Carya illinoinensis cispl3 gene and application thereof in regulating flowering time and stress tolerance of plants

Abstract

The application discloses a Carya illinoinensis CiSPL3 gene, and a nucleotide sequence of the CiSPL3 gene is shown as SEQ ID NO. 1. The application provides an important gene affecting flowering time and drought and salt stress tolerance of Carya illinoinensis CiSPL3 , and overexpression of the gene makes plants bloom earlier, enhances drought tolerance, and enhances salt stress sensitivity, thereby expanding the identification and application of the gene family CiSPL3 . SPL ​

Description

Technical Field

[0001] This invention relates to the separation of from thin-shelled pecans CiSPL3 The application of genes, especially in thin-shelled pecans CiSPL3 The application of genes in regulating plant flowering time and stress falls under the field of plant genetic engineering technology. Background Technology

[0002] Thin-shelled pecans Carya illinoinensis [Wangehn.) K. Koch] is a plant belonging to the genus *Caragana* in the family Juglandaceae. Also known as American pecan, it is a type of pecan. Its nuts are large, thin-shelled, and have a high kernel yield. The kernels are flavorful, aromatic, and nutritious, making them an ideal health food. Thin-shelled pecans are also an important woody oilseed tree species, with kernel oil content exceeding 70%, of which unsaturated fatty acids account for up to 97%. They are also highly storable and make for a superior edible oil. With economic development and improved living standards, the demand for thin-shelled pecans in domestic and international markets is increasing daily, indicating a huge market potential for the thin-shelled pecan industry. Currently, it is being cultivated on a large scale in suitable areas of Jiangsu, Yunnan, Zhejiang, and Anhui provinces.

[0003] Thin-shelled pecans have a long vegetative growth period, late flowering and fruiting, low early yields, and poor economic benefits, which is a bottleneck restricting the development of the industry. Under ideal conditions, young thin-shelled pecan trees (grafted seedlings) need 4-5 years to bear fruit (yield of only 2.8-3.5 kg / mu), and 7-8 years to 10-12.5 kg / mu. The yield and economic benefits per tree in the first 10 years are far lower than those of pecans of the same age. Therefore, to improve the early output and benefits of thin-shelled pecan cultivation and promote the healthy and rapid development of the industry, it is urgent to regulate the balance between vegetative and reproductive growth, promote the transformation of vegetative growth into reproductive growth (flowering transition), activate the expression of flowering factors, shorten the fruiting period, promote flower bud differentiation, and increase fruit yield. Drought stress is one of the most significant abiotic stresses for plants. Drought stress negatively impacts plant morphology, development, metabolism, and various physiological regulation processes. Vast areas in southern China are seasonally arid regions, mainly including Jiangsu, Anhui, Zhejiang, Jiangxi, Hunan, Hubei, and Sichuan provinces in the Yangtze River basin. These regions experience frequent and intense seasonal droughts, which often occur simultaneously with heat, resulting in high evaporation rates. This severely impacts the physiological metabolism, growth, development, fruit yield, and quality of economic trees. Thin-shelled pecans are particularly vulnerable to water deficit during their growth and development; therefore, only by meeting their water requirements in the introduced regions can their normal growth and the attainment of good fruit yield and quality be guaranteed.

[0004] SPL transcription factors are a family of genes unique to plants and widely distributed throughout the plant community. The SPL gene family is extensively involved in regulating plant growth and development and responding to abiotic stress. Switch millet... SPL7 and SPL8 Overexpression of [a substance] can promote flowering, while downregulation of [a substance] alone can [promote flowering]. SPL7 or SPL8 Gene expression can delay flowering to some extent. ZmSPL13

[0005] and ZmSPL29 It can significantly promote corn flowering, and the two have a certain degree of functional redundancy. SPL is a key regulator of plant stress response. The miR156 / SPL module promotes flowering in apples by upregulating SPL. MdWRKY100 To adjust salt tolerance. OsSPL10 The variation directly regulates the rice OsNAC2 The expression of SPL and the production of ROS confer drought resistance. Currently, most reported studies on the function of conserved SPL genes focus on plants such as Arabidopsis thaliana and rice. However, no studies on the function of the SPL gene have been reported in the economically important pecan tree. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a method for influencing the flowering time and abiotic stress resistance of the thin-shelled pecan 'Bonnie'. CiSPL3 Gene, CiSPL3 It is an important gene affecting the flowering time of thin-shelled pecan, as well as its drought and salt stress tolerance. Overexpression CiSPL3 Genes enable plants to flower earlier, increase drought resistance, and enhance their sensitivity to salt stress.

