A gene for negatively regulating gossypium hirsutum fruit branch length and application

Abstract

The application belongs to the field of plant molecular biology and genetic engineering, and specifically discloses a negative regulation Gossypium hirsutum fruit branch length trait gene Gh_A08G254200, which is located on A08 chromosome 122390509~122395098, the gene is composed of two exons and one intron, there is a SNP site at A08:122392954, which is a non-synonymous mutation, the gene is named as GhCSLD3, the CDS sequence length is 3432 bp, the nucleotide sequence of the gene comprises SEQ ID No.1; the amino acid sequence of the encoded protein comprises SEQ ID No.2. In the extreme Gossypium hirsutum non-zero type cotton variety, the gene is silenced, and it is found that the Gossypium hirsutum fruit branch length is elongated, so the gene has a negative regulation effect on the Gossypium hirsutum fruit branch elongation. The verification of the molecular function of the gene has important significance for improving the breeding of Gossypium hirsutum plant type change.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and in particular relates to a gene that negatively regulates the fruit branch length trait of upland cotton and its application. Background Technology

[0002] Cotton, belonging to the Malvaceae family and the Malvaceae genus, is an important economic crop closely related to people's lives, and the cotton industry occupies a significant position in the national economy. However, due to the unique characteristics of cotton cultivation and harvesting, the high labor intensity and low level of mechanization in cotton farming have become pressing issues that need to be addressed in my country's cotton industry. Currently, the widespread application of machine-harvested cotton has greatly improved harvesting efficiency, alleviated the labor shortage, and promoted mechanized cotton cultivation and harvesting models, reducing labor costs. However, the promotion of machine-harvested cotton places certain requirements on cotton plant type; a suitable plant type can better adapt to mechanized harvesting.

[0003] Fruiting branches, as an important morphological feature and boll attachment point in cotton, not only affect mechanized operations and harvesting but also planting density and yield. As a crucial component of the cotton plant architecture, fruiting branches, along with leaf branches (also known as "vegetative branches"), constitute the branching structure of cotton. Cotton fruiting branches grow in the upper-middle part of the main stem, curving outwards horizontally. Currently, cotton fruiting branch types can be divided into two categories: O-type fruiting branches and non-O-type fruiting branches. Existing research indicates that type O fruiting branches are a trait controlled by a recessive single gene. Due to the dosage effect of the gene, different fruiting branch phenotypes are formed in Sea Island cotton and Upland cotton, resulting in determinate fruiting branches. Non-type O fruiting branches exhibit indeterminate growth habits and are classified according to internode length: internode length of 2-5cm is type I fruiting branch (compact type), internode length of 5-10cm is type II fruiting branch (relatively compact type), internode length of 10-15cm is type III fruiting branch (relatively loose type), and internode length of more than 15cm is type IV fruiting branch (loose type). Internode length exhibits the genetic characteristics of a quantitative trait, which is significantly different from the single-gene recessive inheritance pattern of type O fruiting branches, and consequently, the genetic regulatory sites are also different.

[0004] Plant hormones such as gibberellin (GA), cytokinin (CK), salicylic acid (SA), ethylene, strigolactone, and jasmonate acid (JA) play important regulatory roles in plant branch elongation and growth development. Exogenous application of auxins and gibberellin can promote internode elongation in cotton fruiting branches, while ethylene and jasmonate inhibit it. The WRKY, ERF, and BHLH transcription factor families have been identified as important inhibitory regulators of internode elongation in cotton. Other WRKY, ERF, BHLH, MYB, and NAC transcription factor family genes also participate in branch elongation and flowering time in upland cotton, affecting the first node of the fruiting branch. Previous studies have shown that the regulatory sites for zero-type fruiting branches are often located on chromosomes A07 and D07, while related genes on chromosomes D01 and D08 have been shown to be associated with the node position or length of non-zero-type fruiting branches.

