Ghlec1-1a gene and the protein encoded by the same in cotton leaf shape development

Abstract

This invention belongs to the field of molecular biology, and particularly relates to the application of the GhLEC1-1A gene and its encoded protein in cotton leaf development. The amino acid sequence of the encoded protein is shown in SEQ ID NO.2. This invention successfully introduced the GhLEC1-1A overexpression vector into plants and screened out positive overexpression plants. Compared with wild-type plants, the leaf growth and development morphology of the positive overexpression plants differed from that of the wild type, exhibiting increased leaf area and a final leaf shape resembling a chicken-foot leaf. These morphological changes provide important gene targets and germplasm resources for cotton molecular breeding, and offer technical support for the widespread application of the chicken-foot leaf technique in cotton production.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to the application of the GhLEC1-1A gene and its encoded protein in cotton leaf development. Background Technology

[0002] Plant leaves are the primary organs for photosynthesis, and their morphological characteristics directly affect photosynthetic efficiency, transpiration, and the plant's utilization of environmental resources. Leaf shape, as a key phenotypic trait in plant growth and development, has a decisive impact on the overall canopy structure, light-trapping capacity, ventilation, and ultimately, biomass accumulation and yield formation. For example, deeply lobed leaves or leaves of different shapes can optimize photosynthetic efficiency by altering light distribution and airflow within the canopy, while also influencing the occurrence and development of pests and diseases. Therefore, a deep understanding of the molecular mechanisms regulating plant leaf shape development has significant theoretical guidance and practical application value for cultivating high-yielding, high-quality, and stress-resistant crop varieties.

[0003] Cotton, as a globally important economic crop, has its leaf morphology directly impacting key agronomic traits such as canopy structure, photosynthetic efficiency, yield, and stress resistance. Cotton leaf shapes exhibit diversity, including normal leaves, sub-foot leaves, foot leaves, and super-foot leaves, among which the foot and super-foot leaves have attracted widespread attention due to their unique deep-lobed morphology. Compared to normal leaves, deeply lobed leaves, such as foot leaves, effectively improve canopy ventilation and light penetration, reduce self-shading by lower leaves, and thus increase canopy photosynthetic efficiency. This mechanism has been proven to be directly related to cotton's yield potential. For example, studies have shown that sub-foot leaf chromosomal segments introduced from Sea Island cotton significantly enhance the photosynthetic productivity of short-season cotton.

[0004] In recent years, significant progress has been made in the study of the molecular regulatory network of cotton leaf development. Precise leaf morphology is a process governed by a complex molecular regulatory network, involving the synergistic effects of multiple gene families. These gene families include, but are not limited to, KNOTTED1-like homeobox (KNOX) genes that regulate cell division and differentiation, as well as genes involved in the auxin signaling pathway. For example, research has revealed that the GhARF16-1 gene participates in cotton by transcriptionally regulating the GhKNOX2-1 gene. Furthermore, auxin plays a crucial role in the periodic formation of leaf primordia, and its local concentration gradient is the basis for driving leaf tooth formation. Specifically, the auxin maxima model indicates that the peak local auxin concentration in the shoot apical meristem is the initiation signal for inducing leaf primordia formation, and the directional flow of auxin can self-reinforce and form network3. Recent studies have also found that the curled leaf trait in cotton is associated with specific gene mutations; for example, the G301A single nucleotide mutation in the GaIAA14 gene in Asian cotton can lead to leaf curling. Furthermore, the molecular chaperone GHCU regulates leaf curling by modulating the distribution of KNGH1 in abaxial boundary cells, affecting auxin distribution and thus influencing leaf morphology. The elucidation of these molecular biological mechanisms provides a theoretical basis and technical approach for the precise regulation of cotton leaf shape through genetic engineering.

[0005] Given the complexity and multi-gene regulatory nature of leaf development, those skilled in the art anticipate that other transcription factor families, besides the known KNOX and auxin-related genes, may also play crucial roles. The Nuclear Factor Y (NFY) gene family, as conserved transcription factors, plays an important role in plant growth and development, stress response, and morphogenesis. NFY transcription factors typically consist of three subunits: NFYA, NFYB, and NFYC, and function by forming heterotrimers to regulate the expression of downstream genes. Existing research has shown that members of the NFY gene family are involved in multiple processes in plants, including flowering time, embryonic development, and seed maturation.

