Mirophyllum gene mfbhlll gene, encoding protein and use in plant drought improvement

Abstract

The application discloses a Myrobalan gene MfbHLH11, a coding protein and application in plant drought resistance improvement, and belongs to the technical field of genetic engineering.The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is shown as SEQ ID NO.2.The application obtains transgenic ground cover chrysanthemum and dwarf petunia with higher drought tolerance.Meanwhile, under the drought and water loss stress, the Myrobalan gene MfbHLH11 can quickly respond and enhance the drought resistance of plants, thereby providing a theoretical basis and utilization value for improving the drought tolerance of other plants by using the gene.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a Myrothamnus gene MfbHLH11 gene, a coding protein and application thereof in plant drought resistance improvement. BACKGROUND

[0002] Drought stress refers to that a plant cannot obtain water required for its life activities from the environment, so that its physiological activities are inhibited. Water is the source of life and plays an irreplaceable role in the whole life history of a plant. Water deficiency will affect the growth and development of a plant, thereby affecting economic benefits brought by the plant. A plant will cope with drought stress through various mechanisms, such as accumulating more soluble sugar and proline or increasing its drought tolerance by improving the activity of antioxidant enzymes. In a plant cell, the expression amount of some stress response genes will also change. Among the stress response genes, transcription factors (TFs) play an important regulatory role. So far, many transcription factors related to drought stress have been found.

[0003] The bHLH (Basic Helix-Loop-Helix) transcription factor family is the second largest family in plants next to the MYB family, and has been found in eukaryotes such as animals, plants and fungi. The bHLH domain usually contains about 60 amino acids and has two different functional regions, i.e., an alkaline region and a helix-loop-helix (HLH) region. The bHLH family transcription factor coping mechanism for drought stress mainly involves regulation from aspects such as stomatal opening, root hair and leaf hair development and abscisic acid sensitivity. The survival rate of transgenic tobacco (Nicotiana tabacum) with overexpressed apple MdbHLH130 gene is improved under drought stress, and the ABA-induced stomatal closure degree and ROS accumulation amount are significantly reduced. It is shown that MdbHLH130 increases the drought tolerance of a plant by controlling the stomatal opening degree and removing ROS. The overexpression of bHLHm1 transcription factor MdSAT1 in apple can enhance the drought resistance of apple callus, and the transgenic Arabidopsis thaliana also has increased sensitivity to ABA. OsbHLH148 regulates the jasmonic acid signal related pathway by interacting with OsJAZ1 to improve the drought tolerance of a transgenic plant. It can be seen that the bHLH transcription factor plays an important role in the drought stress response of various plants and effectively improves the drought tolerance of a plant.

[0004] Myrothamnus flabellifolia, native to southern Africa, is the only woody plant known to be drought-resistant and resilient. Due to its unique physiological structure, Myrothamnus flabellifolia is highly resistant to drought. Its leaves and stems curl in response to drought but quickly recover upon watering, allowing it to survive water shortages for 9-12 months. However, the molecular mechanisms underlying its drought tolerance remain largely unknown. Therefore, in-depth research into its drought defense mechanisms is a highly valuable research topic. Summary of the Invention

[0005] In response to the above-mentioned deficiencies in the prior art, the present invention provides a MfbHLH11 gene of the Myrica chinensis plant, its encoded protein, and its use in improving plant drought resistance. During drought and water loss, the gene can quickly respond and adapt to stress, thereby enhancing the drought resistance of plants.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] A Myrica chinensis gene MfbHLH11 was cloned from Myrica chinensis leaf RNA by PCR. The core coding region is 768 bp in length and the nucleotide sequence is shown in SEQ ID NO.1.

[0008] The protein encoded by the M. miltiorrhiza gene MfbHLH11 consists of 255 amino acids, has a molecular weight of 63.812 kDa, an isoelectric point (pI) of 5.12, and contains the longest and most complete open reading frame, including a nuclear localization signal peptide "RRSPPNKKNQVKVPKKIHKAEREKLK" beginning at 24 aa. Domain analysis of the MfbHLH11 amino acid sequence revealed bHLH domains located at positions 27-39 aa and 235-243 aa, with an additional HLH domain at positions 43-93 aa. This indicates that MfbHLH11 possesses a bHLH domain and belongs to the bHLH class of transcription factors. Its amino acid sequence is shown in SEQ ID NO. 2.

[0009] A plasmid comprising the above-mentioned M. mellifera gene MfbHLH11 gene.

[0010] A recombinant expression vector comprising the above-mentioned Myrtlesia serrata gene MfbHLH11 gene.

[0011] A transgenic cell line comprising the above-mentioned M. mellifera gene MfbHLH11 gene.

[0012] An engineered bacterium comprising the above-mentioned Myrica ovata gene MfbHLH11 gene.