[0005] Therefore, analyzing the elongation mechanism and regulation mechanism of non-zero fruiting branches in upland cotton, and exploring its superior control sites, is of great research significance for improving cotton planting density and yield, as well as for breeding new cotton varieties. Summary of the Invention

[0006] The purpose of this invention is to provide a method and application for verifying the function of a cellulose synthase-like protein (CSLD3) gene in the effect on fruiting branch length in upland cotton. GhCSLD3 is a gene cloned from cotton by the inventors. Using molecular biology techniques, interfering with this GhCSLD3 gene in cotton significantly increases the fruiting branch length of upland cotton. Therefore, cloning the GhCSLD3 gene is of great significance for improving cotton plant architecture and increasing cotton yield. A review of existing literature revealed that no functions of the GhCSLD3 gene related to this invention have been reported to date.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A gene that negatively regulates the fruit branch length trait of upland cotton, named GhCSLD3, is located on chromosome A08 from 122390509 to 122395098. This gene consists of two exons and one intron. There is a non-synonymous SNP mutation at position A08:122392954. The CDS sequence length is 3432 bp. The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0009] A protein encoded by a gene that negatively regulates the fruit branch length trait of upland cotton. The gene encoding this protein is a tetraploid cotton gene, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2 of the sequence listing.

[0010] A primer pair for cloning the upland cotton fruit branch length gene GhCSLD3 from an upland cotton cDNA library includes a forward primer F: 5'-TGCATATACCGCCCACCCCA-3' and a reverse primer R: 5'-ATCCAAATCGTCCCCCCCAG-3'.

[0011] A plant silencing vector for the gene GhCSLD3, which negatively regulates the fruit branch length trait of upland cotton.

[0012] A subcellular localization expression vector for GhCSLD3, a gene that negatively regulates the fruit branch length trait of upland cotton.

[0013] An Arabidopsis overexpression vector for GhCSLD3, a gene that negatively regulates the fruit branch length trait of upland cotton.

[0014] The application of a protein encoded by a gene that negatively regulates the fruit branch length trait in upland cotton in regulating upland cotton fruit branch elongation and in short-fruit branch cotton varieties.

[0015] The advantages of this invention are:

[0016] 1. This invention provides a novel upland cotton fruit branch regulatory gene GhCSLD3 sequence (CDS sequence), which is highly expressed in the shoot tip and internode of short fruit branch material, indicating that the gene plays an important role in controlling the elongation of non-zero fruit branches;

[0017] 2. By silencing the GhCSLD3 gene in cotton, the length of the fruiting branches of the extremely short fruiting branch material Blanco and the extremely long fruiting branch material Sanjiang Bajiang Dahua was significantly increased.

[0018] 3. Through overexpression experiments of this gene in Arabidopsis thaliana, it was found that the lateral branches of Arabidopsis thaliana were significantly shortened when the gene expression level was high;

[0019] 4. Through subcellular localization experiments, it was found that the gene is located on the cell membrane. This invention is the first to elucidate the biological function of the gene at the gene level, which is of great significance for exploring the elongation and development mechanism of non-zero fruiting branches in upland cotton and for cultivating cotton varieties with suitable fruiting branches. Attached Figure Description

[0020] Figure 1 Cloning of the GhCSLD3 gene cDNA sequence. In the figure, M represents DNA Marker, and the red arrow indicates the size of the target gene band.

[0021] Figure 2Subcellular localization analysis of GhCSLD3 gene in tobacco leaf epidermal cells

[0022] Figure 3 Gene structure and phylogenetic tree analysis of GhCSLD3 homologs using TM-1-CRI as the reference genome.

