Hybrid taxodium LhTRX-h3 gene, expression protein and application thereof

Abstract

The application discloses a hybrid Liriodendron chinense LhTRX-h3 gene, an expression protein thereof and application, relates to the technical field of plant genetic engineering.The hybrid Liriodendron chinense LhTRX-h3 gene disclosed by the application has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2.The application constructs an expression vector of the hybrid Liriodendron chinense LhTRX-h3 gene, transforms the expression vector of the hybrid Liriodendron chinense LhTRX-h3 gene into hybrid Liriodendron chinense, and cultivates, screens and obtains a transgenic hybrid Liriodendron chinense plant with improved non-biological stress resistance.The hybrid Liriodendron chinense LhTRX-h3 gene disclosed by the application can promote the plant to improve non-biological stress resistance, regulate plant growth and regulate the length-width ratio of plant leaves.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and more particularly relates to a hybrid tulip tree LhTRX-h3 gene, an expression protein thereof and application thereof. BACKGROUND

[0002] Liriodendron is a Magnoliaceae plant belonging to the third interglacial species. At present, the genus only has a pair of sister species of Liriodendron chinense and Liriodendron tulipifera. The former is naturally distributed in China and northern Vietnam, while the latter is naturally distributed in the eastern United States to southern Canada, showing a typical East Asia-Eastern North America intercontinental disjunct distribution. The earliest research hybridizes Liriodendron chinense and Liriodendron tulipifera to obtain a hybrid tulip tree with significant heterosis, which retains the phenotype of the parent and also shows stronger growth adaptability, the leaf type has the phenotype of the parent, the flower is beautiful and the flowering period is long, and it is an excellent landscape, road greening and fast-growing timber tree species. Later, a large-scale industrialized propagation of hybrid tulip tree was realized by using somatic embryogenesis technology, but due to the limitation of natural geographical distribution range, different clones show different adaptability to adversity such as high temperature, water stress and drought, which brings certain difficulties to the large-area popularization and application of hybrid tulip tree. Therefore, it is very important to explore the stress resistance of Liriodendron.

[0003] Drought, as one of the important factors affecting crop growth and development, gene expression, distribution and yield and quality, seriously limits the large-area expansion of crops. The adaptability of plants to drought is not only related to the intensity and speed of drought, but also more affected by its own genes. Within a certain drought threshold stress range, many plants can express related drought-resistant genes, and a series of physiological, biochemical and morphological changes occur, thereby showing the comprehensive traits of drought resistance. Therefore, starting from the plant itself, in-depth study of the drought resistance mechanism of plants, revealing the drought resistance characteristics, and improving the drought resistance and drought tolerance of plant varieties, can reduce the water consumption of crop cultivation, maximize the yield and quality of crops, and scientifically select excellent crop varieties suitable for planting in large areas of drought and semi-arid areas, which has become a hot issue of special concern and research by domestic and foreign experts and scholars, and has important significance for the rational use of water resources and the improvement of ecological environment.

[0004] Thioredoxin (TRXs) widely exist in plants, bacteria, yeast and animals, are small proteins (12-14 kDa) existing in every organelle, are first discovered in bacteria, have a classic redox active site Trp-Cys-Gly-Pro-Cys (WC(G / P)PC), have a conserved tertiary structure, and modify target proteins through post-translational disulfide bonds. Thioredoxin plays an important function in plant stress response, including antioxidant effect, participation in signal transduction pathway, protection of protein structure and function, and regulation of metabolic pathways. Its function helps plants to maintain survival and growth under stress conditions, and improves their ability to adapt to environmental changes.

[0005] TRXh is the most numerous member in the family, and its function is also more complex. TRXh plays a role in maintaining the reserve decomposition of the early growth of germination period cereal seed seedlings. The known thioredoxin h target protein in the seed is the glutelin and gliadin storage protein in barley and wheat, which are insoluble in disulfide bonds during the maturation and drying process. At germination, these proteins are reduced to the hydroxyl state. Analysis of wheat TRXh expression shows that it is involved in the transfer of compounds from plants to developing seeds. In addition, TRXh responds to various environmental signals, including drought, salt, high temperature, low temperature, heavy metals, light, diseases, etc., and plays an important role in improving plant tolerance under stress conditions. Studies have shown that in Arabidopsis under cold conditions, CBF and TRXh2 jointly regulate the level of active oxygen in the body, reducing the damage of abiotic stress to plants. Studies on rice TRXh1 found that overexpression of OsTRXh1 plants produced less hydrogen peroxide under salt stress, and knockout of OsTRXh1 had the opposite effect, which was consistent with the results of alfalfa, fully illustrating that the TRXh subclass plays an important role in oxidative stress response under abiotic stress.

[0006] As an important part of the redox system, thioredoxin is involved in the regulation of cellular redox state and plays a role in various stress processes. However, the response of TRXs to drought stress in Schima superba is not clear. SUMMARY

[0007] In view of the above problems existing in the prior art, the technical problems to be solved by the present application are to provide a hybrid Schima superba LhTRX-h3 gene. Another technical problem to be solved by the present application is to provide an expression protein of the hybrid Schima superba LhTRX-h3 gene. The present application also solves the technical problem of providing an application of the hybrid Schima superba LhTRX-h3 gene for obtaining a new plant germplasm with improved abiotic stress tolerance.

[0008] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0009] A hybrid Liriodendron tulipifera LhTRX-h3 gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.

[0010] An expression protein of the hybrid Liriodendron tulipifera LhTRX-h3 gene, the amino acid sequence of which is shown as SEQ ID NO. 2.

[0011] A vector, a recombinant bacterium or a host cell containing the hybrid Liriodendron tulipifera LhTRX-h3 gene.

[0012] Application of the hybrid Liriodendron tulipifera LhTRX-h3 gene in promoting plants to improve the ability to resist abiotic stress.

[0013] The improved ability to resist abiotic stress is to reduce the content of active oxygen in the plant body.

[0014] The application of the hybrid Liriodendron tulipifera LhTRX-h3 gene in promoting plants to improve the ability to resist abiotic stress, comprising:

[0015] 1) Constructing an expression vector of the hybrid Liriodendron tulipifera LhTRX-h3 gene;

[0016] 2) Transforming the constructed expression vector of the hybrid Liriodendron tulipifera LhTRX-h3 gene into the hybrid Liriodendron tulipifera;

[0017] 3) Cultivating, screening and obtaining a transgenic hybrid Liriodendron tulipifera plant with improved ability to resist abiotic stress.

[0018] Application of the hybrid Liriodendron tulipifera LhTRX-h3 gene in regulating plant growth.

[0019] The regulation of plant growth is to reduce the plant height, reduce the petiole length, reduce the main root length, reduce the leaf area, reduce the chlorophyll content and make the leaf color lighter.

[0020] Application of the hybrid Liriodendron tulipifera LhTRX-h3 gene in regulating the stomatal conductance of plant leaves.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] 1) The hybrid Liriodendron tulipifera LhTRX-h3 gene disclosed for the first time in the application, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the amino acid sequence of which is shown as SEQ ID NO. 2. The application constructs an expression vector of the hybrid Liriodendron tulipifera LhTRX-h3 gene, transforms the constructed expression vector of the hybrid Liriodendron tulipifera LhTRX-h3 gene into the hybrid Liriodendron tulipifera, and cultivates, screens and obtains a transgenic hybrid Liriodendron tulipifera plant with improved ability to resist abiotic stress.

