenotypic photo of the 4th leaf of the Arabidopsis transgenic positive strain heterologously expressing IbGH3.4b; C is a phenotypic photo of the plant height of the Arabidopsis transgenic positive strain heterologously expressing IbGH3.4b; D is a comparison of leaf areas of the Arabidopsis transgenic positive strain heterologously expressing IbGH3.4b; E is a comparison of plant heights of the Arabidopsis transgenic positive strain heterologously expressing IbGH3.4b;

[0022] Figure 7 The auxin content detection of Arabidopsis positive strains heterologously expressing IbGH3.4b in Example 4; in the figure, A is a comparison of the endogenous free indoleacetic acid content of Arabidopsis transgenic positive strains heterologously expressing IbGH3.4b; B is a comparison of the endogenous free indolepropionic acid content of Arabidopsis transgenic positive strains heterologously expressing IbGH3.4b; C is a comparison of the endogenous bound auxin indoleacetic acid-aspartic acid content of Arabidopsis transgenic positive strains heterologously expressing IbGH3.4b;

[0023] Figure 8 This is a diagram of the interaction between the promoter of the IbGH3.4b gene and the sweet potato IbTCP11 / 17 transcription factor verified by the yeast one-hybrid experiment in Example 5; in the figure, 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 They represent different dilution multiples respectively, and the leftmost side shows the names of different reporter experiment vectors for co-transfection. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0026] Based on the previous analysis of the whole genome of sweet potato in our laboratory, the amino acid sequences of all Arabidopsis AtGH3s, rice OsGH3s and closely related wild-type sweet potato ItGH3s were aligned in the sweet potato hexaploid database (http: / / public-genomes-ngs.molgen.mpg.De / SweetPotato) using Blastp.

[0027] Eight IbGH3 genes were identified and cloned based on the results of the conserved domain analysis (https: / / www.ncbi.nlm.nih.Gov / Structure / cdd / wrpsb.cgi, CDD). The chromosomal distribution of IbGH3s was mapped using MapGene2Chrom web v2, and they were named according to their order on the chromosome. Two IbGH3 genes were located at the same position on chromosome 13, and we named them IbGH3.4a and IbGH3.4b. This invention only focuses on the IbGH3.4b gene.

[0028] Example 1

[0029] The cloning and tissue expression pattern analysis of the sweet potato IbGH3.4b gene are as follows:

[0030] 1) The fresh leaves of "Xushu 22" were selected as materials to extract total RNA, and reverse transcribed to synthesize cDNA. The cDNA was used as a template and primers were designed using the IbGH3.4b sequence to amplify the complete gene coding region;

[0031] 2) The specific PCR reaction adopts a 50 μl system: Max Super-Fidelity DNA Polymerase (Vazyme, China) 1μl, 2*PhantaMaxbuffer 25μl, dNTP Mix (10mM each) 1μl;

[0032] 1 μl of upstream primer IbGH3.4b-F (5′-ATGCCTGAAGCACCAAAGAGTCTC-3′), 10 μM of downstream primer IbGH3.4b-R (5′-TCAGAGACGCGGCCCTGC-3′), 1 μl of cDNA, and 20 μl of ddH2O;

[0033] The amplification program is: 95℃5min; 95℃30s, 60℃30s, 72℃1min, 35 cycles in total; 72℃ extension for 10min. Take 2μl of PCR product, detect by 1% agarose gel electrophoresis, use gel recovery kit (TaKaRaMiniBESTAgarose Gel DNAExtraction Kit Ver.4.0) to cut the gel to recover the above PCR product, use the ligase in TaKaRaDNALigation Kit Ver.2.1, connect with T-vector (T-VectorpMDTM 20), heat transform into Escherichia coli competent cells DH5α, spread on the plate, and culture at 37℃ overnight; select positive colonies for bacterial cultivation and send to Shanghai Bioengineering Technology Co., Ltd. for sequencing to obtain the full-length CDS sequence:

[0034]

[0035] As shown in SEQ ID NO.2, the IbGH3.4b gene encodes a protein of 535 amino acids, specifically:

[0036] 3) The amino acid sequence shown in SEQ ID NO.2 was compared with the NCBI protein data (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to obtain plant species homologous genes with similar protein sequences to IbGH3.4b. Multiple sequence alignment was performed using DNAMAN software. The results are shown in Figure 1 As shown, IbGH3.4b has two conserved domains typical of GH3 genes, β-turn-β and P-Loop;