[0023] Figure 4 A: Expression analysis of the GhCSLD3 gene in the internodes and shoot tips of cotton fruiting branches. The expression level of the GhCSLD3 gene in Blanco and Sanjiang Bajiang large-flowered cotton was determined using TPM values. B: Quantitative fluorescence analysis of the GhCSLD3 gene. In the figure, Blanco 3363 and Sanjiang Bajiang large-flowered cotton represent very short and very long fruiting branches, respectively; BA: shoot tip of Blanco fruiting branch, SA: shoot tip of Sanjiang Bajiang large-flowered cotton fruiting branch, BF: internode of Blanco fruiting branch, SF: internode of Sanjiang Bajiang large-flowered cotton fruiting branch. Error bars represent the mean (±SEM) of three biological replicates for each sample. C: qRT-PCR silencing efficiency of GhCSLD3 gene in *Arabidopsis thaliana* plants from Sanjiang Bajiang; D: qRT-PCR silencing efficiency of GhCSLD3 gene in *Blanco* plants; E: Phenotype of *Blanco* plants with silencing GhCSLD3 gene and *CLCRV:00* empty vector; F: Phenotype of *Arabidopsis thaliana* plants with silencing GhCSLD3 gene and *CLCRV:00* empty vector; G: Statistical survey of fruit branch length in *Blanco* 3363 plants with silencing GhCSLD3 gene and *CLCRV:00* empty vector, seven biological replicates for each; H: Statistical survey of fruit branch length in *Arabidopsis thaliana* plants with silencing GhCSLD3 gene and *CLCRV:00* empty vector, seven biological replicates for each; I: Wild-type *Arabidopsis thaliana* and T3 generation homozygous line 35S::GhCSLD3(OE1OE2) Phenotypic analysis of OE3; J: T-test analysis of the 1st, 2nd, and 3rd lateral branches of wild-type Arabidopsis and T3 generation homozygous line (35S::GhCSLD3); K: CLCRV:V 阳 The yellowing phenotype of silent cotton plants. Using a t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Detailed Implementation

[0024] The Agrobacterium involved in this invention is the competent Agrobacterium tumefaciens GV3101 strain, which can be purchased publicly on the market (Shanghai Weidi Biotechnology Co., Ltd.), and the competent Escherichia coli DH5α strain was purchased from Kangwei Century Co., Ltd.

[0025] Example

[0026] A gene that negatively regulates the fruit branch length trait of upland cotton, named GhCSLD3, is located on chromosome A08 from 122390509 to 122395098. This gene consists of two exons and one intron. There is a non-synonymous SNP mutation at position A08:122392954. The CDS sequence length is 3432 bp. The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0027] Furthermore, the gene encoding the protein is a tetraploid cotton gene, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No. 2 of the sequence listing.

[0028] A plant silencing vector for the gene GhCSLD3, which negatively regulates the fruit branch length trait of upland cotton.

[0029] A subcellular localization expression vector for GhCSLD3, a gene that negatively regulates the fruit branch length trait of upland cotton.

[0030] An Arabidopsis overexpression vector for GhCSLD3, a gene that negatively regulates the fruit branch length trait of upland cotton.

[0031] A primer pair for cloning the upland cotton fruit branch length gene GhCSLD3 from an upland cotton cDNA library includes a forward primer F: 5'-TGCATATACCGCCCACCCCA-3' and a reverse primer R: 5'-ATCCAAATCGTCCCCCCCAG-3'.

[0032] Experimental Example

[0033] In a recent study, we constructed a mixed pool of extreme fruit branches and used BSA mapping technology to determine QTL intervals. Through transcriptome sequencing of internodes and shoot tips at key stages of fruit branch elongation, combined with KEGG enrichment pathway analysis, we screened a candidate gene, Gh_A08G254200, regulating fruit branch length, and named this gene GhCSLD3. Quantitative real-time PCR revealed that the expression level of this gene in the internodes and shoot tips of extremely short fruit branches was significantly higher than that in extremely long fruit branches. Figure 4 -B) indicates that this gene may be a candidate gene for regulating the length elongation of fruiting branches. Using upland cotton leaf genomic DNA and cDNA as templates, the sequence of the target gene was obtained. Sequence alignment revealed a SNP site within the gene, which is a non-synonymous mutation. This gene is a 3432 bp cellulose synthase-like protein gene, as shown in SEQ ID No. 1. The protein encoded by this gene consists of 1143 amino acids (aa), as shown in SEQ ID No. 2.