[0023] 2) The plant height, petiole length, main root length, leaf area, and chlorophyll content of the LhTRX-h3-OE strain of the application are significantly reduced, the leaf color is lighter, and the whole plant is dwarfed; and the LhTRX-h3-KO plant has no obvious difference from the wild type, but there is no obvious difference in the number of leaves between the overexpression and knockout. The results show that LhTRX-h3 plays a function in plant growth and development, resulting in a significant decrease in the plant growth phenotype of the overexpression strain, but the knockout strain does not show a significant increase, which may be due to the function of other genes in the LhTRX-h3 gene family, resulting in gene redundancy.

[0024] 3) After 5 days of stress, different strains of the transgenic strain constructed in the application all show wilting, yellowing and shedding of leaves, but compared with the wild type, the LhTRX-h3-OE is in good condition, the wilting degree is lower and there is no leaf shedding, and the LhTRX-h3-KO positive plant has yellowing and shedding of leaves.

[0025] 4) The peroxide content of the transgenic strain LhTRX-h3-OE constructed in the application is significantly lower than that of the wild type and LhTRX-h3-KO, and as the stress time increases, the peroxide content of WT increases, but LhTRX-h3-OE does not show a significant increase, so it is speculated that LhTRX-h3 plays a role in oxidative stress under stress, and the peroxide content of LhTRX-h3-KO increases, but the increase is less than that of the wild type, which is speculated to be the effect of gene redundancy of TRX gene family members.

[0026] 5) After drought stress, the F0 and Fv / Fm of WT, LhTRX-h3-OE and LhTRX-h3-KO positive plants change in opposite directions, the F0 of WT, LhTRX-h3-OE-2 and LhTRX-h3-KO-6 all increases after drought stress, indicating that the center of the plant's photosystem is inhibited and has a certain tolerance to drought stress. Fv / Fm has no significant difference among the strains before stress, but all show a slight decrease after 5 days of stress, and LhTRX-h3-KO-6 decreases more obviously. The changes of non-photochemical quenching (NPQ) and photochemical quenching (qP) before and after stress show opposite trends, but whether it is LhTRX-h3-OE or LhTRX-h3-KO strain, the overall change trend is consistent with that of the wild type. At 0d of stress, the NPQ coefficient of WT is the lowest, while the qp is the opposite, the qp coefficient of LhTRX-h3-OE strain is higher, after 5 days of stress, the NPQ coefficient of all strains tends to increase, while the qp coefficient tends to decrease. The above data show that the photosynthetic activity of plant leaves decreases under drought stress, and the photosynthetic capacity decreases.

[0027] 6) After drought stress for 5d, the water loss rate of detached leaves of each line gradually increased with the increase of the time of being detached, and the water loss rate gradually slowed down with the increase of the time of being detached. At 1h of being detached, the water loss rate of WT and LhTRX-h3-KO leaves was higher, the water loss rate of WT was 46.86%, which was 1.50 times of LhTRX-h3-OE-2, 1.33 times of LhTRX-h3-OE-10, 1.28 times of LhTRX-h3-KO-6, and 1.25 times of LhTRX-h3-KO-8. Compared with WT and LhTRX-h3-KO, the water loss rate of LhTRX-h3-OE leaves was significantly lower. After 5h of being detached, the water loss rate of WT leaves reached 63.01%, that of LhTRX-h3-OE-2 reached 53.91%, which was 0.86 times of WT, that of LhTRX-h3-OE-10 reached 54.58%, which was 1.01 times of WT, that of LhTRX-h3-KO-6 reached 62.94%, which was 1.15 times of WT, and that of LhTRX-h3-KO-8 reached 60.68%, which was 0.96 times of WT. The results showed that LhTRX-h3-OE had a lower water loss rate. After 5d of stress, the relative water content of WT leaves was 65.99%, which was similar to that of LhTRX-h3-KO-6 and LhTRX-h3-KO-8, which were 65% and 63.79% respectively, while the relative water content of LhTRX-h3-OE-2 and LhTRX-h3-OE-2 was 78.91% and 76.21% respectively, which was 1.19 times and 1.15 times of WT. Therefore, LhTRX-h3-OE had a higher water retention capacity and water loss rate, and under drought stress, it could protect the plant by reducing water loss and enhance the drought resistance of the plant.

[0028] 7) Before drought stress, there was no significant difference in the ratio of stomatal length to width of WT, LhTRX-h3-OE-2, LhTRX-h3-OE-10 and LhTRX-h3-KO-8 leaves, while after drought stress, the ratio of stomatal length to width of WT, LhTRX-h3-OE-2, LhTRX-h3-OE-10 and LhTRX-h3-KO-8 leaves all increased significantly, and the increase of LhTRX-h3-OE-2, LhTRX-h3-OE-10 was more significant compared with WT and LhTRX-h3-KO-8, indicating that the stomata of LhTRX-h3-OE plants closed faster under drought stress, indicating that LhTRX-h3-OE plants were more sensitive to drought stress and could close stomata more quickly to reduce water loss. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Figure for expression pattern analysis of LcTRX-h3 gene under drought stress;

[0030] Figure 2 Identification of LhTRX-h3 gene overexpression callus (a, LhTRX-h3-OE vector construction map; b, LhTRX-h3-OE callus screening process map; c, LhTRX-h3-OE callus DNA gel electrophoresis map, "+" represents positive control, "-" represents negative control; d, relative expression of LhTRX-h3 in wild type and transgenic callus, 18S rRNA was used as internal reference gene, three biological replicates for each strain);

[0031] Figure 3 Identification of LhTRX-h3 gene knockout (LhTRX-h3-KO) callus (a, LhTRX-h3-KO vector construction map; b, c, sanger sequencing map of LhTRX-h3-KO callus);

[0032] Figure 4 LhTRX-h3 transgenic strain phenotype and growth trait analysis map (a, b, c are WT, LhTRX-h3-OE and LhTRX-h3-KO plant phenotype map respectively; d-i are the statistical results of plant height (d), petiole length (e), main root length (f), leaf number (g), leaf area (h) and chlorophyll content (i) of WT, LhTRX-h3-OE, LhTRX-h3-KO transgenic strains respectively; data analysis uses one-way ANOVA, and multiple comparisons use Duncan method);

[0033] Figure 5 WT, LhTRX-h3-OE, LhTRX-h3-KO plant phenotype map under drought stress (drought stress 0 days, 5 days WT, LhTRX-h3-OE, LhTRX-h3-KO plant phenotype);

[0034] Figure 6 WT, LhTRX-h3-OE, LhTRX-h3-KO plant physiological index determination map under drought stress;

[0035] Figure 7 WT, LhTRX-h3-OE, LhTRX-h3-KO plant DAB (a), NBT (b) staining map under drought stress;