[0037] 4) RNA was extracted from eight different tissues of sweet potato (including buds, young leaves, first mature leaves, mature leaves, stems, fibrous roots, pencil roots, and storage roots) using RNAprep Pure Plant Plus Kit (TIANGEN, China), and its quality was verified by agarose gel electrophoresis, and its concentration was detected using an ultramicrospectrophotometer;

[0038] 5) The RNA that meets the quality and concentration standards is reverse transcribed into cDNA using the HiScript IIQ Select RT SuperMix (Vazyme, China) kit;

[0039] 6) The instrument used for real-time fluorescence quantitative PCR was CFX Connect (BIO-RAD, USA), and the cDNA of each tissue formed by reverse transcription was used as the detection template, and IbActin was used as the internal reference gene, IbActin-F (5′-CTGGTGTTATGGTTGGGATGG-3′), IbActin-R (5′-GGGGTGCCTCGGTAAGAAG-3′);

[0040] The qRT-PCR reaction system was as follows: 2×SYBR Premix Ex Taq II, 5 μl; 0.5 μl each of IbGH3.4b-qF (5′-CGTCAAATTTGCGCCCATTG-3′) and IbGH3.4b-qR (5′-GTTGCTCCATTGCTTTTGGC-3′); 3 μl of template cDNA; 1 μl of ddH2O; the final total system was 10 μl;

[0041] The qRT-PCR reaction program was designed as follows:

[0042] ① Pre-denaturation temperature is 95℃, time is 30s; ② Denaturation temperature is 95℃, time is 10s; ③ Annealing temperature is 65℃, time is 30s; ②-③ cycle number is 40c.

[0043] Adoption 2 -ΔΔCT The data were processed by the method. During the whole experiment, each gene contained three biological replicates and three technical replicates. Figure 2 As shown, IbGH3.4b was highly expressed in mature leaves (ML), stems (ST) and fibrous roots (FR) of sweet potato, but was less expressed in young leaves (YL) and storage roots (SR).

[0044] Example 2

[0045] The subcellular localization analysis of IbGH3.4b is as follows:

[0046] 1) Specific restriction enzyme cleavage sites were selected, and specific primers 35S::IbGH3.4b-GFP-F (5′-ggacagggtacccggggatccATGCCTGAAGCACCAAAGAGTC-3′) and 35S::IbGH3.4b-GFP-R (5′-caccatggtactagtgtcgacTCAATCCTGTTTGTTGCTCCATT-3′) were designed to construct the IbGH3.4b subcellular localization vector, and the IbGH3.4b gene sequence with BamHI and SalⅠ restriction enzyme cleavage sites was amplified, and the recovered and purified target sequence was inserted into the 35S-pCAMBIA2300-GFP subcellular localization vector; finally, the constructed 35S::GFP-IbGH3.4b vector was double-digested and sequenced for verification;

[0047] 2) The successfully constructed 35S::GFP-IbGH3.4b plasmid was transformed into Agrobacterium GV3101, OD 600 Adjust to about 0.8. Inject the lower surface of tobacco leaves about 4 weeks old, culture in the dark for 12 hours and then culture normally for 2-3 days, tear off the leaf epidermis of the infected part, stain with DAPI in dark for 2 minutes, rinse with ddH2O 3 times for preparation, observe under the DAPI and GFP channels of fluorescence microscope, according to Figure 3 The results showed that IbGH3.4b protein was localized in the cytoplasm and nucleus.

[0048] Example 3

[0049] The construction of IbGH3.4b overexpression vector and genetic transformation of Arabidopsis thaliana are as follows:

[0050] 1) Select specific restriction enzyme cleavage sites, design and construct specific primers GH3.4bOE-F (5′-gacgataagggatcctctagaATGCCTGAAGCACCAAAGAGTC-3′) and GH3.4bOE-R (5′-tctaagcttgcatgcctgcagTCAATCCTGTTTGTTGCTCCATT-3′) for overexpression vector of IbGH3.4b, amplify the IbGH3.4b gene sequence with Xba I and Pst I restriction enzyme cleavage sites, insert the recovered and purified target sequence into p1305-35S vector, and finally perform double restriction enzyme digestion and sequencing verification on the constructed vector 35S::IbGH3.4b, as shown in FIG. Figure 4 As shown;