[0007] To solve the above-mentioned technical problems, this invention discloses a thin-shelled pecan. CiSPL3 Genes, the ones mentioned CiSPL3 The nucleotide sequence of the gene is shown in SEQ ID NO.1:

[0008] ATGGAAACAGGCAGAGCTGAGGGAAAGAGGAGCTTAATTACTACAATAACATGGTGGAGGAGGCTGAAGAGGAGGAGGATGAAGCTAGCGAGTTAGGATTTGGCGAGGATGAGAAGAAGAGAAATGACTCCCGCATCTAGCAGGAGAACATCATCTGGCAGTGGAGGGTCAACGCTGATTACTTGCCAGGCGGATAATTGCAATGCTGATTTCACCGAAGCC AAGCCGTACCACCGGCGCCATAAGGTCTGCGAATTTCATGCCAAGGCTCCGGTTGTCAATGTTGGTGGACTCCATAAAAGGTTCTGCCAGCAATGCAGCAGGTTCCATATGCTATCAGAGTTTGATGAAAACAAAAAGAGTTGTCGGAAACGTCTAGCTGGTCACAATGAGAGGCGTCGGAAAAGCTCATCCGATTCTTATGGAGAAGGTTCACATTGA (SEQ ID NO.1)

[0009] The present invention also provides the above. CiSPL3 The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO.2:

[0010] METGRAEGKRSLNYYNNMVEEAEEEEDEASELGFGEDEKKREMTPASSRRTSSGGSGGSTLITCQADNCNADFTEAKPYHRRHKVCEFHAKAPVVNVGGLHKRFCQQCSRFHMLSEFDENKKSCRKRLAGHNERRRKSSSDSYGEGSH (SEQ ID NO.2)

[0011] Furthermore, the present invention also provides an amplification method for the above-mentioned... CiSPL3 Gene primer pairs include CiSPL3 -F and CiSPL3 -R; the CiSPL3 The nucleotide sequence of -F is shown in SEQ ID NO.3; CiSPL3 The nucleotide sequence of -R is shown in SEQ ID NO.4.

[0012] Meanwhile, including the above CiSPL3 Recombinant gene expression vectors, expression kits, transgenic strains, or recombinant bacteria are also within the scope of protection of this invention.

[0013] This application also provides a thin-shelled pecan. CiSPL3

[0014] This application also provides a method for cloning the above-mentioned promoter, wherein the sequence used for promoter cloning is:

[0015] Upstream primer: TGCCTGCAGGTCGACTCTAGATAAGCATATTGGTACGTGTGCTC;

[0016] Downstream primer: GGACTGACCACCCGGGGATCCTTTGGCAAAACAAGAGTAGACAG.

[0017] In one specific implementation, by saying CiSPL3 The gene was ligated with the PHB vector to obtain PHB- CiSPL3 The overexpression vector was then transformed into *E. coli* DH5α competent cells for amplification and screening to obtain PHB- with correct sequencing results. CiSPL3 The overexpression vector was further transformed into Agrobacterium GV3101, ultimately yielding GV3101-PHB- CiSPL3 Engineered bacteria.

[0018] This invention further proposes the above-mentioned thin-shelled pecan. CiSPL3 Gene, CiSPL3 Proteins encoded by genes, or containing CiSPL3 Recombinant gene expression vectors, expression kits, recombinant bacteria and transgenic lines, and the application of the above promoters in regulating plant flowering time and abiotic stress.

[0019] Specifically, the regulation of plant flowering involves using thin-shelled pecans... CiSPL3 When the gene is overexpressed in plants, the resulting transgenic plants flower earlier, have increased resistance to drought stress, and reduced tolerance to salt stress.