[0013] A gene chip comprises the above-mentioned M. philadelphica gene MfbHLH11.

[0014] The use of the above-mentioned Myrica ovata gene MfbHLH11 in screening or identifying drought-resistant varieties.

[0015] The above-mentioned melamine gene MfbHLH11, plasmid, recombinant expression vector, transgenic cell line, engineered bacteria or gene chip are used in improving the drought-resistant quality or germplasm resources of melamine, or in cultivating transgenic drought-resistant melamine varieties.

[0016] A preparation for improving the drought resistance of Milo, comprising a component that promotes the expression of the Milo gene MfbHLH11 as an active ingredient.

[0017] Furthermore, the active ingredient is a small molecule compound, shRNA, gRNA or short peptide.

[0018] Beneficial effects of the present invention:

[0019] The present invention has produced transgenic ground cover chrysanthemums and petunias with high tolerance to drought. Furthermore, under drought and water loss stress, the MfbHLH11 gene of the genus Myrtillus can rapidly respond and enhance the plant's drought resistance, providing a theoretical basis and utility for using this gene to improve drought tolerance in other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of homologous sequence alignment of the MfbHLH11 gene of the present invention;

[0021] Figure 2 is a phylogenetic tree of the M. milanorrhiza gene MfbHLH11 of the present invention;

[0022] Figure 3 This is the result of the subcellular localization of the Myrtlesia serrata gene MfbHLH11 of the present invention;

[0023] Figure 4 This is the positive identification result of the transgenic ground cover chrysanthemum plant carrying the MfbHLH11 gene;

[0024] Figure 5 This is the positive identification result of the transgenic petunia plant with MfbHLH11 gene;

[0025] Figure 6 The growth status of wild-type and transgenic ground cover chrysanthemum plants after drought stress treatment;

[0026] Figure 7 The growth status of wild-type and transgenic petunia plants after drought stress treatment;

[0027] Figure 8 To determine the chlorophyll content of wild-type and transgenic ground cover chrysanthemum plants;

[0028] Figure 9 Chlorophyll content determination of wild-type and transgenic petunia plants;

[0029] Figure 10 To determine the proline content in wild-type and transgenic ground cover chrysanthemum plants;

[0030] Figure 11 To determine the malondialdehyde (MDA) content in wild-type and transgenic ground cover chrysanthemum plants under stress;

[0031] Figure 12 To determine the SOD activity of wild-type and transgenic ground cover chrysanthemum plants under stress;

[0032] Figure 13 To determine the POD activity of wild-type and transgenic ground cover chrysanthemum plants under stress;

[0033] Figure 14 To determine the relative water content of wild-type and transgenic petunia;

[0034] Figure 15 The stomatal aperture of wild type and transgenic petunia was measured;

[0035] Figure 16 Determination of electrical conductivity of wild type and transgenic petunia;

[0036] Figure 17 Determination of soluble sugars in wild-type and transgenic petunia;

[0037] Figure 18 For the determination of soluble protein in wild type and transgenic petunia;

[0038] Figure 19 To determine the hydrogen peroxide (H2O2) content in wild-type and transgenic petunia plants;

[0039] Figure 20 Diaminobenzidine (DAB) staining of wild-type and transgenic petunia plants under drought stress;

[0040] Figure 21 Nitroblue tetrazolium (NBT) staining of wild-type and transgenic petunia plants under drought stress;

[0041] Figure 22 The superoxide anion (O 2- ) content. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0043] Example 1: Extraction of the MfbHLH11 gene from the Milo

[0044] 1. Obtain leaves of Mironia serrata, snap-frozen with liquid nitrogen, and stored in a -80°C freezer for total RNA extraction. Total RNA was extracted using a polysaccharide and polyphenol sample plant RNA extraction kit purchased from Chengdu Lanbo Biological Company. cDNA was synthesized using innovagene's inNova UScript II All in One First-Strand cDNA Synthesis SuperMix according to the product instructions for first-strand synthesis. The first-strand cDNA synthesized by the above kit was used as the amplification template, and the designed primers F: 5'-TCCCCCGGGA TGAGCATGGACTCTGAGGT-3' and R: 5'-GACTAGTTTACAAATTGTCAACT GCGAGT-3' were used. The added restriction sites were F: SmaI and R: SpeI, respectively. PCR was used to amplify the cDNA as a template. The amplification system is shown in Table 1. The amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles, and final extension at 72°C for 5 min.