[0006] Given the complexity of leaf morphogenesis, especially its three-dimensional structure and specific tissue differentiation, the potential role of the NFY gene family in cotton leaf development warrants further investigation. Therefore, this invention proposes the application of the GhLEC1-1A gene and its encoded protein in cotton leaf development. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the GhLEC1-1A gene and the protein it encodes in cotton leaf development.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention first provides the application of the GhLEC1-1A gene and its encoded protein in cotton leaf development. The amino acid sequence of the encoded protein is shown in SEQ ID NO.2: MERGDEFSRFPKLAKSNSGLGIIQHGDSSDSINNVNNIFNITDSVSNAGNIFNITANVSNAGNISNTSNTNAVSTMPPPGPVLREQDQYMPIANVIRIMRRILPPHAKISDEAKETIQECVSEFISFITGEANERCQSEQRKTVTAEDILCAMGKLGFDDYMEPLTVYLTRYRQSENERTSLRGDTFLKRGNAYGPMMTPPHGVAPFNAGFQEGMTDATSAAARAIMGGYNHGAPPGGAAGSSSQQAPFDNNLDPFDVFK.

[0010] The nucleotide sequence of the GhLEC1-1A gene is shown in SEQ ID No. 1.

[0011] Specifically, the cotton in question is upland cotton.

[0012] More specifically, this is achieved by overexpressing the GhLEC1-1A gene in cotton, resulting in increased leaf area and reduced leaf clefts in the main stem leaves.

[0013] This invention provides a method for preparing transgenic cotton, comprising the following steps: introducing the GhLEC1-1A gene into cotton through gene transformation to achieve overexpression; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0014] Specifically, the cotton in question is upland cotton.

[0015] More specifically, it is introduced through Agrobacterium-mediated transformation.

[0016] Optionally, this further includes screening for transgenic plants with increased leaf area and reduced leaf clefts in the main stem leaves.

[0017] The present invention also provides a method for obtaining transgenic cotton or its progeny using the method described herein.

[0018] Compared with existing technologies, the beneficial effects of this invention are: This invention successfully introduces the GhLEC1-1A overexpression vector into plants and screens out positive overexpression plants. Compared with wild-type plants, the leaf growth and development morphology of positive overexpression plants differs from that of wild-type plants, exhibiting increased leaf area and a final leaf shape resembling a chicken-foot leaf. These morphological changes provide important gene targets and germplasm resources for cotton molecular breeding, and provide technical support for the widespread application of the chicken-foot leaf technique in cotton production. Attached Figure Description

[0019] Figure 1 Phylogenetic analysis of the LEC1 genus of Gossypium.

[0020] Figure 2 The overall phenotype of GhLEC1-1A-OE and wild-type plants

[0021] Figure 3 Comparison of leaf shape of main stem leaves between GhLEC1-1A-OE and wild type.

[0022] Figure 4 The leaf area statistics for the main stem leaves of GhLEC1-1A-OE and wild type are presented.

[0023] Figure 5 Statistical results of leaf lobes on the main stem leaves of GhLEC1-1A-OE and wild type.

[0024] Figure 6 The photosynthetic statistics of the GhLEC1-1A-OE and wild-type populations are presented.

[0025] Figure 7 The results show the number of bolls per plant for GhLEC1-1A-OE and wild-type plants. Detailed Implementation

[0026] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described, unless otherwise specified, are commercially available.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0028] Example 1: Identification and evolutionary analysis of the LEC1 gene in the genus *Gossypium*;

[0029] The LEC1 gene of all Gossypium species was identified by blastn using the cds sequence of AtLEC1 (AT1G21970) in Arabidopsis thaliana (E value set at 1e-50). The results are shown in Table 1.

[0030] Table 1. Identification and analysis of LEC1 members of the genus *Gossypium*.

[0031]

[0032]

[0033] AA: Number of amino acids, MW: Molecular weight, PI: Theoretical isoelectric point.

[0034] The LEC1 sequence was obtained and multiple sequence alignment was performed using the l-INS-i algorithm with MAFFT software. A phylogenetic tree was constructed using iqtree2 software, and finally, Figtree software was used for visualization. The results are as follows: Figure 1 As shown, phylogenetic analysis revealed two pairs of LEC1 genes within the genus *Gossypium*. LEC1-2 is closely related to AtLEC1 in *Arabidopsis*, while LEC1-1 is distantly related and highly conserved within the genus *Gossypium*. The nucleotide sequence of the GhLEC1-1A gene is shown in SEQ ID NO.2, and the encoded amino acid sequence is also shown in SEQ ID NO.2.