[0020] Preferably, the plant is a thin-shelled pecan or Arabidopsis thaliana.

[0021] The application process includes the following steps:

[0022] 1) Provide the thin-shelled pecan CiSPL3 Gene;

[0023] 2) The thin-shelled hickory nuts CiSPL3 Genes are ligated with vectors to obtain recombinant vectors;

[0024] 3) The recombinant vector was transformed into Agrobacterium to obtain recombinant Agrobacterium;

[0025] 4) Infect the plants with the recombinant Agrobacterium to obtain overexpressed thin-shelled pecans. CiSPL3 Gene-based plants.

[0026] Beneficial effects: The present invention provides CiSPL3 It is an important gene affecting the flowering time of thin-shelled pecan, as well as its drought and salt stress tolerance. Overexpression CiSPL3 Genes enable plants to flower earlier, increase drought tolerance, enhance sensitivity to salt stress, and expand [the range of genes]. SPL Identification and application of gene families. Attached Figure Description

[0027] Figure 1 Phylogenetic analysis of plant SPL proteins;

[0028] Figure 2 for CiSPL3 Gene expression at different stages of female flower bud differentiation in pecan and in different tissues of pecan;

[0029] Figure 3 for CiSPL3 Functional analysis of genes in Arabidopsis thaliana, A: CiSPL3 Overexpression of the gene in Arabidopsis thaliana can significantly advance flowering. (B:) CiSPL3 Analysis of flowering days in transgenic Arabidopsis thaliana and wild-type plants;

[0030] Figure 4 for CiSPL3 Electrophoresis results of gene promoter PCR amplification (primers with restriction enzyme sites);

[0031] Figure 5 for CiSPL3 Analysis of cis-regulatory elements in gene promoters;

[0032] Figure 6 For GUS staining detection CiSPL3 Activity analysis of gene promoters in different tissues of Arabidopsis thaliana;

[0033] Figure 7 for CiSPL3 Functional analysis of genes in Arabidopsis thaliana: A: Growth of wild-type and transgenic Arabidopsis thaliana seedlings in MS medium containing 300 mM mannitol; B: Root length statistics after seedling germination; C: Plant phenotype after drought treatment.

[0034] Figure 8 for CiSPL3 Functional analysis of genes in Arabidopsis thaliana: A: Phenotypic results of wild-type and transgenic Arabidopsis thaliana seedlings 15 days after germination in MS medium containing 100 mM and 200 mM NaCl; B: Statistical analysis of root length after seedling germination; C: Phenotypic results of wild-type and transgenic Arabidopsis thaliana lines 20 days after germination in MS medium containing 100 mM NaCl; D: Phenotypic results of plants treated with 300 mM NaCl. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0036] Example 1 CiSPL3 Gene cloning.

[0037] Based on the thin-shelled pecan genome database CiSPL3 The cds sequence of the gene was cloned using the cDNA of female flower buds of pecan as a template. CiSPL3 Gene.

[0038] Specifically, female flower buds of 7-8 year old Carya illinoinensis 'Pawnee' were collected. Total RNA was extracted from the female flower buds using the TransZol Up Plus RNA Kit, and the RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit (Takara). Based on the CDS sequence of the Carya illinoinensis gene published in Phytozome 13.0 (Carya illinoinensis Pawnee v1.1), amplification primers were designed.

[0039] Upstream primer CiSPL3-F (SEQ ID NO.3): ATGGAAACAGGCAGAGCTGAGGG;

[0040] Downstream primer CiSPL3-R (SEQ ID NO.4): TCAATGTGAACCTTCTCCATAAG.