[0045] Table 1 PCR amplification system

[0046]

[0047] 2. After PCR, electrophoresis analysis was performed. The amplified fragment was recovered using a gel recovery and purification kit from Nanjing Novozymes. The amplified fragment was ligated into the Peasy-T1 Simple cloning vector and transformed into Escherichia coli DH5α competent cells. White colonies were picked for colony PCR to identify positive clones. The positive clones were sent to Chengdu Qingke Biotechnology Co., Ltd. for determination of the coding sequence of the MfbHLH11 gene of the milosia serrata. The sequence is shown in SEQ ID NO. 1. The open reading frame of the obtained gene was then translated into an amino acid sequence using ORF Finder software. The amino acid sequence is shown in SEQ ID NO. 2.

[0048] Example 2: Sequence homology and homology analysis of M. milanorrhiza MfbHLH11

[0049] According to the sequence results, sequence alignment was performed in the NCBI database, and it was found that the cloned gene sequence was most homologous to the bHLH family transcription factor. The protein sequence of the transcription factor and other members of the bHLH transcription factor family were aligned, and the SMART was used to analyze the domain of the MfbHLH11 amino acid sequence. The two low complexity domains were at 27aa-39aa and 235aa-243aa, and the HLH domain was at 43aa-93aa. The multiple sequence alignment results showed that the MfbHLH11 gene had high consistency with the bHLH genes with high homology in other plants, which further confirmed that the MfbHLH11 gene belonged to the bHLH class of transcription factors (such as Figure 1 ). Further construction of a phylogenetic tree using the aligned amino acid sequences of species with high homology (such as Figure 2 ) found that the target gene was clustered into a branch alone and had a close genetic relationship with AcbHLH47-like of Actinidia chinensis, VcbHLH011 of Vaccinium myrtillus, VrbHLH47-like of Vitis riparia, CsbHLH47-like of Camellia sinensis, ClbHLH47 of Camellia reticulata, and VvbHLH62 of Vitis vinifera.

[0050] Example 3: Construction of MfbHLH11 plant expression vector of Myrothamnus flabellifolia

[0051] The above sequenced plasmid was extracted, and the MfbHLH11 gene-containing cloning vector plasmid was digested with SmaI and SpeI. The DNA fragment was recovered using a gel recovery kit, and the fragment was connected with the linearized pCAMBIA1300 vector recovered after digestion. The obtained vector was named 35S: : pCAMBIA1300-MfbHLH11.

[0052] Example 4: Subcellular localization of MfbHLH11

[0053] According to the gene sequence of MfbHLH11 and the structural characteristics of the plant expression vector pCAMBIA1300-YFP, two appropriate enzyme digestion sites SacI and KpnI were selected, and the stop codon at the end of the MfbHLH11 gene was removed. The upstream and downstream primers were designed to construct the YFP fusion protein, and the primers were synthesized by Chengdu Oke Biological Company. The specific sequences of the primers are as follows:

[0054] SacI-F: 5'-GAGAACACGGGGGAC GAGCTC ATGAGCATGGACTCTGAGG T-3';

[0055] KpnI-R: 5'-GCCTTGCTCACCA GGTACCCAAATTGTCAACTGCGAGT-3';

[0056] The MfbHLH11 gene was ligated with the linearized fragment of the pCAMBI A1300 vector recovered after enzyme digestion using the REⅢMix homologous recombination kit to construct the fusion vector 35S::pCAMBIA1300-MfbHLH11-YFP. The pCAMBIA1300-MfbHLH11-YFP fusion protein plus the nuclear localization protein NLS-RFP was used as the experimental group, and the pCAMBIA1300-YFP empty vector plus the nuclear localization protein was used as the blank control group. They were injected into Nicotiana benthamiana and cultured for 48-72 hours. The transfected leaf preparations were observed under a confocal microscope and it was found that the blank control group could detect fluorescence signals at all locations of the cells, while the experimental group could only detect fluorescence signals in the cell nucleus. This result shows that the MfbHLH11 gene is located in the cell nucleus, which is consistent with the bioinformatics prediction results. The observation results are as follows Figure 3 shown.

[0057] Example 4: Genetic transformation of the MfbHLH11 gene

[0058] Thaw the Agrobacterium GV3101 competent cells on ice, take 5 μL of the recombinant plasmid 35S:: pCAMBIA1300-MfbHLH11 into 200 μL competent cells, mix gently with a pipette tip, place on ice for 30 minutes, freeze in liquid nitrogen for 1 minute, and place in a 37°C water bath for 5 minutes. After placing on ice for 2 minutes, inoculate the positive bacterial solution into 1 mL (40 μg / mL Chl and 20 μg / mL Rif) YEP liquid medium and culture at 28°C for 2 days until turbid. Pipette 100 μL of the turbid bacterial solution and add it to 50 mL (40 μg / mL Chl and 20 μg / mL Rif) YEP liquid medium and culture at 28°C overnight with shaking. Pipette 2 mL of the cultured bacterial solution using a UV spectrophotometer to measure the OD value of the bacterial solution. 600 The final concentration was diluted to OD 0 using antibiotic-free 1 / 2MS liquid medium in a clean bench. 600 =0.3, as a reserve for subsequent infection.