[0051] 2) Genetic transformation of Arabidopsis thaliana

[0052] ① First, place a small amount of Arabidopsis seeds (Col) in a 1.5ml centrifuge tube; add 1ml 70% anhydrous ethanol, let stand for 1min, and absorb the anhydrous ethanol; add 1ml 2% sodium hypochlorite (containing 0.1% Trition 100), let stand for 8min, and absorb the sodium hypochlorite; rinse with sterile water 5 times, then suspend the seeds with sterile water, apply them on 1 / 2MS solid culture medium (without hygromycin), and vernalize for 3 days at 4℃ dark treatment; place in a light incubator at 23℃, culture for about 1 week, and transplant into soil with a ratio of soil, vermiculite and perlite of 8:1:1 and a water content of 70%;

[0053] ② When the plant is about 3 weeks old, the top of the main stem can be cut off to inhibit the growth of the top. When the plant is about 4 weeks old, the side branches will bloom in large numbers and can be used for transgenic by infiltration. The activated 35S::IbGH3.4b Agrobacterium culture was centrifuged at 4000 rpm for 10 min, collected at the bottom of the centrifuge tube, and diluted with infiltration solution (containing 1 / 2MS liquid culture medium, 5% sucrose solution, and 0.02% Silwet L-77) to OD 600 The inflorescence of Arabidopsis thaliana was soaked in the transformation solution for 60 seconds, taken out and kept away from light for 48 hours, and then placed in a 23°C light incubator for normal culture. After the seeds matured, they were collected in paper bags and dried. These seeds were the T1 generation.

[0054] ③ Conventional planting and hygromycin resistance screening were performed to obtain homozygous strains in the T3 generation.

[0055] Example 4

[0056] The transgenic positive plants were obtained, phenotypes were observed, and auxin content was detected as follows:

[0057] 1) Take leaves of T3 transgenic positive homozygous plants and extract total DNA using TPS extraction method;

[0058] The total DNA of wild-type Arabidopsis leaves was used as a negative control, and HYG-F (5′-GCTTCTGCGGGCGATTTGTGT-3′) and HYG-R (5′-GGTCGCGGAGGCTATGGATGC-3′) were used as primers for PCR amplification. Figure 5 A shows that Arabidopsis transgenic lines 1-10 have obvious bands at around 500 bp like the positive control, while the negative control with H2O as the template has no band at around 500 bp. It is preliminarily determined that Arabidopsis transgenic lines 1-10 are transgenic lines, and they are named IbGH3.4bOE-1 to IbGH3.4bOE-10 respectively;

[0059] 2) Take the leaves of the T3 transgenic positive homozygous plants, extract the total RNA and reverse transcribe it into cDNA. The wild-type Arabidopsis leaf cDNA was used as a negative control, and IbGH3.4b-qF (5′-CGTCAAATTTGCGCCCATTG-3′) and IbGH3.4b-qR (5′-GTTGCTCCATTGCTTTTGGC-3′) were used as primers for real-time fluorescence quantitative PCR analysis. Three technical replicates were set for each sample, and 2 -ΔΔCt The results are shown in Figure 5 As shown in Figure 2B, there was no significant difference in the expression levels of the transgenic lines IbGH3.4bOE-4 and IbGH3.4bOE-8 compared with the wild type, while the expression levels of IbGH3.4b in the remaining eight lines (IbGH3.4bOE-1, IbGH3.4bOE-2, IbGH3.4bOE-3, IbGH3.4bOE-5, IbGH3.4bOE-6, IbGH3.4bOE-7, IbGH3.4bOE-9 and IbGH3.4bOE-10) were significantly increased compared with the wild type, further verifying that these eight transgenic lines were indeed positive lines for overexpression of IbGH3.4b. We selected four strains with higher expression levels, IbGH3.4bOE-2, IbGH3.4bOE-6, IbGH3.4bOE-7, and IbGH3.4bOE-10, for subsequent experiments.

[0060] 3) Phenotypic observation of IbGH3.4b overexpression transgenic positive plants

[0061] Compared with the wild type, the leaves of the IbGH3.4b overexpressing Arabidopsis transgenic lines were curled and the leaf area was reduced ( Figure 6 A, B, D), plant height reduction ( Figure 6 C, E), while the number of leaves per plant did not change significantly, indicating that overexpression of IbGH3.4b affected the growth and development of plants.