[0041] PCR amplification products were detected by 1% agarose gel electrophoresis and photographed using a gel imaging system. The target DNA band was excised, and the target fragment was recovered and purified using a gel extraction kit. It was then ligated into the pMD19-T vector to obtain the pMD19-CiSPL3 recombinant vector, which was then transformed into *E. coli* DH5α competent cells. Positive clones were selected and sent to the company for sequencing. The ligation reaction of the target gene was performed on a PCR instrument. The reaction system consisted of: 0.5 µl of pMD19-T Simple Vector, 4.5 µl of DNA fragment, and 5 µl of Solution I. After thorough mixing, the mixture was placed in a PCR instrument at 16°C for approximately 10 hours or overnight. The transformation procedure was as follows: 10 µl of the complete system was added to 100 µl of DH5α *E. coli* competent cells and incubated on ice for 30 minutes. The cells were then incubated at 42°C for 2 minutes, followed by incubation on ice for 2-3 minutes (do not shake the centrifuge tube during this process). On a clean bench, add 800 µl of LB liquid medium (without antibiotics) to the competent cell culture solution and incubate at 37°C with shaking for about 1 hour. Centrifuge at 37°C, 8000g for 5 minutes to precipitate the cells. Discard most of the supernatant on a sterile bench, leaving about 100 µl. Use a pipette to resuspend the cells and spread them evenly on LB solid medium containing kanamycin. Seal the plate with sealing film and incubate overnight at 37°C until single colonies grow. Pick a single colony and transfer it to LB liquid medium containing kanamycin. Incubate at 37°C, 200 rpm for about 5 hours with shaking. Use the bacterial culture as a template for PCR detection. If the target fragment is found, send the culture for sequencing.

[0042] The CiSPL3 gene sequence, 444 bp in length, encoding 147 amino acids, was cloned. Its base sequence is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2.

[0043] The relative molecular weight, theoretical isoelectric point, and hydrophilicity of CiSPL3 were analyzed using the online website ExPASy (https: / / web.expasy.org / protparam / ). Conserved domains of the protein were analyzed using the NCBI Conserved Domain Search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). Homologous sequences of CiSPL3 were searched in the NCBI database using BLAST, and a phylogenetic tree was constructed using MEGA 5.0 software.

[0044] Based on online predictions using ExPASy ProtParam, the CiSPL3 protein has a molecular weight of 16.67 kDa, a theoretical isoelectric point (pI) of 7.05, an instability coefficient of 73.36, a fatty acid amino acid index of 35.24, and an average GRAVY of -1.227, thus it is presumed to be an unstable hydrophilic protein. Analysis using NCBI Conserved Domain Database (CDD) shows that the CiSPL3 protein sequence contains an SBP domain between amino acids 63 and 137. Phylogenetic analysis of SPL3 proteins from different species shows... Figure 1 CiSPL3 and walnut JrSPL3 proteins are on the same small branch and are genetically close to walnuts.

[0045] Our previous study investigated the external morphology and anatomical structure of female flower buds in pecans, dividing them into five stages: FB1 (initial stage of female flower development), FB2 (female inflorescence formation stage), FB3 (female flower primordia formation stage), FL1 (early flowering stage), and FL2 (peak flowering stage). Then, we performed transcriptome sequencing analysis on the different differentiation stages of pecan female flower buds, such as... Figure 2 As shown in A, CiSPL3 The gene is continuously highly expressed during the transition from shoot apical meristem to floral meristem (undifferentiated stage - early differentiation stage - inflorescence formation stage), that is, its expression is significantly upregulated during the development of flower buds into female flowers. CiSPL3 The gene is expressed in all tissues of pecan, with significantly higher expression levels in female flowers and fruits than in other tissues, and the highest expression level in female flowers. Figure 2 B). This application provides CiSPL3 The gene holds promise for application in thin-shelled pecans to regulate the plant's flowering time and resilience to abiotic stress.

[0046] Example 2 CiSPL3 Construction of gene overexpression vectors.

[0047] Using the pMD19-CiSPL3 recombinant vector as a template, primers containing restriction enzyme sites were designed:

[0048] BamHI (SEQ ID NO.5): CTCTCTCTCAAGCTT GGATCC ATGGAAACAGGCAGAGCTGAGGG;

[0049] PstⅠ(SEQ ID NO.6):GATCAATTCGAGCTC CTGCAG TCAATGTGAACCTTCTCCATAAG. PCR amplification was performed using kod DNA polymerase (KOD-401, TOYOBO). The 20 ml PCR reaction system consisted of: 2 ml 10× buffer, 2 ml 2 mM dNTPs, 2 ml DNA template, 1 ml DMSO, 0.8 ml 10 pmol / ml forward and reverse primers, and 0.5 U kod DNA polymerase (KOD-401, TOYOBO). Water was added to make up to 20 ml. The PCR amplification program was: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, 50–64℃ annealing for 30 s, 68℃ extension for 1 min / kb, 40 cycles; incubation at 68℃ for 8 min. DNA electrophoresis was performed, and the target fragment was recovered.