[0059] Ground cover chrysanthemum: Select healthy wild-type ground cover chrysanthemum sterile seedlings propagated in tissue culture bottles, and on a clean bench, cut the middle and upper leaves to make the edges of the leaves into a square of one square centimeter. Place the back of the leaf on the pre-culture medium and culture under light for 48 hours. Remove the pre-cultured leaf disc from the culture medium and soak it in the prepared Agrobacterium infection solution for 10 minutes to ensure that each leaf is in full contact with the bacterial solution. After taking it out, place it on a sterilized filter paper disc, first use the sterilized filter paper to absorb excess bacterial solution, and then air-dry for about 2 minutes. Place the leaves without excessive bacterial solution (to prevent excessive reproduction of Agrobacterium) on the co-culture medium and culture in the dark for 48 hours. After the co-culture is completed, transfer the leaf disc to the delay culture medium on the clean bench and carry out delayed culture for 10 days. After 10 days, transfer the leaf disc to the meristematic culture medium and then change it every two weeks until young shoots grow. When the young shoots grow from the leaf disc to about 2-3 cm in height, cut off the young shoots (one per bottle) and transfer them to the rooting screening medium (Kan, 50 μg / mL) to wait for rooting and culture into seedlings. Some ground cover chrysanthemum seedlings that have not been successfully transferred with the recombinant vector will stop growing or even turn yellow and die at this stage. DNA is extracted from surviving plants and tested by PCR (such as Figure 4 ), obtain T1 generation transgenic plants, and repeat the screening steps until positive overexpression pure line plants are screened out.

[0060] Petunia: Select young petunia leaves and disinfect them by soaking them in 20% Bleach for 15 minutes. Rinse them three times with sterile water. Cut the leaves into small squares, avoiding the veins and any damaged areas. Place the leaves, facing upward, on the pre-incubation medium and incubate for 2 days. Ensure that the wound edges of the leaves are fully exposed to the medium. Remove the pre-incubated leaf disc from the medium and soak it in the prepared Agrobacterium infection solution, shaking it constantly to ensure full contact for 10 minutes. Then, use tweezers to remove the leaf disc and place it on a sterilized filter paper disc. Use sterile filter paper to remove the bacterial solution and allow it to dry. Transfer the dried leaf disc to the co-cultivation medium and incubate in the dark for 2 days. After the co-cultivation, rinse the leaf with a 250 mg / L Cef solution and then rinse three times with sterile water. Use sterile filter paper to remove excess water and allow it to dry. Transfer the leaf disc to the regeneration medium and incubate at room temperature for 4 weeks. The regeneration medium was changed every 4 weeks until callus seedlings were grown, and then transferred to rooting screening medium (Kan, 50μg / mL) to wait for rooting and culture into seedlings. Some seedlings that were not successfully transferred with the recombinant vector will stop growing or even turn yellow and die at this stage. DNA was extracted from surviving plants and tested positive by PCR (such as Figure 5 ), obtain T1 generation transgenic plants, and repeat the screening steps until positive overexpression pure line plants are screened out.

[0061] Example 5: Stress treatment of transgenic ground cover chrysanthemum and petunia

[0062] Drought stress treatment: The control group (denoted as control) was watered normally, while the experimental group was watered and subjected to natural drought stress. Each treatment was repeated three times. The degree of wilting of the plants during drought treatment was observed, and rewatering was performed uniformly when severe wilting occurred. All plants were observed during the drought stress period, and photos were taken to record the differences in phenotypic characteristics between WT and transgenic lines (the phenotypes of ground cover chrysanthemum and petunia are as follows: Figure 6 、 7 ).

[0063] By studying the phenotypes of the roots and adult plants of wild-type and overexpressing ground cover chrysanthemum and petunia seedlings under drought, it was found that the ground cover chrysanthemum overexpressing MfbHLH11 gene lines Line-24, Line-27 and Line-28 and the petunia overexpressing MfbHLH11 gene lines Line1 and Line4 were more tolerant to drought than WT, indicating that overexpression of the MfbHLH11 gene improved the tolerance of ground cover chrysanthemum and petunia to drought stress.

[0064] Example 6: Determination of Physiological Indicators of Ground Chrysanthemum

[0065] 1. Cultivation of ground cover chrysanthemum

[0066] After acclimatization, WT and transgenic plants were transplanted into pots filled with an equal mass of mixed soil (humus soil: vermiculite = 1:1). One plant was grown per pot in a greenhouse (22 ± 2°C, 12 / 12h photoperiod) until they had 7–8 leaves. Before stress treatment, the pots were flooded with an equal amount of water 24 hours in advance to saturate all plants with water. After the start of treatment, watering was stopped in the stress-treated experimental group, and natural drought stress was applied. The conventionally cultured control group was watered and maintained normally. Each treatment was replicated three times.