[0036] Figure 8 WT, LhTRX-h3-OE, LhTRX-h3-KO plant chlorophyll fluorescence parameter change map under drought stress (a, initial fluorescence change; b, maximum photochemical efficiency change; c, non-photochemical quenching change; d, photochemical quenching change; data analysis uses one-way ANOVA, and multiple comparisons use Duncan method);

[0037] Figure 9 Figure 9 is a graph showing the water change of WT, LhTRX-h3-OE, LhTRX-h3-KO leaves under drought stress (a is the leaf in vitro water loss rate; b is the leaf relative water content RWC; data analysis uses single factor ANOVA, and multiple comparisons use Duncan method);

[0038] Figure 10 Figure 10 is a graph showing the stomata change of WT, LhTRX-h3-OE, LhTRX-h3-KO plants under drought stress (a is the stomata graph of drought stress 0d, 5d, bar = 5μm; b is the stomata conductance (pore length / pore width) of WT, LhTRX-h3-OE, LhTRX-h3-KO lines under drought stress 0, 5d; data analysis uses single factor ANOVA, and multiple comparisons use Duncan method). DETAILED DESCRIPTION

[0039] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described below in combination with specific examples. In the following examples, if not specifically stated, the technical means used are conventional means well known to those skilled in the art.

[0040] The hybrid Liriodendron plants used in the following examples were derived from hybrid Liriodendron plants cultivated in the previous laboratory.

[0041] The intermediate vector competent cells used in the following examples were purchased from Genview Biotech Co., Ltd.; the E. coli competent cells DH5a were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; and the Agrobacterium competent cells EHA105 were purchased from Shanghai Weidi Biological Technology Co., Ltd.

[0042] The main reagents used in the following examples are as follows:

[0043] The plant total RNA extraction kit is a product of Plumbio (Beijing) Biotechnology Co., Ltd. SuperTotal RNA Extraction Kit kit; cDNA reverse reagent kit is HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit produced by Nanjing Novelz Biological Technology Co., Ltd.; 2x Rapid TaqMaster Mix, Phanta Max Super-Fidelity DNA Polymerase, Clone express II one step cloning kit are purchased from Nanjing Novelz Biological Technology Co., Ltd.; AceQ Universal SYBR qPCR Master Mix, DNA gel recovery reagent kit are purchased from TaKaRa (Beijing) Co., Ltd.; TSINGKET SV-007 VS pClone007 Versatile Simple Vector Kit is purchased from Nanjing Tsingke Biological Technology Co., Ltd.; Plasmid extraction reagent kit is purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Restriction endonuclease is purchased from NEB official website; Cellulase (Cellucase R-10), Macerozyme (Macerozyme R10), Pectolyase (Pectolyase Y-23) are purchased from YakμLt company; Ampicillin (Amp), Kanamycin (Kna) are purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The reagent formula used in the following examples is:

[0044] The formula of the antibiotic mother liquor is: ampicillin (Amp) 50 mg / mL, kanamycin (Kan) 100 mg / mL.

[0045] The formula of LB medium is: yeast extract (Yeast Extract) 5 g / L, tryptone (Tryptone) 10 g / L, sodium chloride (NaCl) 10 g / L.

[0046] The formula of W5 solution is: NaCl 154 mM, CaCl2 125 mM, KCl 5 mM, MES (pH 5.7) 2 mM, glucose 5 mM.

[0047] The formula of MMg solution is: mannitol 0.4 M, MgCl2 15 mM, MES (pH 5.7) 4 mM.

[0048] The formula of WI solution is: MES (pH 5.7) 4 mM, mannitol 0.5 M, KCl 20 mM.

[0049] The formula of enzyme solution is: cellulase (Cellucase R-10) 0.15 g, macerozyme (Macerozyme R-10)

[0050] 0.1 g, Pectolyase (Pectolyase Y-23) 0.01 g, Mannitol 0.8 M, KCl 80 mM, MES 200 mM, CaCl2 10 mM, BSA 0.1%, ddH2O To 10.

[0051] The GUS dye solution preparation method is as follows: 0.5 mL of X-Gluc stock solution, 1 mL of 0.5 M phosphate buffer, 0.1 mL of 10% Triton X-100, 0.2 mL of 100 mM potassium ferricyanide, 0.2 mL of 100 mM potassium ferrocyanide, 0.2 mL of 0.5 M EDTA, and distilled water to 10 mL.

[0052] The X-Gluc stock solution preparation method is as follows: dissolve 30 mg of X-Gluc powder in 1.5 mL of DMF to a final concentration of 20 mg / mL. Mix well by vortexing, wrap in aluminum foil, aliquot, and store at -20°C.

[0053] The preparation method of the transparent liquid is as follows: weigh 80 g of chloral hydrate and 10 mL of glycerol, and add distilled water to make up to 100 mL.

[0054] The preparation method of the sodium phosphate buffer is as follows: 1 M NaH2PO4 solution: dissolve 12 g of NaH2PO4 in 100 mL of distilled water (final volume); 1 M Na2HPO4 solution: dissolve 14.2 g of Na2HPO4 in 100 mL (final volume) of distilled water; mix 16 mL of 1 M NaH2PO4 and 84 mL of 1 M Na2HPO4, and make up to 2 L with distilled water to prepare 50 mM sodium phosphate buffer (pH 7.5).

[0055] The preparation method of NBT dye is as follows: in an amber bottle (avoid light), dissolve 0.1 g of NBT in 50 mM sodium phosphate buffer (pH 7.5) to make up to 50 mL; mix the solution thoroughly with a magnetic stirrer to obtain a 0.2 mmol / L solution.

[0056] The preparation method of DAB dye is as follows: dissolve 50 mg of DAB in about 45 mL of distilled water, and place it in an amber bottle (avoid light); adjust the pH to 3.8 with 0.1 N hydrochloric acid while mixing appropriately on a magnetic stirrer (stir overnight); after complete dissolution, the solution will be clear light brown. Make up to 50 mL to obtain a 1 mg / mL solution.

[0057] Example 1

[0058] 1. Identification of members of the TRX gene family in Catalpa bungeana

[0059] The Hidden Markov Model (PF00085) of TRX gene domain was downloaded from the pfam database (http: / / pfam.sanger.ac.uk / ), and the gene family members were identified using HMMER software with an E-value less than e-10, obtaining 45 sequences. The TRX gene family member sequences were downloaded from the Arabidopsis database (https: / / www.arabidopsis.org / ), and local Blast was performed using BLAST software, obtaining 55 sequences; based on the sequences obtained by the two methods, the union set was obtained, obtaining 90 sequences. Then the NCBI CD-Search (https: / / www.ncbi.nlm.nih.gov / cdd) was used to analyze the conservative domain of the candidate protein sequence, and after screening out the sequences without TRX structure or containing incomplete TRX domain, the LcTRX-h3 gene meeting the conditions was finally identified.