[0034] Subcellular localization results showed that GhCSLD3 was expressed in the cell membrane. Figure 2 Phylogenetic analysis showed that Gh_A08G245200 and Gh_D08G245400 are homologous genes, most closely related, with approximately 61% sequence homology, encoding CSLD3, which consists of two exons and one intron. Gene annotation showed that this gene is located on the endoplasmic reticulum and cytoplasmic membrane, encoding cellulose synthase-like protein 3.

[0035] To investigate the function of GhCSLD3 in regulating fruit branch elongation, a VIGS vector for GhCSLD3 was constructed and injected into Blanco cotton (with extremely short fruit branch length) and Sanjiang Bajiang cotton (with extremely long fruit branch length). Yellowing phenotype began to appear in cotton seedlings two weeks after injection. Figure 4 -K), indicating the success of this VIGS experiment. To detect the silencing efficiency of GhCSLD3 in the two materials, after yellowing occurred, the inventors' control plants (CK) and positive plants (CLCRV) were compared. 阳 RNA was extracted from leaves of the negative control plant (CLCRV:00) and the gene-silenced plant (CLCRV:GhCSLD3), and gene expression levels were detected using qRT-PCR. The results showed that the gene expression level of the CLRRV:GhCSLD3 silenced line was significantly lower than that of the CLRRV:00 and CK lines, while there was no significant difference in gene expression levels between CK and CLRRV:00. Figure 4 C, D). Two months after injection into cotton seedlings, fruiting branches began to elongate. Phenotypic observation and fruiting branch length measurement of plants with high gene silencing efficiency revealed that the fruiting branch length of the silencing lines was significantly higher than that of the CLCRV:00 plants. Figure 4 E and F). We then identified transgenic Arabidopsis thaliana and observed the phenotypic characteristics of the T3 generation homozygous lines. We found that after overexpression of this gene in Arabidopsis thaliana, the overexpressing plants had significantly shorter lateral branches compared to wild-type Arabidopsis thaliana. Figure 4 The above results further demonstrate that GhCSLD3 plays a negative regulatory role in the development of fruiting branch length in upland cotton.

[0036] Cloning, identification, and functional analysis of the full-length sequence of GhCSLD3, a gene negatively regulating fruit branch length in upland cotton.

[0037] 1. Extraction of total RNA from cotton

[0038] About two months after sowing the cotton plants in the field, the third node from the bottom and young leaves were taken from the cotton plants during the critical period of fruit branch elongation. The leaves were wrapped in tin foil, placed in liquid nitrogen and quickly ground. Total RNA was extracted using the RNA extraction kit from Novizan. The quality and concentration of RNA were detected by 1.2% agarose gel electrophoresis and spectrophotometry. The plants were stored at -80℃.

[0039] 2. Cloning of the cotton GhCSLD3 gene

[0040] Using total RNA as a template, cDNA was synthesized using the reverse transcription kit (AG11728) from Acry Biotech Ltd., following the manufacturer's instructions. Gene-specific primers were designed based on the GhCSLD3 gene sequence. The primer sequences were: forward primer F: 5'-TGCATATACCGCCCACCCCA-3' and reverse primer R: 5'-ATCCAAATCGTCCCCCCCAG-3'.

[0041] The GhCSLD3 gene was obtained by PCR amplification using total cDNA as a template, and the results are as follows: Figure 1 As shown, the target gene with the corresponding band size was obtained and the PCR product was recovered. Through the above steps, the full-length 3432bp coding sequence of this gene in cotton was obtained (SEQ ID NO:1), and its protein coding sequence was deduced (SEQ ID NO:2), wherein the start codon is ATG and the stop codon is TAA.