[0050] CiSPL3 fragment and PHB vector (purchased from Shanghai Pujie Biotechnology Co., Ltd.) were double-digested using BamHI and PstⅠ restriction endonucleases. The digested fragments were recovered and ligated into the EasyGeno DNA recombination system (#VI201-02, Tiangen Biotech). The 10 μL recombination system consisted of 5 μL 2×EasyGeno Assembly Mix, 2.5 μL digested vector DNA, and 2.5 μL fragment DNA. The reaction mixture was added to a 250 μL EP tube, incubated at 50°C for 30 minutes, transformed into E. coli, plated, and incubated at 37°C for 16 hours. Bacteria were picked and sequenced. Positive bacterial cultures were selected for sequencing confirmation, ultimately yielding PHB-. CiSPL3 Overexpression vector. PHB- CiSPL3 The overexpression vector was transformed into Agrobacterium GV3101, ultimately yielding GV3101-PHB- CiSPL3 Engineered bacteria. Specifically, take competent Agrobacterium cells stored at -80℃ and hold them in your hand at room temperature for a short time until partially thawed. While in an ice-water mixture, insert them into ice. Add 0.1 μg (no more than 10 μl) of plasmid DNA to every 100 μl of competent cells, mix thoroughly by hand, and incubate sequentially on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37℃ water bath for 5 minutes, and in an ice bath for 5 minutes. Add 700 μl of antibiotic-free LB broth and incubate at 28℃ with shaking at 200 rpm for 2-3 hours. Centrifuge at 6000 rpm for one minute to collect the bacteria. Retain approximately 100 μl of the supernatant, gently resuspend the bacterial block, and spread it onto an LB agar plate containing 50 μg / ml kanamycin. Invert the plate and incubate at 28℃ for 2-3 days. Randomly select one single colony for colony PCR. Label the correctly identified Agrobacterium single clone for later use.

[0051] Example 3: Functional identification of transgenic Arabidopsis thaliana.

[0052] The GV3101-PHB- obtained in Example 3 CiSPL3 Engineered bacteria were used to infect wild-type Arabidopsis thaliana using an inflorescence infection method to obtain T0 generation transgenic seeds 35S. CiSPL3 .

[0053] The inflorescence infection procedure is as follows: First, disinfect wild-type Arabidopsis seeds with a 5% sodium hypochlorite solution for 8 minutes, rinse 5 times with sterile water, and then spread the disinfected seeds evenly in a Petri dish containing MS medium. Vernalize the seeds at 4°C for 2-3 days, and then place them in an artificial climate chamber to begin germination and growth. 7-12 days after germination, select robust and uniformly growing seedlings and transplant them into potting soil pre-soaked with Flower-Free Compound Fertilizer. Cover the seedlings with plastic wrap, and remove the wrap once the seedlings have established themselves.

[0054] Water the flowering plants thoroughly one day in advance. Invert the small pot and immerse all the flower clusters in the pre-suspended bacterial solution for about 30 seconds. Repeat the above method once after 7 days. After 2-3 weeks, reduce the amount of nutrient solution applied to accelerate aging. Collect the mature seeds in a paper bag and store them in a desiccator for 7 days.

[0055] The obtained T0 generation transgenic seeds were screened for hygromycin resistance (30 μg / ml HYG) to identify resistant plants, and positive plants were obtained by PCR. After obtaining T1 seeds from the positive plants, the same method was used until stable T3 generation transgenic plants were obtained. Three [seeds were taken]. CiSPL3 The transgenic lines, officially named lines OE1, OE3, and OE10, incorporate wild-type Arabidopsis thaliana and 35S: CiSPL3 Transgenic T3 generation seeds were directly sown in a nutrient substrate (a mixture of peat moss and vermiculite in a 2:1 ratio) and cultured under normal conditions (16h light / 8h darkness, temperature 22℃, light intensity 150μmol / m²). 2 (·s), observe the bolting and flowering time of each plant.