[0060] 3. Total RNA extraction and cDNA synthesis

[0061] The total RNA of the hybrid Liriodendron seedlings was extracted using the Super Total RNA Extraction Kit (Promega Company) according to the instructions. The extracted total RNA was used as a template to obtain the cDNA strand by reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit (Vazyme Company). 2. Expression pattern analysis of LhTRX-h3 gene of hybrid Liriodendron under drought stress

[0062] The Liriodendron somatic embryo seedlings were generated by Liriodendron somatic embryogenesis. Before the experiment, the Liriodendron somatic embryo seedlings were acclimated in the greenhouse for 2 weeks (22°C, 16 hours of light, 8 hours of darkness, and relative humidity of 75%). The seedlings with consistent growth and good growth were selected and transplanted into the seedling pots for culture. When the Liriodendron seedlings grew to the 5th fully expanded true leaf, drought stress treatment was performed. The drought treatment was simulated by PEG6000, and 15% PEG6000 was poured into the seedling pots as the experimental group, and the plants irrigated with nutrient solution were selected as the control group. Each treatment was set up with 3 biological replicates, and the plant leaf samples were collected after 0d, 1h, 12h, and 3d of treatment, washed with sterile water, and immediately frozen in liquid nitrogen, and stored in a -80°C freezer for standby.

[0063] The qRT-PCR primer sequences are as follows:

[0064]

[0065] ​qLhTRX-h2-F: 5'-TGATCGATTTCACGGCCAA-3',

[0066] qLhTRX-h2-R: 5'-TCCGTCCTTATTCGCACCT-3';

[0067] qLhTRX-h2.2-F: 5'-CCACCATGAACGAATTGGCTA-3',

[0068] qLhTRX-h2.2-R: 5'-TGAAGTTGCTCCCTATGCTTG-3';

[0069] qLhTRX-h3-F: 5'-CCACTTTCAACACGGAGCTG-3',

[0070] qLhTRX-h3-R: 5'-CTCCCACTCTTCCGCAACA-3';

[0071] qLhNrx-h1.6-F: 5'-CCATTTTCTGATTCGGCCACC-3',

[0072] qLhNrx-h1.6-R: 5'-CTTCCTTCTTCAGCTCGTTG-3';

[0073] qLhNrx-h1.7-F: 5'-CTACTGGCAAGACGATCACA-3',

[0074] qLhNrx-h1.7-R: 5'-GACCAATTACACCCGTCCT-3';

[0075] qLhClot-F: 5'-CTTAGCCGACAAAGACCCATC-3',

[0076] qLhClot-R: 5'-CATGGGTGGTTAGGATTTCTC-3'.

[0077] The fluorescence quantitative PCR reaction system was as follows: 2x AceQ qPCR SYBR Green Master Mix 10 μL, Forward Primer 0.4 μL, Reverse Primer 0.4 μL, cDNA 2 μL, ddH2O To 20 μL.

[0078] The fluorescence quantitative PCR reaction conditions were as follows: 95 °C denaturation for 30 s; 95 °C for 5 s, 60 °C for 10 s, 40 cycles; 96 °C for 5 s; 65 °C for 1 min; 95 °C for 15 s; 50 °C for 30 s.

[0079] The relative expression of genes was calculated by 2 -△△C The data were analyzed by EXCEL 2023 and IBM SPSS Statistic, and the graphs were drawn by GraphPad Prism 8.0.2.

[0080] The results are shown in Figure 1 LhTRX-h3 gene was up-regulated, and reached the peak at 12h with the increase of stress time.

[0081] Example 2

[0082] 1. Cloning of target genes

[0083] The CDS sequence of LcTRX-h3 gene was used as the reference sequence to design primers using snapgene software. The wild-type hybrid larch cDNA was used as the template, and the high-fidelity DNA polymerase from Nanjing Novozyme was used for amplification. The primer sequences are as follows:

[0084] PBI121-TRX-h3-F:

[0085] 5'-agaacacgggggactctagaATGGCAACGGTTGAAGTAGGAAC-3',

[0086] PBI121-TRX-h3-R:

[0087] 5'-cggggaaattcgagctcCTAAGCAGCGGACGTGGAAG-3'.

[0088] The PCR reaction system was as follows: 25μL 2×Phanta Max Buffer, 1μL dNTP Mix (10mM each), 2μL Forward primer (10mM each), 2μL Reverse primer (10mM each), 1μL Phanta Max Super-Fidelity DNA Polymerase, 2μL DNA, ddH2O To 50μL.

[0089] The PCR reaction program was as follows: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 57℃ annealing for 15s, 72℃ extension for 30-60s / kb, 35 cycles; 72℃ for 5min; 10℃∞.

[0090] PCR amplification products using 1.5% agarose gel electrophoresis, 25 min detection results. Clear and bright bands. Electrophoresis fragments of the target gene under UV light cut out the purpose of the segment gel recovery.

[0091] The gel recovery fragments were connected to the pClone007 intermediate vector, and the connection system was mixed and placed in a metal bath at 25°C for 5 min, and then transformed immediately. The transformation method was as follows:

[0092] ① Take 100 μL of melted competent cells in ice bath, add 10 μL of ligation product, mix gently, and incubate on ice for 25 min. ② 42°C water bath heat shock for 45-60 s, quickly transfer to ice bath, incubate for 2 min (do not shake the sample during ice incubation, otherwise the transformation efficiency will be reduced). ③ Add 700 μL of antibiotic-free LB culture solution to the centrifuge tube, mix well, and incubate at 37°C, 200 rpm for 1 h. ④ According to the experimental needs, different volumes of recovery solution were taken and evenly coated on LB medium containing Amp antibiotic, and the plate was inverted and placed in a 37°C incubator for overnight culture.

[0093] The intermediate vector connection system was: target fragment 4 μL, 5 × pClone007 Versatile Simple Vector Mix 2 μL, ddH2O To 10 μL.

[0094] Pick the single colony of the plate into a 1.5 mL centrifuge tube, add 600 μL of Amp liquid LB, incubate at 37°C on a shaker at 250 rpm for 6-8 h, and then perform bacterial liquid PCR verification. The primer sequence is as follows:

[0095] M13-F: 5'-TGTAAAACGACGGCCAGT-3',

[0096] M13-R: 5'-CAGGAAACAGC TATGACC-3'.

[0097] The PCR reaction system was: 7.5 μL 2 × Rapid Taq Master Mix, 0.6 μL Forward primer (10 mM each), 0.6 μL Reverse primer (10 mM each), 2 μL DNA, ddH2O To 15 μL.

[0098] The PCR reaction program was: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 57°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; 72°C for 5 min; 10°C ∞.

[0099] The correct bacterial liquid was sent to a company for sequencing. After the sequencing was correct, the culture was expanded. 3 mL Amp-resistant LB liquid medium and 200 μL bacterial liquid were added to a 10 mL centrifuge tube and placed in a 37°C, 250 rpm shaker for 16h.

[0100] The plasmid extraction kit from TIANGEN was used to extract the plasmid from the expanded bacterial liquid. The intermediate vector plasmid containing the target fragment was prepared and stored in a -20°C refrigerator.

[0101] According to the sequencing results, the nucleotide sequence of the target gene is shown in SEQ ID NO. 1. The gene is named LcTRX-h3 gene. The amino acid sequence of the expressed protein is shown in SEQ ID NO. 2.

[0102] Example 3

[0103] 1. Construction of overexpression vector

[0104] The p35S:GUS plasmid vector was digested with Xba I and Sac I as the enzyme cutting site. The vector digestion product was recovered using a DNA recovery kit.