[0042] The PCR procedure is shown in Table 1:

[0043]

[0044] The PCR system is shown in Table 2.

[0045] Primer F 1μl Primer R 1μl cDNA 2μl DdH2O 21μl

[0046] 3. Analysis of GhCSLD3 gene expression in cotton

[0047] Using cDNA from the shoot tips and internodes of the fruiting branches of the *Sanjiang Bajiang Dahua* (a material with extremely long fruiting branches) and the *Blanco* (a material with extremely short fruiting branches) as templates, respectively, the GhCSLD3 gene was validated using the PerfectStart Green qPCR SuperMix kit from TRANS Biosciences, with histone3 as an internal control gene. The forward primer F: 5'-TTACCGAAGATGTGGTCACTGG-3' and the reverse primer R: 5'-TCAGGAAAATCGAGGTGAAGGG-3' were used. Three technical replicates and three biological replicates were designed. The relative expression levels of GhCSLD3 in the extremely long fruiting branch material (*Sanjiang Bajiang Dahua*) and the extremely short fruiting branch material (*Blanco*) were analyzed, and the relative expression levels were calculated using the 2–ΔΔCT method. Figure 2 The GhCSLD3 gene is highly expressed in the shoot tips and internodes of the short-fruited branch material Blanco.

[0048] 4. Subcellular localization and analysis of the GhCSLD3 gene

[0049] Using leaf cDNA from upland cotton as a template, specific primers were designed using the TOYOBO high-fidelity enzyme KODone. The full-length CDS of the GhCSLD3 gene, with the stop codon removed, was cloned using primers containing corresponding adapters and restriction sites. The empty vector pCAMBIA2300_35S_GFP-HA was double-digested with BamHI and EcoRI restriction enzymes to obtain a linearized vector. The target gene was ligated to the linearized vector to obtain the 35S::GhCSLD3-GFP plant expression vector. The recombinant plasmid was transformed into Agrobacterium competent cells GV3101 via liquid nitrogen freeze-thaw method to achieve transient transformation of tobacco leaves. The injected tobacco plants were cultured overnight in darkness and then grown normally in a culture room for 2 days. Subcellular localization results were then observed using laser confocal microscopy. Figure 3 As shown: Green fluorescent signals are expressed in the cell membrane.

[0050] 5. Overexpression of the GhCSLD3 gene in Arabidopsis thaliana

[0051] Using leaf cDNA from upland cotton as a template, specific primers with corresponding adapters and restriction sites were designed using the TOYOBO high-fidelity enzyme KODone to clone the full-length CDS of the GhCSLD3 gene (with the stop codon removed). The empty vector pCAMBIA2300_35S_GFP-HA was double-digested with BamHI and EcoRI restriction enzymes to obtain a linearized vector. The target gene was ligated to the linearized vector to obtain the 35S::GhCSLD3-GFP plant expression vector. The recombinant plasmid was transformed into Agrobacterium competent cells GV3101 via liquid nitrogen freeze-thaw method to infect Arabidopsis inflorescences. Seeds were harvested from infected Arabidopsis, and positive seedlings were selected on K+-resistant 1 / 2 MS solid medium. Positive seedlings were identified in the T1 generation, and gene expression levels were detected in the T2 generation. Lines with a green seedling:yellow seedling ratio of 3:1 were selected for the next generation culture until the T3 generation homozygous lines were observed for phenotypic data.