[0067] 2. Determination of chlorophyll content under stress

[0068] After 17 days of cultivation of ground cover chrysanthemum, 0.3 g of fresh leaf samples of WT, Line-24, Line-27, and Line-28 plants with normal growth and drought treatment were taken respectively. The leaves were completely immersed in 95% ethanol solution and placed in a 25°C constant temperature incubator in the dark for 48 hours. Use a pipette to draw 200 μL of the pigment extract after light protection treatment and add it to the wells of the enzyme labeling plate. Use 95% ethanol solution as a blank control. Set the enzyme reader to measure the absorbance of each sample at wavelengths of 665 nm and 649 nm. Repeat this step three times. The results of chlorophyll content of ground cover chrysanthemum and petunia are shown in the figure. Figure 8 、 9 .

[0069] like Figure 8 、 9As shown in the figure, the chlorophyll content of each transgenic line overexpressing MfbHLH11 increased after stress treatment, and the increase was significantly greater than that of the WT. This indicates that the ground cover chrysanthemum and petunia plants overexpressing the MfbHLH11 gene have better resistance to drought stress than the wild type.

[0070] 3. Determination of MDA content under stress

[0071] After culturing ground cover chrysanthemum for 17 days, 0.3 g fresh leaf samples were taken from the normal growing and drought treated WT, Line-24, Line-27 and Line-28 plants respectively.

[0072] Preparation of enzyme solution: Place the sample leaf in a mortar and pestle and add 1.5 mL of phosphate buffer twice. Grind the leaf thoroughly until a homogenate is formed without leaf fragments (grinding should be performed on ice and as quickly as possible to prevent sample oxidation). Centrifuge at 10,000°C for 15 minutes. The supernatant is the malondialdehyde extract.

[0073] Prepare the MDA reaction solution: Weigh 0.6 g of thiobarbituric acid (TBA) into a small beaker. Add a small amount of 1 M NaOH solution and stir with a glass rod until the TBA is completely dissolved and no particles are present. Transfer the solution to a 100 mL volumetric flask, protected from light, and then add a sufficient amount of 10% trichloroacetic acid (TCA) solution to make the volume 100 mL. Shake well, wrap in tin foil, and store in a dark place.

[0074] Take 5mL centrifuge tubes and number them, first draw 2mL of reaction solution and add it to each tube. Then quickly add 1mL of each sample enzyme solution according to the number, shake the reaction solution and enzyme solution, place them in a water bath and boil for 15 minutes. Cool quickly and then centrifuge again. Take the supernatant and inject it into the enzyme plate and put it into the enzyme reader, and measure the absorbance (A) value at wavelengths of 450, 532 and 600nm respectively. Calculate the MDA content according to the formula, repeat 3 times, and the results are shown in the table. Figure 10 .

[0075] like Figure 10 As shown in the figure, under stress conditions, the MDA content of each line increased, but the MDA content of the leaves of WT plants was significantly higher than that of Line24, Line27 and Line28, indicating that the transgenic line overexpressing MbHLH11 can minimize the damage to its own membrane system under stress.

[0076] 4. Determination of superoxide dismutase (SOD) activity under stress

[0077] After 17 days of cultivation of ground cover chrysanthemum, 0.1 g of fresh leaf samples of WT, Line-24, Line-27, and Line-28 plants under normal conditions and drought treatment were collected, 5 mL centrifuge tubes were taken and numbered, and 3 mL of reaction solution was first aspirated and added to each tube. Then, 20 μL of the corresponding sample enzyme solution was quickly added according to the number, and the reaction solution and enzyme solution were mixed. Two blank controls, one and two, were set up, and 3 mL of reaction solution was added as with the sample tubes, of which 20 μL of enzyme solution was replaced with 20 μL of PBS; the experimental groups of blank control one and sample tube were placed in light reaction (4000 Lux 30 min), and blank control two was placed in a dark place for 30 min. The microplate reader was zeroed with 200 μL of the dark-treated blank control two solution, and then the absorbance of blank control one (control tube) and each sample (sample tube) at a wavelength of 560 nm was measured. The SOD activity unit is an enzyme activity unit (u) based on the inhibition of 50% of NBT photoreduction. The SOD activity was calculated according to the formula, and repeated 3 times. The results are shown in the table. Figure 11 .

[0078] like Figure 11 As shown, superoxide dismutase can scavenge superoxide anion free radicals in plants and improve plant resistance to stress. Under drought stress, the SOD activity in the leaves of plants of each line increased to varying degrees, and the SOD activity of plants of Line 24, Line 27, and Line 28 lines was higher than that of the WT line.