[0062] 4) Detection of auxin content in Arabidopsis transgenic positive lines expressing IbGH3.4b

[0063] 4-1 Sample pretreatment

[0064] 4-1-1 Solid Sample

[0065] (1) Taking out cryopreserved stem and leaf samples of Arabidopsis thaliana transgenic positive for heterologous expression of IbGH3.4b;

[0066] (2) Weigh 50 mg of sample, add 10 μL of 100 ng / mL internal standard mixed solution and 1 mL of methanol / water / formic acid (15:4:1, v / v / v) extractant, and mix well;

[0067] (3) Vortex for 10 min, centrifuge for 5 min at 4°C and 12,000 rpm, and transfer the supernatant to a new centrifuge tube for concentration;

[0068] (4) After concentration, the sample was reconstituted with 100 μL of 80% methanol / water solution, filtered through a 0.22 μm filter membrane, and placed in an injection vial for LC-MS / MS analysis;

[0069] 4-2 Chromatographic mass spectrometry acquisition conditions

[0070] The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) (ExionLC TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (Tandem Mass Spectrometry, MS / MS) ( 6500+, https: / / sciex.com.cn / );

[0071] The liquid phase conditions mainly include:

[0072] ①Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm id);

[0073] ② Mobile phase: Phase A, ultrapure water (add 0.04% acetic acid); Phase B, acetonitrile (add 0.04% acetic acid);

[0074] ③ Gradient elution program: 0 min A / B is 95:5 (V / V), 1.0 min A / B is 95:5 (V / V), 8.0 min is 5:95 (V / V), 9.0 min is 5:95 (V / V), 9.1 min is 95:5 (V / V), 12.0 min is 95:5 (V / V);

[0075] ④Flow rate 0.35mL / min; column temperature 40℃; injection volume 2μL;

[0076] Mass spectrometry conditions mainly include:

[0077] The temperature of the electrospray ionization source (ESI) was 550°C, the mass spectrometer voltage was 5500 V in positive ion mode, -4500 V in negative ion mode, and the curtain gas (CUR) was 35 psi. In the Q-Trap 6500+, each ion pair was scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0078] 4-3 Standard Curve

[0079] Prepare standard solutions of different concentrations of 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL, and obtain the mass spectrometry peak intensity data of the corresponding quantitative signal of the standard of each concentration; draw standard curves for different substances with the external standard to internal standard concentration ratio (Concentration Ratio) as the abscissa and the external standard to internal standard peak area ratio (Area Ratio) as the ordinate.

[0080] 4-4 Quantitative analysis

[0081] Quantification is completed by using the multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry. In the MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, and excludes the ions corresponding to other molecular weight substances to preliminarily eliminate interference; the precursor ions are induced by the collision chamber and then broken to form multiple fragment ions, and the fragment ions are then filtered through the triple quadrupole to select the required characteristic fragment ions, eliminating non-target ion interference, making quantification more accurate and repeatable. After obtaining the mass spectrometry analysis data of different samples, the chromatographic peaks of all target substances are integrated and quantitative analysis is performed using the standard curve.

[0082] The results showed that the content of free auxin (IAA and IPA) in the Arabidopsis transgenic positive lines expressing IbGH3.4b was significantly lower than that in the wild type ( Figure 7 A, B), while the bound auxin indoleacetic acid-aspartic acid (IAA-Asp) was significantly higher than that of the wild type ( Figure 7 C), the results show that overexpression of IbGH3.4b increases the content of bound auxin.