[0052] 5. Phenotypic analysis of CLCRV:GhCSLD3 silent lines

[0053] Using internode cDNA from extremophile branches as a template and CLCRV as a vector, the GhCSLD3 gene silencing fragment was amplified using the KOD one high-fidelity enzyme provided by TOYOBO according to the PCR program. The corresponding restriction enzyme sites (SpeI and AscI) were selected, and the fragment was then processed... The recombinase was added and ligated into the CLCRV vector digested with SpeI and AscI to obtain the CLCRV:GhCSLD3 vector. After transformation with *E. coli*, positive single clones were selected, identified by colony PCR, and sent to Qingke Biotechnology Co., Ltd. for first-generation sequencing. The correctly sequenced recombinant plasmid was transformed into *Agrobacterium* GV3101 for subsequent injection. *Agrobacterium* GV3101 cultures containing *CLCRVB* (helper bacteria), *CLCRV:00* (negative control), *CLCRV*-positive (positive control), and *CLCRV:GhCSLD3* were inoculated into K+ and R+ liquid LB medium and cultured overnight at 28°C and 200 rpm. The cultured *Agrobacterium* culture was then transferred to 50 mL centrifuge tubes, centrifuged at 6000 rpm for 10 min to collect the cells, and thoroughly resuspended in an equal volume of resuspension buffer (200 μmol / LAS + 10 mmol / L MgCl2 + 1 mmol / L LMES), adjusting the OD600 to approximately 1.0, and incubated in the dark for 3 h. When injecting the bacterial culture, CLCRV:GhCSLD3, empty vector CLCRV:00, and CLCRV positivity were mixed with the auxiliary bacterium CLCRVB at a 1:1 ratio, and injected into two cotyledons of cotton using a 1 mL syringe. After injection, the mixture was cultured in the dark for 24 hours, and then allowed to grow normally in an artificial climate chamber.

[0054] To detect the silencing efficiency of the GhCSLD3 gene in cotton: When CLCRV-positive plants showed a yellowing phenotype, RNA was extracted from young cotton leaves containing CK, CLCRV:00, CLCRV-positive, and CLCRV:GhCSLD3, and reverse transcribed into cDNA. The gene silencing efficiency was detected by qRT-PCR, and plants with high silencing efficiency were selected for subsequent phenotypic observation.

[0055] Fruit branch length measurement: Seven GhCSLD3 gene-silenced plants and five CLCRV plants were taken from each of the Blanco and Sanjiang Bajiang large-flowered plants. A The length of the fruiting branches of the plants was measured with a ruler approximately every 7 days. Finally, a T-test was performed on the second fruiting branch of the gene-silenced plants and the unloaded plants.

[0056] 6. Phylogenetic analysis of the GhCSLD3 gene

[0057] Using TM-1-CRI as the reference genome, genomic, CDS, and protein sequence data of homologous genes were downloaded from the Cottongen website (https: / / www.cottongen.org / ), and closely related homologous genes were selected. GhCSLD3 proteins were clustered using MEGA.11 software, and the gene structure of the GhCSLD3 gene was plotted using the online website GSDS (http: / / gsds.gao-lab.org / ). Gh_A08G245200 and Gh_D08G245400 were identified as homologous genes, most closely related, with approximately 61% sequence homology, encoding CSLD3, both consisting of two exons and one intron. Gene annotation showed that this gene is located on the endoplasmic reticulum and plasma membrane, encoding cellulose synthase-like protein 3.

Claims

1. A gene that negatively regulates the fruit branch length trait of upland cotton, characterized in that: The gene is named GhCSLD3, and its nucleotide sequence is shown in SEQ ID No.

1.

2. The protein encoded by the gene negatively regulating the fruit branch length trait of upland cotton as described in claim 1, characterized in that: The gene encoding this protein is a tetraploid cotton gene, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2 of the sequence listing.

3. The subcellular localization expression vector for GhCSLD3, the gene that negatively regulates the fruit branch length trait of upland cotton, as described in claim 1.

4. The Arabidopsis overexpression vector for GhCSLD3, the gene that negatively regulates the fruit branch length trait of upland cotton, as described in claim 1.

5. The application of silencing the gene for negatively regulating the fruit branch length trait of upland cotton as described in claim 1 in promoting the elongation of fruit branches in upland cotton.

Images