[0056] The results are as follows Figure 3 As shown, compared to wild-type Arabidopsis, the flowering time of the three transgenic Arabidopsis lines (OE1, OE3, and OE10) was significantly earlier, by about 5 days, indicating that overexpression... CiSPL3 It has the function of promoting plant flowering.

[0057] Example 4 GUS staining detection CiSPL3 Activity analysis of gene promoters in different tissues of Arabidopsis thaliana.

[0058] (one) CiSPL3 Cloning of gene promoters:

[0059] Using the DNA of the thin-shelled pecan 'Boni' as a template, the following primer sequence was used:

[0060] Upstream primer (with XbaI restriction site, SEQ ID NO.7): TGCCTGCAGGTCGACTCTAGATAAGCATATTGGTACGTGTGCTC;

[0061] Downstream primer (with BamHI restriction site, SEQ ID NO.8): GGACTGACCACCCGGGGATCCTTTGGCAAAACAAGAGTAGACAG;

[0062] Amplification CiSPL3 The sequence in the 1500-2000 bp region upstream of the start codon (ATG) of a gene is used as the promoter. CiSPL The amplified sequence was 1994 bp, as follows: Figure 4 As shown in SEQ ID NO.9, the promoter sequence was sequenced and compared with the pecan genome data, revealing that both sequences were the expected promoter sequences.

[0063] Using the online program PlantCARE CiSPL3 Analyzing the promoter sequence of a gene, such as... Figure 5 As shown, CiSPL3 The promoter sequence contains 18 functionally annotated cis-acting elements, including ABRE, LTR, ARE, CGTCA-motif, RY-element, TGA-element, TGACG-motif, CAT-box, TCT-motif, MBS, and TC-rich repeats. Among them are 5 cis-acting elements related to light response: Box 4, G-box, I-box, G-Box, and TCT-motif, which are widely present in plant gene promoters. Nine cis-acting elements with other functions, five abscisic acid response-related elements (ABRE), four MeJA hormone response elements (CGTCA-motif, TGACG-motif), one auxin response element (TGA-element), one low-temperature response element (LTR), two drought-induced elements (MBS), one defense and stress response cis-acting element (TC-rich repeats), one zein metabolism regulatory element (O2-site), three anaerobic induction regulatory elements (ARE), one seed-specific regulatory element (RY-element), and one meristem expression regulation-related element (CAT-box).

[0064] Using the P1300GN carrier, thin-shelled pecans were constructed. CiSPL3 GUS fusion expression vector for gene promoter ( CiSPL3Pro::GUS recombinant expression vector), constructed using Agrobacterium-mediated inflorescence staining method. CiSPL3Pro ::GUS recombinant expression vector was transformed into Arabidopsis thaliana. T0 generation seeds were sown on a resistance medium, and two weeks later, T1 generation transgenic seedlings with hygromycin resistance were obtained. Wild-type Arabidopsis thaliana and CiSPL3Pro ::GUS transgenic line T1 seeds were sown in nutrient pots. After approximately 35 days of growth, histochemical staining was performed on leaves, roots, stem segments, flowers, and pods. Results showed that the staining was very deep and strong in the roots; staining was also present in the leaves, but lighter than in the roots; the staining was very light in the stems; and the staining was very deep in the flower buds and petals. Staining was also present in the pods. Figure 6 ).

[0065] Example 5: Phenotypic observation and root length measurement of Arabidopsis thaliana seedlings under drought stress.

[0066] Sterilized wild-type and transgenic Arabidopsis thaliana T3 seeds were sown onto MS plates and MS+300mM mannitol plates, respectively, and vernalized for 2 days at 4°C in the dark. They were then placed vertically in a growth chamber at 23°C with 16 hours of light and 8 hours of darkness. Root length was measured after 15 days. Figure 7 (A and B), in a mannitol-containing medium, CiSPL3 The root length of the OE3 gene line seedlings is still significantly longer than that of wild-type Arabidopsis.