[0105] The enzyme cutting reaction system was: Cutsmart Buffer 2 μL, Xba I 1 μL, Sac I 1 μL, plasmid 10 μL, ddH2O 6 μL

[0106] The enzyme cutting reaction program was: 37°C for 3h, 65°C for 20min, 16°C for ∞.

[0107] The recombinant vector was constructed by homologous recombination method. The recombination connection system was: 5×CE II Buffer 4 μL, II 1 μL, linearized vector 4 μL, target fragment 2 μL, ddH2O 9 μL.

[0108] The recombination connection reaction program was: 30°C for 60min, 4°C for ∞.

[0109] The recombination connection product was transformed into E. coli competent DH5α. After the bacterial plaque grew, PCR verification was performed. The correct bacterial liquid was used for subsequent experiments. The LhTRX-h3 gene overexpression vector LhTRX-h3-OE(A) was prepared. Figure 2 The primer sequence is as follows:

[0110] YZ-LhTRX-h3-F:

[0111] 5'-gtggaaaaagaagacgttcc-3',

[0112] YZ-LhTRX-h3-R:

[0113] 5'-ggactctaatcataaaaacccatc-3'.

[0114] 2, infecting hybrid Liriodendron chinense embryogenic callus

[0115] Take the above prepared LhTRX-h3 OE The bacterial solution of the vector is cultured in a 250 mL conical flask with 2 mL of bacterial solution, 50 mL of LB resistance, 200 rpm, 28°C for 4 hours.

[0116] ① Take the bacterial solution cultured on the shaking table for 4 hours to measure the OD 600 value, stop shaking the bacteria when the OD 600 value is between 0.6 and 1.0, transfer to a 50 mL Beckman centrifuge tube and centrifuge at 6000 rpm for 10 min to collect the bacteria. Remove the supernatant and resuspend the bacterial solution with M13 liquid medium. ② Take the embryogenic callus and place it in a 100 mL Erlenmeyer flask, add the bacteria solution after reselection, and gently shake the flask for 10 min. ③ After the infection is completed, collect the callus with a sterilized cell sieve and absorb the excess bacterial solution with sterile filter paper. Then transfer the callus to M13 solid medium for co-culture of the callus and Agrobacterium. ④ Place the co-cultured callus in a 100 mL Erlenmeyer flask and wash it alternately with sterile water and M13 liquid medium containing cef antibiotic to remove bacteria. After the bacteria are removed, collect the callus with a cell sieve and dry it with sterile filter paper, then place it in M13 medium containing Cef for recovery culture.

[0117] The DNA of the fine new callus growing on the selection medium is extracted by the CTAB method, and the 2x Rapid Taq Master Mix of Vazyme Company is used for PCR amplification to verify whether it is positive Figure 2 B).

[0118] ① Place the wild type callus and overexpression positive callus in a 250 mL conical flask, add 50 mL of M13 liquid medium for liquid culture. ② Replace the new M13 liquid medium after a week to prevent callus browning, continue to culture for a week to obtain sufficient single cells. ③ Filter the single cells of liquid culture using a 150 mesh and 400 mesh cell sieve, collect the single cells on the 400 mesh sieve in a new 250 mL conical flask, and add 50 mL of Z14 high osmotic transition medium. ④ After one day of transition culture, use a 1 mL syringe to absorb each suspension line uniformly and spread it on Z36 somatic embryogenesis solid medium with filter paper. ⑤ Finally, place it in a constant temperature dark incubator for culture, and after 3 months, new fine callus is obtained.

[0119] Fine callus tissue was cultured to a single line in 3 dishes, and DNA was extracted. The target band was amplified by PCR and sent to the company for sequencing, confirming it as a transgenic positive callus. Figure 2 C).

[0120] RNA was extracted from wild-type and transgenic positive RNA using Prometheus' RNA extraction kit. After reverse mixing, quantitative real-time PCR was performed to detect the relative expression level of LhTRX-h3. The results are as follows: Figure 2 As shown in Figure D, the expression level of the LhTRX-h3 gene varied among different lines, but compared with the control, the expression level of LhTRX-h3 in LhTRX-h3-OE was significantly increased. Among the lines, LhTRX-h3-OE-2 and LhTRX-h3-OE-16 showed the highest expression levels, with their LhTRX-h3 expression levels being 6 times that of the wild-type control. The expression levels of LhTRX-h3-OE-1, LhTRX-h3-OE-10, and LhTRX-h3-OE-17 were lower, being 2-3 times that of the wild-type control. After identifying them as transgenic lines, subsequent experiments were conducted.

[0121] Example 4

[0122] 1. Constructing CRISPR knockout vectors

[0123] 1) Target site selection

[0124] The full-length target gene was identified based on transcriptome or genome sequence. Primers were designed, and the gene was cloned using target plant DNA / cDNA as a template. An intermediate vector was ligated, and the gene was transformed into *E. coli*. Positive strains were identified by PCR, and sequencing was performed. The complete genome sequence of the gene was determined, and introns, exons, extremely conserved sites, WUSBOX sites, and SNP sites were marked. All matching target sequences were searched from the website (CRISPR-P v2.0 (hzau.edu.cn) or http: / / crispr.mit.edu / ). The GC content of the target sites was selected to be as high as possible (otherwise, the targeting efficiency would be affected). The target sequence was ligated to the 5' end of the sgRNA, and secondary structure analysis was performed using online software. Target sequences with consecutive pairings longer than 7 bp were avoided. Specificity analysis was then performed on the target. The final identified target sequence is as follows:

[0125] Target 1: 5'-GCTGCCACAGCGTGGATGAA-3',

[0126] Target 2: 5'-TGCGGAAGAGTGGGAGGTGG-3',

[0127] Target 3: 5'-GCCCACCTTTCTTGTTCCTCA-3'.

[0128] 2) Design primers

[0129] Primers were designed using online website primerDesign (http: / / skl.scau.edu.cn / primerdesign / ), Gibson Assembly was used for connection, the target sequence was input, the corresponding promoter was selected, and the primers containing overlapping were automatically formed.

[0130] The first round of PCR primer sequences are as follows:

[0131] U-F: 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0132] gR-R: 5'-CGGAGGAAAATTCCATCCAC-3',

[0133] TRX-h3-gRT1: 5'-GCTGCCACAGCGTGGATGAAGTTTTAGAGCTAGAAAT-3',

[0134] U3dT1: 5'-TTCATCCACGCTGTGGCAGCTGACCAATGGTGCTTTG-3',

[0135] TRX-h3-gRT2: 5'-TGCGGAAGAGTGGGAGGTGGGTTTTAGAGCTAGAAAT-3',

[0136] U3bT2: 5'-CCACCTCCCACTCTTCCGCATGACCAATGTTGCTCC-3',

[0137] TRX-h3-gRT3: 5'-CCCACCTTCTTGTTCCTCAGTTTTAGAGCTAGAAAT-3',

[0138] U6-1T3: 5'-TGAGGAACAAGAAGGTGGGCAATCACTACTTCGTCT-3'.