[0035] Example 2: Construction of GhLEC1-1A overexpression vector;

[0036] 1. Cloning of the full-length GhLEC1-1A gene: RNA was extracted from samples of cotton variety Yuzao 1, and cDNA was obtained by reverse transcription. Specific primers were designed, and a fragment of approximately 783 bp was amplified from the cDNA template by PCR. This fragment was ligated into a T vector and transformed into DH5α. After 12 hours, single colonies were picked and shaken for testing, and then sent for sequencing.

[0037] The primers for gene cloning and amplification are as follows:

[0038] SEQ ID NO.3: F-terminal primer: 5'-acgggggacgagctcATGGAACGTGGTGATGAGTTC-3';

[0039] SEQ ID NO.4: R-terminal primer: 5'-gctctgcaggtcgacTCATTTGAACACATCAAATGGATCC-3';

[0040] The successfully validated recombinant T-target fragment was double-digested with SacI and SalI enzymes. The digestion efficiency was observed using agarose gel electrophoresis, and the digested products (target fragment and vector fragment) were recovered using a gel extraction kit. After gel extraction, the target gene was ligated into the pCAMBIA1300-EGFP-MCS expression vector, and the ligation product was transformed into *E. coli* competent cells DH5α. In the process, after selecting single clones for bacterial culture verification, the recombinant plasmid with correct sequencing was transferred into Agrobacterium GV3101. After selecting positive clones and shaking them, 60% glycerol was added and stored at -80℃.

[0041] Example 3: Agrobacterium-mediated genetic transformation of cotton;

[0042] 1) Remove the husks from the cottonseeds of Yuzao No. 1 cotton, sterilize by shaking with HgCl2 for 10 minutes, and rinse with sterile water 3-5 times;

[0043] 2) Place 10 seeds in each bottle into a seedling culture medium (macronutrient A: 25 ml / L (CaCl2·2H2O 60 mg / L, MgSO4·7H2O 100 mg / L, (NH4)2SO4 53.6 mg / L, NaH2PO4·H2O 60 mg / L), macronutrient B: 25 ml / L (potassium nitrate stock solution concentration 38000 mg / L), glucose 15 g / L, coagulant Agar 8 g / L, pH adjusted to 6.0, sterilized at 121℃ for 16 min), and culture at 28℃ in the dark. After 48 hours, transplant the seedlings.

[0044] 3) After 56 days, when the hypocotyl has grown to about 10cm, preparations for inoculation can begin;

[0045] 4) Shake the bacteria in advance, collect 50ml of bacterial solution on the same day, and adjust the OD600 to 0.6 with MGL resuspension (acetylsylgenone final concentration 50mg / L);

[0046] 5) Remove the etiolated seedlings and place them on sterile filter paper in a petri dish. Cut off the roots and cotyledons, leaving only the hypocotyl. Cut the hypocotyl into 0.5cm segments with a smooth cut surface.

[0047] 6) Immerse the cut hypocotyls in the MGL resuspended bacterial solution, ensuring the solution completely covers the hypocotyls, and leave for 10 minutes. Discard the bacterial solution and place the hypocotyls on clean filter paper to air dry. Arrange the hypocotyls neatly on a co-culture medium covered with clean filter paper (MS medium 4.74 g / L, glucose 30 g / L, plant growth regulator 200 µL / L 2,4-D (stock solution concentration 0.1 mg / mL) and 200 µL / L KT (stock solution concentration 0.1 mg / mL), solidifying agent phytage 2.5 g / L, pH adjusted to 5.8, sterilized at 121℃ for 16 minutes), and incubate in the dark for 36-48 hours.