[0083] Example 5

[0084] Yeast one-hybrid experiments confirmed that IbGH3.4b is regulated by IbTCP11 / 17, as follows:

[0085] 1) Select specific restriction enzyme sites and design specific primers for constructing the yeast single hybrid vector of IbTCP11 / 17: pGADT7-AD-TCP17-BamHI (5′-gtgggcatcgatacgggatccATGGAGGTTGAAAACCCAAGAA-3′) and pGADT7-AD-TCP17-SacI (5′-attcatctgcagctcgagctcTCAGGTTTTTCCTTTGCCGTT-3′), pGADT7-AD-TCP11-BamHI (5′-gtgggcatcgatacgggatccATGGAGGTTGAAAACCCAAGAA-3′) and pGADT7-AD-TCP17-SacI (5′-attcatctgcagctcgagctcTCAGGTTTTTCCTTTGCCGTT-3′). ′-gtgggcatcgatacgggatccATGGGGGAGAATCGTCAGCA-3′) and pGADT7-AD-TCP11-SacI (5′-attcatctgcagctcgagctcTCAACGACGGGAGTCAGAGG-3′), amplify the IbTCP11 / 17 gene sequence with BamHI and SacI restriction sites, insert the recovered and purified target sequence into the pGAD7 vector, and finally perform double restriction digestion and sequencing verification on the constructed pGADT7-IbTCP11 / 17 vector;

[0086] 2) Select specific restriction enzyme sites and design specific primers for constructing the yeast single hybrid vector of IbGH3.4b, pAbAi-GH3.4b-Hind-F

[0087] (5′-TGATGAATTGAAAAGCTTGACAATGGTAGAAGGATGACCTTTAC-3′) and pAbAi-GH3.4b-Xhol-R

[0088] (5′-CAGAGCACATGCCTCGAGGGTTTTTTTAGCGCTTTTACAGC-3′), amplify the IbGH3.4b gene promoter sequence with HindIII and Xhol restriction sites, insert the recovered and purified target sequence into the pAbAi vector, and finally perform double restriction digestion and sequencing verification on the constructed pAbAi-IbGH3.4b vector;

[0089] 3) pGADT7-IbTCP11 / 17, pGADT7 (negative plasmid), pGADT7-53 (positive plasmid) and linearized pAbAi-53 (positive plasmid), pAbAi (negative plasmid), and pAbAi-IbGH3.4b were co-transfected into yeast. Only when the promoter of IbGH3.4b can be activated by IbTCP11 / 17 can the yeast strain have Aba resistance and can grow normally in Aba medium. The results are shown in Figure 8 As shown, the promoter of IbGH3.4b can be bound by IbTCP11 / 17.

[0090] The present invention isolates a complete cDNA of a GH3-type auxin early response gene IbGH3.4b from sweet potato, connects it to a plant expression vector, transforms Arabidopsis thaliana by Agrobacterium infection, and obtains transgenic plants. Plant phenotypic results show that Arabidopsis thaliana transformed with pCAMBIA1301-35S-IbGH3.4b vector has curled leaves, a significantly reduced leaf area compared with a control group (WT), and an increased content of bound auxin. At the same time, the expression of the IbGH3.4b gene is regulated by TCP transcription factors IbTCP11 and IbTCP17. The acquisition of the auxin early response gene IbGH3.4b in the present invention provides a precious gene resource for molecular plant breeding, and is of great significance for analyzing the molecular mechanism of plant growth and development and auxin synthesis and metabolism. This gene can be applied to plant genetic improvement.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A IbGH3.4b gene encoding sweet potato auxinamide synthase, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein, characterized in that The protein is encoded by the IbGH3.4b gene encoding the sweet potato auxinamide synthase according to claim 1, and the amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: It comprises the IbGH3.4b gene encoding the sweet potato auxinamide synthase as described in claim 1.

4. A host cell, characterized in that It comprises the IbGH3.4b gene encoding sweet potato auxinamide synthase as described in claim 1 or the recombinant vector as described in claim 3.

5. Use of any one of the IbGH3.4b gene encoding sweet potato auxinamide synthase according to claim 1, the protein according to claim 2, the recombinant vector according to claim 3, and the host cell according to claim 4 in regulating plant growth and development and / or regulating auxin anabolism.

6. The use according to claim 5, characterized in that: The regulating plant growth and development includes regulating plant leaf curling and leaf area.

7. The use according to claim 5, characterized in that: The regulating auxin anabolism includes regulating the content of auxin.

8. The use according to claim 7, characterized in that: The TCP-type transcription factors that regulate the expression of the IbGH3.4b gene include IbTCP11 and IbTCP17.

9. A plant cultivation method for changing leaf morphology, plant type and auxin content, characterized in that: include: Constructing a plant expression vector containing the IbGH3.4b gene of claim 1; transforming plant cells with the constructed plant expression vector; The transformed plant cells are grown into transgenic plants.

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