[0139] The second round of PCR primer sequences are as follows:

[0140] U-GAL:

[0141] 5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG-3',

[0142] Pgs-GA2:

[0143] 5'-CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTC TTG-3',

[0144] U-GA2:

[0145] 5'-GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAG G-3',

[0146] Pgs-GA3:

[0147] 5'-CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTC TTG-3',

[0148] U-GA3:

[0149] 5'-CGCTATAGTGTCACCTAAATCGTATGCGTGGTGGAATCGGCAGCAAA GG-3',

[0150] Pgs-GAR:

[0151] 5'-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCC AAGCTCTTG.

[0152] 3) Construction of sgRNA expression cassette

[0153] ① First round of PCR (insert Target site between each promoter and sgRNA by PCR)

[0154] The first round of PCR reaction system was: 12.5 μL 2x Phanta Max Buffer, 0.5 μL dNTP Mix (10 mM each), 1 μL Forward Primer (10 μM) (U-F) & (gRT1 / gRT2 / gRT3), 1 μL Reverse Primer (10 μM) (U3dT1 / U3bT2 / U6-1T3) & (gR-R), 0.5 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL plasmid DNA, 8.5 μL ddH2O.

[0155] The PCR reaction program was: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 30 s, 35 cycles; 72 °C for 5 min; 10 °C ∞.

[0156] Agarose gel electrophoresis (the concentration of the gel is 2%) U = 200 V, I = 400 mA, P = 80 w, 5 μL PCR product is run, 7 μL 2000 Marker, the running time is not less than 25 min

[0157] 2. The second round of PCR (connecting the short sequences amplified by the first round of PCR to produce a 2-fragment combined sgRNA expression cassette fragment)

[0158] The template used in the second round of PCR is 1 μL of the first round of PCR product diluted 10 times with water

[0159] The second round of PCR reaction system is: 25 μL 2x Phanta Max Buffer, 1 μL dNTP Mix (10 mM each), 2 μL Forward Primer (10 μM) (U-F), 2 μL Reverse Primer (10 μM) (gR-R), 1 μL PhantaMax Super-Fidelity DNA Polymerase, 1+1 μL plasmid DNA, 17 μL ddH2O.

[0160] The PCR reaction program is: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 36 s, 35 cycles; 72°C for 5 min; 10°C ∞.

[0161] After running the 2% gel, the gel is cut and recovered, and the concentration is measured, 10 μL of the recovered product and two end primers are taken in two directions for measurement, and the sequences of the AtU3d, AtU3b, and AtU6-1 expression cassettes with the target gene inserted are obtained.

[0162] 3. The third round of PCR (adding the overlap site required for ligation to the gRNA expression cassette amplified by the second round of PCR)

[0163] The template for the third round of PCR is the second round of gel recovery product, and the concentration is diluted to 30 ng / μL.

[0164] The template used in the third round of PCR is 1 μL of the first round of PCR product diluted 10 times with water

[0165] The third round of PCR reaction system is: 25 μL 2xPhanta Max Buffer, 1 μL dNTP Mix (10 mM each), 2 μL Forward Primer (U-GAL / U-GA2 / U-GA3), 2 μL Reverse Primer (Pgs-GA2 / Pgs-GA3 / Pgs-GAR), 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL gel recovery product, 18 μL ddH2O.

[0166] The PCR reaction program is: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 65°C annealing for 15 s, 72°C extension for 42 s, 35 cycles; 72°C for 5 min; 10°C∞.

[0167] After 2% gel running and gel recovery, the concentration is measured, 10 μL of the recovered product is taken and two-end primers are sent in both directions for measurement, and each expression cassette sequence of the amplified overlap site is obtained.

[0168] 4) Final vector construction

[0169] The plasmid pYLCRISPR / Cas9P35S-N stored in the laboratory is used as a template, BsaI-HF2 is selected as the enzyme cutting site, and Gibson assembly premix of NEB company is used to recombine and connect the three expression cassettes with the enzyme cutting product.

[0170] The enzyme cutting reaction system is: Cutsmart Buffer 5 μL, BsaI-HF2 1 μL, pYLCRISPR / Cas9P35S-N (2 ug) 8 μL, ddH2O 32 μL

[0171] The enzyme cutting reaction program is: 37°C for 1 h, 80°C for 30 min, 10°C∞.

[0172] The recombinant vector is constructed by homologous recombination method, and the recombination connection system is: ddH2O 4 μL, pYLCRISPR / Cas9 vector 3 μL, AtU3dT1 1 μL, AtU3bT2 1 μL, AtU6-1T3 1 μL, 2xmaster mixture 10 μL.

[0173] The recombination connection reaction program is: 50°C for 30 min, 4°C∞.

[0174] Take 10 μL of the ligation product, dilute 1 μL of the vector plasmid to 3 μL for running, and the gel concentration is 0.8%, the band length is correct, and it is clear and bright. Take the remaining 10 μL of the ligation product to transform E. coli DH5α. Pick a single colony and add 600 μL of resistant LB, shake at 220 rpm and 37°C for 8 h, then perform PCR verification. The verification primers are as follows:

[0175] M13-F: 5'-TGTAAAACGACGGCCAGT-3',

[0176] SP-L2-New: 5'-GTCGTGCTCCACATGTTGACCGGTAA-3',

[0177] Reverse plus SP6-F: 5'-CATACGATTTAGGTGACACTATAG-3'.

[0178] Select the correct bacterial liquid to extract plasmid, and transfer the plasmid into Tian Gen EHA105 Agrobacterium. The LhTRX-h3 gene knockout vector LhTRX-h3 KO ( Figure 3 A).

[0179] 2, Infected hybrid liriodendron embryo callus

[0180] Take the above prepared bacterial liquid containing LhTRX-h3 vector for expansion culture, add 2 mL of bacterial liquid to a 250 mL conical flask, add 50 mL of resistant LB, shake at 200 rpm and 28°C for 4 hours.

[0181] ①Take the bacterial liquid cultured on the shaking table for 4 hours to measure the OD600 value, stop shaking when the OD600 value is between 0.6-1.0, transfer to a 50 mL Beckman centrifuge tube and centrifuge at 6000 rpm for 10 min to collect the bacteria. Remove the supernatant, add M13 liquid medium to resuspend the bacterial liquid. ②Take the embryogenic callus and place it in a 100 mL Erlenmeyer flask, add the bacteria liquid after reselection, and gently shake the Erlenmeyer flask for 10 min. ③After the infection is completed, collect the callus with a sterilized cell sieve, and use sterile filter paper to absorb the excess bacterial liquid. Then transfer the callus to M13 solid medium for co-culture of the callus and Agrobacterium. ④Place the co-cultured callus in a 100 mL Erlenmeyer flask, and wash it with sterile water and M13 liquid medium with added cephalosporin antibiotic alternately. After the decontamination is completed, collect the callus with a cell sieve, and use sterile filter paper to dry the callus, and then place it in M13 medium containing Cef for recovery culture.

[0182] The DNA of the fine new callus growing on the screening medium was extracted by CTAB method, and PCR amplification was performed using 2x Rapid Taq Master Mix of Vazyme Company to verify whether it was positive Figure 2 B).