[0048] 7) After co-culturing, the hypocotyls were removed and dispersed on selective medium (MS medium 4.74 g / L, glucose 30 g / L, plant growth regulators 200 µL / L 2,4-D (stock solution concentration 0.1 mg / mL) and 200 µL / L KT (stock solution concentration 0.1 mg / mL), coagulant phytage 2.5 g / L, pH adjusted to 5.8, sterilized at 121℃ for 16 min, and antibiotics 2 mL / L cephalosporin (stock solution concentration 200 mg / mL) and 1 mL / L kanamycin (stock solution concentration 50 mg / mL) were added. The medium was then cultured at 28℃ for 24 h under light. Subculture was performed every 3 weeks until embryogenic callus was produced. The embryogenic callus tissue was then transferred to differentiation medium for further culture (differentiation medium 4.684 g / L, glucose 30 g / L, glutamine 1.0 g / L, asparagine 0.5 g / L, with 1 mL of cephalosporin added). IBA (1 mg / mL stock solution) and 200 µL / L KT (0.1 mg / mL stock solution) were used as plant growth regulators, with Phytage 2.5 g / L as the solidifying agent, pH adjusted to 5.9, and sterilized at 121℃ for 16 min. The culture conditions remained unchanged until seedlings were grown.

[0049] 8) Seedling rooting: Transfer the emerging seedlings to rooting medium (1 / 2 MS medium, glucose 15g / L, solidifying agent phytage 2.5g / L, pH adjusted to 5.9, sterilized at 121℃ for 16min). When the seedlings reach the size of the culture bottle opening, transfer them to a clean culture bottle, add water and open the cap, harden the seedlings for 12 days, and culture the seedlings in sterile water until a large number of white new roots grow out, then transfer them to a greenhouse or transplant them to the field.

[0050] Example 4: Identification and phenotypic observation of GhLEC1-1A transgenic plants;

[0051] Wild-type and GhLEC1-1A-OE cotton materials were planted at the cotton experimental base of the College of Agriculture, Shihezi University, Xinjiang. Whole-plant photography was conducted on July 1st, and 12 main stem leaves of the cotton plants were also photographed for observation. The results are as follows: Figure 1As shown. Leaf area and leaf lobe ratio were calculated using ImageJ software. The results are as follows. Figure 2 and Figure 3 As shown, compared to the wild type, the leaf shape of the main stem leaves of the GhLEC1-1A-OE plant stopped changing after developing into chicken-foot leaves. The results are as follows... Figure 4 and Figure 5 As shown, compared with the wild type, the main stem leaves of the GhLEC1-1A-OE plant have larger leaf area and smaller leaf lobes.

[0052] In addition, the photosynthetic rate of the cotton population was determined using a LI-840A CO2 / H2O analysis system (LI-COR Inc., Lincoln, NE, USA) and a closed-circuit assimilation chamber method. The results are as follows: Figure 6 As shown, the canopy photosynthesis of GhLEC1-1A-OE plants was significantly upregulated compared to wild-type plants. After cotton maturity, the number of effective bolls per plant was counted, and the results are as follows: Figure 7 As shown, compared with the wild type, the number of bolls per plant in GhLEC1-1A-OE plants was significantly increased. This indicates that the change in leaf shape of GhLEC1-1A-OE can improve the photosynthetic rate of the cotton population, thereby increasing the number of effective bolls per plant.

[0053] In summary, the GhLEC1-1A gene, which is related to the leaf shape trait of upland cotton, discovered in this invention, can regulate the leaf shape development, leaf area, and leaf lobing ratio of upland cotton. Therefore, it has great application potential in the breeding of new upland cotton varieties and in regulating the leaf shape of upland cotton.

[0054] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of the GhLEC1-1A gene or its encoded protein in cotton leaf development, characterized in that, The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the GhLEC1-1A gene is shown in SEQ ID No.

1.

3. The application as described in claim 1, characterized in that, The cotton in question is upland cotton.

4. The application as described in claim 1, characterized in that, This is achieved by overexpressing the GhLEC1-1A gene in cotton, resulting in increased leaf area and reduced leaf clefts in the main stem leaves.

5. A method for preparing transgenic cotton, characterized in that, It includes the following steps: introducing the GhLEC1-1A gene into cotton through gene transformation to achieve overexpression; The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

6. The method as described in claim 5, characterized in that, The cotton in question is upland cotton.

7. The method as described in claim 5, characterized in that, It is introduced through Agrobacterium-mediated transformation.

8. The method as described in claim 5, characterized in that, Further steps include screening for transgenic plants with increased leaf area and reduced leaf clefts in the main stem leaves.

9. Obtaining transgenic cotton or progeny by the method of any one of claims 5 to 8.

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