[0067] Potted Arabidopsis thaliana seedlings (35S:) that had been growing normally for 5 weeks and were of wild type and transgenic type were compared. CiSPL3 Transgenic plants were subjected to drought treatment for 15 days, and their morphological characteristics were observed. The results are as follows: Figure 7 As shown in Figure C, under drought conditions, the wild-type control showed obvious wilting and death 15 days after stress. Although the transgenic lines also showed wilting due to water loss, the symptoms were much milder than the control, and they could continue to bolt and flower. Three days after rehydration, most of the transgenic lines began to revive, while most of the wild-type lines still died. Therefore, CiSPL3 Overexpression of the gene enhances the drought tolerance of Arabidopsis plants.

[0068] Example 6: Phenotypic observation and root length measurement of Arabidopsis thaliana under salt stress.

[0069] Wild-type and transgenic Arabidopsis thaliana T3 seeds were sterilized and then plated on MS, MS+100mM NaCl, and MS+200mM NaCl media, respectively. The culture dishes were placed vertically and vernalized at 4°C for 2 days, followed by normal culture in a light incubator. Root length was measured and photographed after 15 days. Figure 8 As shown in A and 8B, in salt-stressed medium, the transfer CiSPL3 The root length of the genetically modified Arabidopsis seedlings was significantly shorter than that of the wild-type control.

[0070] After 20 days of growth on a medium containing NaCl (100 mM), 76.2%, 78.6%, and 52.4% of the transgenic lines, wild-type (WT) Arabidopsis thaliana, respectively, completely albino and died, while the wild-type had a survival rate of over 80%. Figure 8 C).

[0071] Wild-type and transgenic Arabidopsis thaliana plants, which had been growing normally for 5 weeks, were treated with a 300 mM NaCl solution, repeated every three days for four times. After 15 days of salt stress treatment, the transgenic and wild-type plants showed significantly different phenotypic characteristics. The leaves of the transgenic plants gradually withered, and the wild-type plants also showed varying degrees of yellowing, but were better than the transgenic plants. Figure 8 D). In summary, overexpression CiSPL3 The gene significantly reduces the tolerance of transgenic plants to salt stress.

[0072] This invention provides CiSPL3 There are many ideas and methods for genes and their applications, and many specific methods and approaches to implement this technical solution. The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. All components not explicitly stated in this embodiment can be implemented using existing technology.

Claims

1. The application of a thin-shelled pecan gene, CiSPL3, in regulating flowering time and abiotic stress in plants, wherein... The nucleotide sequence of the CiSPL3 gene is shown in SEQ ID NO.1, and the promoter sequence of the CiSPL3 gene of the pecan is shown in SEQ ID NO.

9. The regulation of plant flowering is achieved by overexpressing the CiSPL3 gene of the pecan in the plant. The resulting transgenic plants have earlier flowering time, enhanced resistance to drought stress, and reduced tolerance to salt stress. The plant is either pecan or Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The protein encoded by the CiSPL3 gene has the amino acid sequence shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The primer pair for amplifying the CiSPL3 gene is CiSPL3-F and CiSPL3-R; the nucleotide sequence of CiSPL3-F is shown in SEQ ID NO.3; and the nucleotide sequence of CiSPL3-R is shown in SEQ ID NO.

4.

4. The application according to claim 1, characterized in that, Includes the following steps: (1) Cloning the CiSPL3 gene of the thin-shelled pecan; (2) The CiSPL3 gene of the thin-shelled pecan was ligated to a vector to obtain a recombinant vector; (3) The recombinant vector was transferred into Agrobacterium to obtain recombinant Agrobacterium; (4) Infect the plants with the recombinant Agrobacterium to obtain plants that overexpress the CiSPL3 gene of pecan.

5. The application according to claim 1, characterized in that, Use the following sequence to clone the promoter: Upstream primer: TGCCTGCAGGTCGACTCTAGATAAGCATATTGGTACGTGTGCTC; Downstream primer: GGACTGACCACCCGGGGATCCTTTGGCAAAACAAGAGTAGACAG.

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