[0183] ①Put the wild type callus and knock-out positive callus into 250 mL conical flask, and add 50 mL M13 liquid medium for liquid culture. ②Replace the new M13 liquid medium after a week to prevent callus browning, and continue to culture for one week to obtain sufficient single cells. ③Filter the single cells in liquid culture using 150 mesh and 400 mesh cell screens, collect the single cells on the 400 mesh screen into a new 250 mL conical flask, and add 50 mL Z14 high-osmotic transition medium. ④After transition culture for one day, use 1 mL of pipette gun to evenly spread each suspension line on the Z36 somatic embryogenesis solid culture medium with filter paper. ⑤Finally, put it in a constant temperature dark incubator for culture, and after 3 months, new fine callus tissue is obtained.

[0184] Take the fine callus tissue to expand culture to 3 dishes per strain, extract DNA, and after amplifying the target band by PCR, send it to the company for sequencing to confirm that it is a transgenic positive callus. Take the correct strain for verification to cut the gel and recover the PCR product, and connect 007B to transform E. coli, and pick single clone bacteria liquid to send to the company for sanger sequencing.

[0185] The results are shown in Figure 3 B, LhTRX-h3-KO-1 increases base A at target site 1, LhTRX-h3-KO-4 deletes 3 bases at target site 1, and LhTRX-h3-KO-6 and LhTRX-h3-KO-8 both delete a base G at target site 3. After identification as transgenic knock-out strains, subsequent experiments were carried out.

[0186] Example 5

[0187] 1. Phenotype observation of transgenic plants and determination of chlorophyll content

[0188] WT, LhTRX-h3-OE and LhTRX-h3-KO plants grown on the seedling selection medium for about 90 days, after positive rate verification and expression amount verification, the plant height, root length, petiole length, leaf number, leaf area and other phenotypes were observed and counted. The ruler was used to measure the plant height, root length and petiole length, and the leaf number was counted, and LhTRX-h3-OE and LhTRX-h3-KO mutant two transgenic lines, each line included 10 biological repeats. Image J software was used to count the leaf area of the plant, and each line included 10 biological repeats. The chlorophyll content determination was using Nanjing Jiancheng kit, LhTRX-h3-OE and LhTRX-h3-KO mutant two transgenic lines, each line included 3 biological repeats. All experimental data were subjected to one-way ANOVA analysis, and finally the graph was drawn using GraphPad Prism8 drawing software for analysis.

[0189] The kit was used to determine the chlorophyll content, and the reagent was purchased from Nanjing Jiancheng Technology Co., Ltd.

[0190] The calculation method is as follows:

[0191] Chlorophyll a content (mg / g fresh weight) = (12.7 x A663-2.69 x A645) x V extract x N / m x 1000 Chlorophyll b content (mg / g fresh weight) = (22.9 x A645-4.68 x A645) x V extract x N / m / 1000 Total chlorophyll content (mg / g fresh weight) = (20.21 x A645+8.02 x A663) x V extract x N / m / 1000 Note: V extract: extract volume; N: dilution multiple; m: sample mass (g).

[0192] The results are as follows: Figure 4As shown, the plant height, petiole length, main root length, leaf area, and chlorophyll content of the LhTRX-h3-OE lines were significantly reduced, the leaf color became lighter, and the whole plant was dwarfed, while the LhTRX-h3-KO plants had no obvious difference from the wild type. However, there was no obvious difference in the number of leaves between the overexpression and knockout lines. The plant height of LhTRX-h3-OE-2 was significantly reduced by 37.11% compared with the wild type, and LhTRX-h3-OE-10 was significantly reduced by 32.36%. The plant height of the two LhTRX-h3-KO lines was only increased by 8.8% and 10.14%, respectively. The petiole length of LhTRX-h3-OE-2 was reduced by 19.7% compared with the wild type, and LhTRX-h3-OE-10 was reduced by 22.95%. The petiole length of LhTRX-h3-KO-6 and LhTRX-h3-KO-8 was increased by 5.33% and 2.09%, respectively. The main root length of LhTRX-h3-OE-2 was reduced by 19.89% compared with the wild type, and LhTRX-h3-OE-2 was reduced more significantly by 37.79%. LhTRX-h3-KO-6 and LhTRX-h3-KO-8 were increased by 3.43% and 6.14%, respectively. The leaf area of LhTRX-h3-OE-2 was significantly reduced by 14.37% compared with the wild type, and LhTRX-h3-OE-10 was reduced more significantly by 19.781%. LhTRX-h3-KO-6 and LhTRX-h3-KO-8 were increased by 3.36% and 7.78%, respectively. In general, LhTRX-h3 plays a certain role in plant growth and development, resulting in a significant reduction in the growth phenotype of the overexpression lines. However, the knockout lines did not show a significant increase, which may be due to the action of other genes in the LhTRX-h3 gene family, resulting in gene redundancy.

[0193] 2. Effect of drought stress on the physiological activity of transgenic plants

[0194] A wild type line with consistent growth and healthy growth was selected, and the bottle seedlings of LhTRX-h3-OE and LhTRX-h3-KO were transferred to 20% PEG6000 drought stress medium for 0d, 1d, 3d, and 5d, respectively.

[0195] 1) Take photos to observe the phenotype: as shown in Figure 5 After 5d of stress, different lines showed wilting, yellowing, and shedding of leaves, but compared with the wild type, the LhTRX-h3-OE lines were in good condition, had lower wilting degree, and had no leaf shedding, while the LhTRX-h3-KO positive plants had yellowing and shedding of leaves.

[0196] 2) Measurement of photosynthetic physiological indicators: The contents of H2O2 and MDA were measured using a kit. The H2O2 kit was purchased from Nanjing Jiancheng Technology Co., Ltd., and the MDA kit was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd. One strain of wild type, two strains of LhTRX-h3-OE and two strains of LhTRX-h3-KO after drought stress were taken for measurement. Four seedlings of each strain were taken, and at least five replicates were taken at each time point. The results are shown in Figure 6 MDA content in the plant increased with the increase of stress time, but the growth rate of WT and LhTRX-h3-KO was faster than that of LhTRX-h3-OE, indicating that the cells in the body were more severely oxidatively stressed. The H2O2 content at different time points was measured. It was found that at 0d, the hydrogen peroxide content of LhTRX-h3-OE was significantly lower than that of wild type and LhTRX-h3-KO, and with the increase of stress time, the hydrogen peroxide content of WT increased, but LhTRX-h3-OE did not show obvious increase, so it was speculated that LhTRX-h3 played a role in oxidative stress under stress, and the hydrogen peroxide content of LhTRX-h3-KO increased, but the increase was lighter than that of WT, which was speculated to be the gene redundancy of TRX gene family members.

[0197] 3) Take 0d, 5d of wild type 1 strain, LhTRX-h3-OE, LhTRX-h3-KO two strains of each after drought stress, take the same size leaves for staining, the specific steps are as follows:

[0198] NBT staining: Prepare 0.2 mmol / L NBT staining solution, immerse the leaves in NBT staining solution, 28°C avoid light staining for 16h, discard the staining solution, cook in anhydrous ethanol for 5 minutes, until the chlorophyll is completely faded, observe the staining condition.