[0079] 5. Determination of peroxidase (POD) activity under stress

[0080] After 17 days of cultivation of ground cover chrysanthemum, 0.1 g of fresh leaf samples of WT, Line-24, Line-27, and Line-28 plants under normal conditions and drought treatment were collected, and three replicates of one sample were measured at a time. 300 μL of reaction solution was added to the wells of the enzyme labeling plate to be measured in advance, and 3 μL of enzyme solution and a blank control replaced with distilled water were added to each well using four pipettes for measurement. The wavelength of the enzyme reader was set to 470 nm, and the absorbance value of each sample well was measured once every 1 min, with an interval of 3 minutes, for a total of 4 measurements. A change of 0.01 in △A470 per minute was defined as 1 peroxidase activity unit (u), and the POD activity was calculated according to the formula. Repeat 3 times, and the results are shown in the table. Figure 12 .

[0081] like Figure 12 As shown in the figure, under drought and salt stress, the POD activity in the leaves of plants of each line increased, but the POD activity of plants of Line24, Line27 and Line28 were higher than that of the WT line.

[0082] 6. Determination of proline content under stress

[0083] Prepare a calibration curve: Pipette 0.5mL-3mL (0.5mL intervals) of the prepared proline solution into six labeled 50mL volumetric flasks. Add ddH2O to bring the six flasks to 50mL. The proline concentration in each flask should be 1-6μg / mL. Pipette equal amounts of the proline standard solution, ninhydrin solution, and glacial acetic acid from each flask and boil in a water bath for 30 minutes. Measure the absorbance of each flask at 520nm. Create a calibration curve plot based on the results and calculate the regression equation.

[0084] After 17 days of cultivation of ground cover chrysanthemum, 0.1 g of fresh leaf samples of WT, Line-24, Line-27, and Line-28 plants under normal conditions and drought treatment were collected. 1 mL of sulfosalicylic acid solution was used to grind the sample leaves until there was no particles in the slurry, and then the slurry was boiled in a water bath for 10 minutes. The sample solution was taken out and cooled to room temperature, and then 2 mL of the sample solution, glacial acetic acid, and ninhydrin solution were taken, added to a new centrifuge tube, mixed, and treated in a boiling water bath for 0.5 h. The treated mixed solution was taken out and cooled to room temperature, and 4 mL of toluene was carefully pipetted and added to each sample mixed solution. After mixing with a pipette, the toluene was extracted from the proline in the aqueous phase, and then it was allowed to stand and wait for stratification. 200 μL of the upper colored organic phase was aspirated and added to the wells of the enzyme labeling plate. The absorbance of each sample was measured at a wavelength of 520 nm using the enzyme labeler. This step was repeated three times. The results are shown in the table. Figure 13 .

[0085] like Figure 13 As shown in the figure, under drought stress conditions, the proline content in the Line24, Line27, and Line28 transgenic lines was much higher than that in the wild type, indicating that overexpression of the MfbHLH11 gene can better adapt to drought conditions in ground cover chrysanthemum.

[0086] Example 7: Determination of Physiological Indicators of Petunia

[0087] 1. Cultivation of petunia

[0088] T1-generation positive petunia plants were cultured in a greenhouse (22 ± 2°C, 12 / 12h photoperiod) until they had 7–8 leaves. Before stress treatment, pots were flooded with an equal amount of water 24 hours in advance to saturate all plants with water. After the start of treatment, watering was stopped in the stress-treated experimental group, and natural drought stress was applied; the conventionally cultured control group was watered normally. Each treatment was replicated three times. Plants were rewatered when they showed severe wilting. The degree of wilting during drought treatment was observed.

[0089] 2. Determination of relative water content of petunia

[0090] Take each strain drought stress treatment after the experimental group and the control group under normal culture leaves, respectively, weighing, recorded as the original fresh weight. Then the leaves are completely immersed in deionized water for 24 h, until the leaves are fully saturated with water. Take out the leaves with filter paper to dry the water, weighing recorded as saturated fresh weight. Finally, the leaves are placed in glass dishes, placed in a 90°C oven to dry without water, at this time, the leaf dry weight is taken, and then the relative water content is calculated by the formula, the results are shown in Figure 14 .

[0091] As shown in Figure 14 , under drought stress, the RWC of transgenic petunia lines is significantly higher than that of WT plants. From the above data, it can be concluded that overexpression of MfbHLH11 gene can increase the water retention capacity of petunia to improve the tolerance to drought stress.