[0199] DAB staining: Prepare 1 mg / mL DAB staining solution, adjust pH = 3.8, immerse the leaves in DAB staining solution, 28°C avoid light staining for 16h, discard the DAB staining solution, cook in anhydrous ethanol for 5 minutes, until the chlorophyll is completely faded, observe the staining condition.

[0200] The results are shown in Figure 7As shown, under no stress, WT, LhTRX-h3-KO-6, and LhTRX-h3-KO-8 all developed dark brown patches, while LhTRX-h3-OE-2 and LhTRX-h3-OE-10 only showed slight browning. After 5 days of stress, WT, LhTRX-h3-KO-6, and LhTRX-h3-KO-8 all developed large areas of dark brown patches, while LhTRX-h3-OE-2 only showed a deepening of brown color without developing large areas of dark brown patches, and LhTRX-h3-OE-10 only showed slight browning on the leaf lateral surface. Figure 7 A). NBT staining results showed that WT, LhTRX-h3-KO-6, and LhTRX-h3-KO-8 already had dark blue patches without stress. After stress, the area of ​​dark blue patches increased significantly, and the LhTRX-h3-OE-2 leaves with stress for 0 days and 5 days had the smallest staining area. Figure 7 B). The combined results of physiological indicators and staining studies indicate that LhTRX-h3 plays a role in regulating ROS.

[0201] 4) Chlorophyll fluorescence measurement: Chlorophyll fluorescence was measured in plants subjected to drought stress for 5 days, including one wild-type line, two LhTRX-h3-OE lines, and LhTRX-h3... KO Two strains were used, and eight healthy seedlings of uniform growth were selected from each strain for testing. The results are as follows: Figure 8 As shown, after drought stress, the F0 and Fv / Fm of WT, LhTRX-h3-OE, and LhTRX-h3-KO positive plants showed opposite trends. The F0 of WT, LhTRX-h3-OE-2, and LhTRX-h3-KO-6 all increased after drought stress, indicating that the photosystem center of the plants was suppressed and they had some tolerance to drought stress. Fv / Fm showed no significant difference among the lines at 0 days of stress, but all showed a slight decrease at 5 days, with a more pronounced decrease in LhTRX-h3-KO-6. Non-photochemical quenching (NPQ) and photochemical quenching (qP) showed opposite trends before and after stress, but the overall trend of both LhTRX-h3-OE and LhTRX-h3-KO lines was consistent with the wild type. At 0 days of stress, the NPQ coefficient of WT was the lowest among all lines, while qp was the opposite. The qp coefficient of the LhTRX-h3-OE line was higher. After 5 days of stress, the NPQ coefficient of all lines generally tended to increase, while the qp coefficient showed the opposite trend, generally decreasing. These data indicate that under drought stress, the photosynthetic activity of plant leaves decreased, and the photosynthetic capacity weakened.

[0202] 5) Measurement of leaf water loss rate and relative water content: The relative water content of leaves of plants treated with drought stress for 5 days was measured, including one wild type strain, two LhTRX-h3-OE strains, and two LhTRX-h3-KO strains, with 3 repeats for each strain. The relative water content was measured by cutting the leaves into small pieces and dividing them into two groups. One gram (Wf) of leaves was weighed, and one group was placed in a 75°C oven to dry completely, and then the dry weight (Wd) was measured. The other group was placed in distilled water and soaked for 70 minutes, and then the weight (Wt) was measured. The relative water content of the leaves was calculated according to the following formula: Relative water content RWC (%) = (Wf-Wd) / (Wt-Wd) x 100%. The results are shown in Figure 5, and the water loss rate of leaves of each strain gradually increased with increasing in vitro time after 5 days of drought stress. The water loss rate gradually slowed down with increasing in vitro time. At 1 hour in vitro, the water loss rate of leaves of WT and LhTRX-h3-KO was higher. The water loss rate of leaves of WT was 46.86%, which was 1.50 times that of LhTRX-h3-OE-2, 1.33 times that of LhTRX-h3-OE-10, 1.28 times that of LhTRX-h3-KO-6, and 1.25 times that of LhTRX-h3-KO-8. In comparison, the water loss rate of leaves of LhTRX-h3-OE was significantly lower than that of WT and LhTRX-h3-KO. After 5 hours in vitro, the water loss rate of leaves of WT reached 63.01%, that of LhTRX-h3-OE-2 reached 53.91%, which was 0.86 times that of WT, that of LhTRX-h3-OE-10 reached 54.58%, which was 1.01 times that of WT, that of LhTRX-h3-KO-6 reached 62.94%, which was 1.15 times that of WT, and that of LhTRX-h3-KO-8 reached 60.68%, which was 0.96 times that of WT. The results showed that LhTRX-h3-OE had a lower water loss rate. There was no significant difference in the relative water content of leaves of each strain without stress, but after 5 days of stress, the relative water content of leaves of WT was 65.99%, which was similar to that of LhTRX-h3-KO-6 and LhTRX-h3-KO-8, which were 65% and 63.79%, respectively. The relative water content of leaves of LhTRX-h3-OE-2 and LhTRX-h3-OE-2 was 78.91% and 76.21%, respectively, which was 1.19 times and 1.15 times that of WT. Therefore, LhTRX-h3-OE had a higher water retention capacity and water loss rate, and it could protect the plant by reducing water loss and enhance the drought resistance of the plant under drought stress. Figure 9

[0203] ​6) Stomatal conductance measurement: Stomatal conductance was measured on leaves of plants subjected to drought stress for 5 days, including one wild-type line, two LhTRX-h3-OE lines, and two LhTRX-h3-KO lines. Three seedlings were selected from each line, and leaves of the same size and position were selected from each seedling to prepare slides. Five pictures were taken from each slide for data analysis.

[0204] The results are as follows Figure 10 As shown, before drought stress, there was no significant difference in the stomatal length-to-width ratio of leaves of WT, LhTRX-h3-OE-2, LhTRX-h3-OE-10, and LhTRX-h3-KO-8 plants. However, after drought stress, the stomatal length-to-width ratio of leaves of WT, LhTRX-h3-OE-2, LhTRX-h3-OE-10, and LhTRX-h3-KO-8 plants increased significantly. Compared with WT and LhTRX-h3-KO-8, the increase of LhTRX-h3-OE-2 and LhTRX-h3-OE-10 was more significant. This indicates that the stomatal closure rate of the overexpressing lines was faster under drought stress, and that LhTRX-h3-OE plants were more sensitive to drought stress and could close stomata more quickly to reduce water loss.

[0205] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. The application of hybrid Liriodendron tulipifera LhTRX-h3 gene with nucleotide sequence as shown in SEQ ID NO. 1 in regulating plant growth, characterized in that, The plant growth is regulated to reduce plant height, petiole length, main root length, leaf area, chlorophyll content, and leaf color; and the plant is a hybrid Liriodendron chinense.

2. Application of a hybrid Liriodendron chinense LhTRX-h3 gene with a nucleotide sequence as shown in SEQ ID NO. 1 in accelerating the speed of stomata closure of a strain overexpressing the gene under drought stress; the strain is a hybrid Liriodendron chinense.

Images