[0092] 3. Measurement of stomatal aperture of petunia

[0093] Take 18 pieces of WT and each line plant, respectively, mark and place in 100 mL MES-KCl buffer (50 mM KCl, 0.1 mM CaCl2, 10 mM MES, pH 6.15) (leaf back down, make it fully contact with the liquid surface) light induction 2.5 h, then take 6 pieces of leaves of each line in the MES-KCl buffer containing 300 mM mannitol (50 mM KCl, 0.1 mM CaCl2, 10 mM MES, pH 6.15) under light treatment for 2 h, after treatment, the leaves are taken out, the water on the leaves is wiped dry with filter paper. Then use tweezers and transparent tape to stick the lower epidermis on the glass slide to make a temporary mount. Place the glass slide under the microscope, observe and 42 shoot the closure of the stomata. About 30 stomata diameters of each line of each treatment are counted for data analysis (see Figure 15 ).

[0094] As shown in Figure 15 , under 300 mM mannitol treatment, the stomatal closure of overexpression plants Line1 and Line4 is higher than that of wild type plants ( Figure 15 A). Statistical analysis of 30 stomata data shows that the stomatal closure degree (ratio of width to length) of overexpression plants is significantly smaller than that of WT (as shown in Figure 15 B), which is extremely significant. Therefore, under drought stress treatment, plants overexpressing MfbHLH11 gene can close the stomata in time, minimize the loss of water in the body, and improve the plant's resistance to water loss.

[0095] 4. Measurement of electrical conductivity of petunia

[0096] Weigh 1g of leaves from the drought-treated experimental group plants and the conventionally cultured control group plants respectively, cut them into pieces as much as possible, place them in a 50mL centrifuge tube filled with an equal amount of deionized water, put them in a vacuum box, and use a vacuum pump to evacuate for 30 minutes (the time can be adjusted according to the water saturation of the leaves). After all the leaves in the tube are saturated with water and sink in the solution, take out the centrifuge tube and let it stand at room temperature. Then use a conductivity meter to measure the conductivity of the liquid at this time, recorded as L1, and then put the centrifuge tube into a water bath and heat and boil for 20 minutes, take out the centrifuge tube and let it stand and cool to room temperature. Then measure the conductivity of the liquid at this time, recorded as L2. Use the formula to calculate the relative conductivity of each strain and compare the differences in plasma membrane permeability between strains. See the results. Figure 16 .

[0097] like Figure 16 As shown in the figure, under drought stress, the REC of transgenic petunia plants was significantly lower than that of WT plants. The results indicate that overexpression of the MfbHLH11 gene reduces the damage of the plasma membrane of petunia plants under drought stress and enhances their drought resistance.

[0098] 5. Determination of soluble sugar in petunia

[0099] The plants in the treatment group after drought stress and the control group under normal culture were uniformly sampled, and 0.1g of leaves were weighed for each sample. The leaf samples were added to 1mL of distilled water and ground into a homogenous slurry without particles in a mortar on ice. The homogenate was transferred to a centrifuge tube and sealed to prevent the tube from bursting. It was boiled in a water bath for 10 minutes, cooled at room temperature, and centrifuged at 4000r for 10 minutes. 20μL of the supernatant was diluted 10 times with distilled water to 200μL, and shaken well as the sample solution for the next step. The standard stock solution and the standard diluent were taken at a ratio of 1:9, mixed, and a 100μg / mL standard application solution was prepared. The substrate solution in the kit and the self-prepared concentrated sulfuric acid were added to the 200μL sample diluent in the centrifuge tube in turn, while the blank tube and the standard tube used double distilled water and the standard application solution instead of the sample solution. After mixing, the sample was boiled in a water bath for 10 minutes. The sample absorbance was measured at a wavelength of 620nm, and the soluble sugar content was then calculated using the formula. The results are shown in Figure 17 .

[0100] like Figure 17 As shown in the figure, under drought stress, the soluble sugar content of all plants increased, but the difference between the transgenic plants and the WT plants reached a highly significant level. The results show that overexpression of the MfbHLH11 gene can improve the tolerance of petunia plants to drought stress by increasing the soluble sugar content.

[0101] 6. Determination of soluble protein in petunia

[0102] The plants in the treatment group after drought stress and the control group under normal culture were uniformly sampled, and 0.1g of leaves were weighed for each sample. 0.9mL of pH 7.4 phosphate buffer was used to grind in a mortar on ice until a homogenous slurry without particles was formed. The homogenate was transferred to a 1.5mL centrifuge tube and centrifuged at 4000r for 15min at 4℃, and the supernatant was retained. The sample solution and Coomassie Brilliant Blue colorimetric solution were added to the centrifuge tube in sequence. The blank tube and the standard tube used distilled water and protein standard solution instead of the sample solution. After mixing, the mixture was allowed to stand for 10min. The wavelength of the enzyme reader was set to 595nm, and the absorbance of each sample was measured. The results are shown in the figure. Figure 18 .

[0103] like Figure 18 As shown in the figure, under drought stress, the soluble protein content of all plants showed a downward trend, but the WT plants decreased more than the transgenic plants, indicating that overexpression of the MfbHLH11 gene can maintain the nutrients in the petunia body in a steady state, thereby coping with the drought stress environment.

[0104] 7. Determination of hydrogen peroxide (H2O2) content in petunia

[0105] The plants in the drought stress treatment group and the control group were uniformly sampled, and 0.1g of each sample leaf was weighed. According to the ratio of weight (g): volume (mL) = 1:9, 0.9mL of phosphate buffer (PH7.4) was added and the sample leaves were ground until homogenized without particles. Centrifuge at 4000r for 15min at 4℃ and retain the supernatant. The specific operation was carried out according to the Hydrogen Peroxide assay kit of Nanjing Jiancheng Biology, repeated 3 times, and the results are shown in the table. Figure 19 .

[0106] like Figure 19 As shown in the results, under drought stress, the accumulation of reactive oxygen species in Line1 and Line4 transgenic lines was less than that in the wild type, indicating that overexpression of the MfbHLH11 gene in petunia can help plants eliminate the accumulation of reactive oxygen species, thereby reducing the damage caused by peroxides.

[0107] 8. Petunia diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining

[0108] Prepare a 1mg / mL NBT solution and a 1mg / mL DAB solution in advance. Place 30mL of staining solution in 50mL centrifuge tubes per tube. Place leaves from each drought-stressed and normally cultured control group in the staining solution until completely submerged. Place no more than five leaves per tube to prevent damage. DAB staining should be performed in the dark overnight (staining time can be adjusted depending on the staining situation). When dark brown spots appear on most leaves, remove the leaves and place them in a new 50mL centrifuge tube and add anhydrous ethanol. For NBT staining, place the leaves in the dark overnight and then in the light for an additional 24-48 hours (lighting time can be adjusted depending on the staining situation). When most leaves develop dark blue spots, transfer them to anhydrous ethanol for decolorization. Place the leaves in anhydrous ethanol in a water bath at 80°C for 20 minutes, changing the ethanol several times. Once the leaves have faded to a transparent state, photograph them. The results of diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining were as follows Figure 20 、 21 shown

[0109] 9. Petunia superoxide anion (O 2- ) content

[0110] The plants in the treatment group after drought stress and the control group under normal culture were uniformly sampled, and 0.1g of each sample leaf was weighed. The sample leaves were placed in a mortar and 1mL of extract was added. The leaves were ground on ice until they were homogenous and free of particles. Transferred to a 1.5mL centrifuge tube and centrifuged at 10000r for 20min at 4℃, and the supernatant was retained. The specific operation was carried out according to the superoxide anion content determination kit of Suzhou Keming Biology, repeated 3 times, and 200μL of the upper solution in each sample tube was added to the sample well of the quartz enzyme labeling plate. The absorbance of each sample was measured by setting the enzyme labeling instrument wavelength to 530nm. The results are as follows: Figure 22 shown.

[0111] When plants are subjected to abiotic stress, a large amount of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anions (O2-), accumulate in their cells. Excessive accumulation of ROS leads to imbalance of oxidative metabolism and lipid peroxidation, which in turn increases the degree of cellular oxidative damage. Under drought stress, the O 2- The content of O in WT plants was increased. 2- The content of MfbHLH11 increased more significantly and was significantly different from that of transgenic petunia, indicating that overexpression of the MfbHLH11 gene can reduce the damage caused by ROS accumulation caused by drought stress to plants.

[0112] Finally, it should be noted that the above detailed description is merely illustrative of the technical solutions of the present application and is not limiting, and although the present application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present application, and all should be covered in the scope of the claims of the present application.

Claims

1. A milo gene MfbHLH11 , characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The milo gene of claim 1 MfbHLH11 The encoded protein is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A method comprising the mulberry gene according to claim 1 MfbHLH11 Gene plasmid.

4. A method comprising the mulberry gene according to claim 1 MfbHLH11 Recombinant gene expression vector.

5. A method comprising the mulberry gene according to claim 1 MfbHLH11 A transgenic cell line comprising a gene, characterized in that The transgenic cell lines do not include plant cell lines.

6. A method comprising the mulberry gene according to claim 1 MfbHLH11 Genetically engineered bacteria.

7. A gene chip, characterized in that Comprising the milo gene of claim 1 MfbHLH11 .

8. The milo gene according to claim 1 MfbHLH11 , the use of the plasmid according to claim 3, the recombinant expression vector according to claim 4, the transgenic cell line according to claim 5, the engineered bacteria according to claim 6 or the gene chip according to claim 7 in improving the drought-resistant quality or germplasm resources of Milo, or in cultivating transgenic drought-resistant Milo varieties.

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