Application of GhPDAT gene in regulation of cotton bud and boll abscission

Abstract

The application provides application of a cotton GhPDAT gene in regulation of cotton bud boll abscission, and belongs to the technical field of genetic engineering. The GhPDAT gene negatively regulates cotton bud boll abscission; the nucleotide sequence of the coding region of the GhPDAT gene is shown in SEQ ID NO. 2. The function of the GhPDAT gene is verified by VIGS silencing technology, and silencing the gene can significantly improve the cotton bud boll abscission rate. The application provides a new genetic resource and breeding tool for reducing the cotton bud boll abscission rate and improving the cotton yield, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the cotton GhPDAT gene in regulating cotton boll shedding. Background Technology

[0002] cotton( Gossypium spp. Cotton is a globally important economic crop and source of natural fiber, and its yield and quality directly affect the economic benefits of the textile industry. Boll shedding is one of the key limiting factors in cotton production; statistics show that the boll shedding rate is as high as 40-70%, severely restricting the increase of cotton yield per unit area. Boll shedding is regulated by multiple factors, including genetic factors, environmental conditions, and hormone levels, and is a typical quantitative trait.

[0003] Traditional breeding methods for improving boll shedding traits are time-consuming, inefficient, and easily affected by environmental factors. Marker-assisted selection (MAS) and genetic engineering techniques offer effective ways to accelerate the breeding process. Genome-wide association study (GWAS) is an effective method based on the principle of population linkage disequilibrium. It identifies genes and QTLs controlling complex quantitative traits by analyzing the associations between genetic markers and target traits in natural populations. In recent years, GWAS has been widely used for the genetic analysis of complex agronomic traits in important crops such as cotton.

[0004] However, current research on key genes regulating cotton boll shedding and their functional mechanisms is insufficient, and there is a lack of gene resources and functional molecular markers that can be directly used for molecular breeding. Therefore, identifying key genes closely related to cotton boll shedding is of great significance for elucidating their functions and conducting marker-assisted breeding. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the cotton GhPDAT gene in regulating cotton boll shedding.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of the cotton GhPDAT gene in regulating cotton bud and boll shedding, wherein the GhPDAT gene negatively regulates cotton bud and boll shedding; the nucleotide sequence of the coding region of the GhPDAT gene is shown in SEQ ID NO.2.

[0007] Preferably, the GhPDAT genome nucleotide sequence is shown in SEQ ID NO.1.

[0008] Compared with the prior art, the present invention has the following beneficial effects: Based on the reference genome of high-quality upland cotton Xinluzao 31 (HC04), this invention successfully identified a gene associated with cotton boll shedding using population genome resequencing and genome-wide association analysis (GWAS) techniques. GhPDAT .

[0009] This invention verifies the effectiveness of VIGS silencing technology. GhPDAT The gene's function is to silence it, which can significantly increase cotton boll shedding rate. This invention provides a new genetic resource and breeding tool for reducing cotton boll shedding rate and increasing cotton yield, and has significant application value. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0011] Figure 1 The results of the correlation analysis of bud and boll shedding rate during the peak boll-forming period of natural populations are shown below. Among them, A is the Manhattan plot of AR1 in the 2018-KEL environment, B is the QQ plot of AR1 in the 2018-KEL environment, C is the Manhattan plot of AR1 in the 2018-SHZ environment, D is the QQ plot of AR1 in the 2018-SHZ environment, E is the Manhattan plot of AR1 in the 2019-KEL environment, F is the QQ plot of AR1 in the 2019-KEL environment, G is the Manhattan plot of AR1 in the 2019-SHZ environment, H is the QQ plot of AR1 in the 2019-SHZ environment, I is the Manhattan plot of AR1 in the BLUP environment, and J is the QQ plot of AR1 in the BLUP environment. Figure 2 The results show the assessment of linkage disequilibrium within the candidate intervals of the natural population; where A, B, and C show the linkage disequilibrium analysis results for qAR1-D06, qAR1-A03-1, and qAR1-A03-2, respectively. Figure 3 The results of scanning electron microscopy (SEM) for different growth states of cotton and cotton boll ablation are shown. The scale bar of the overall view in the SEM results is 500 μm, and the scale bar of the local details is 100 μm. Figure 4 for HC04_D06001880 ( GhPDAT Results of gene expression level determination in extreme phenotype materials; Figure 5 For silence GhPDAT The effect of genes on cotton boll shedding; where A represents the phenotype of VIGS cotton and the control, and B represents... GhPDATThe relative expression level of the gene in VIGS plants, where C represents the statistical analysis results of the bud and boll shedding rate in VIGS plants. Detailed Implementation

[0012] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0013] Example 1: Identification of key sites for cotton bud and boll shedding traits

[0014] Whole-genome sequencing was performed on 169 materials (original seeds of Xinjiang upland cotton varieties collected from breeding units, after selection, identification, self-pollination purification, and propagation of typical individual plants; variety information is detailed in Table 1). The sequencing data were aligned to the Xinluzao 31 (HC04) reference genome. The alignment rate for each material was above 99%, with an average sequencing depth of 12.1 kb. A total of 1,458,839 SNPs were detected, with an average density of 0.63 / kb. 36,894 SNPs (1.21%) were located within gene coding regions. By combining bud and boll shedding phenotypic data (bud and boll shedding rate AR1 during flowering, bud and boll shedding rate AR2 during peak boll shedding, and relative shedding rate ARA) under four different environments (Shihezi City in 2018, Korla City in 2018, Shihezi City in 2019, and Korla City in 2019), a genome-wide association analysis (GWAS) was conducted, and significant association loci were screened with a threshold of -Log10 (p-value) > 5. To increase the reliability of candidate intervals, this study only retained SNPs that were significantly associated in at least two environments. Ultimately, four SNPs (HC04_D06_1661821, HC04_D06_1821891, HC04_A03_93236433, and HC04_D01_4627060) were retained. Furthermore, based on LD analysis, the decay distance for this population was determined to be 500 kb. Using 500 kb upstream and downstream of significant SNPs as candidate intervals for this phenotype, four QTL intervals were identified: qAR1-D06-1, qAR1-D06-2, qAR1-A03-1, and qARA-D10-1. Since qAR1-D06-1 and qAR1-D06-2 have an 840 kb overlap, these two QTLs were merged into a single candidate region, qAR1-D06 (see Table 2). Based on genome annotation information, the QTL interval qAR1-D06 was identified as a key site significantly associated with the bud-boll abscission rate (AR1) during flowering. This interval is physically located at chromosome D06, bp 1161821-2321891, with a length of approximately 1.16 Mb, and contains 65 annotated genes. Figure 1 , Figure 2 ).

[0015] Table 1 Cotton Material Information

[0016]

[0017]

[0018]

[0019]

[0020] Table 2. QTL analysis of cotton boll shedding traits

[0021]

[0022] Example 2: Transcriptome analysis of extreme cotton boll shedding materials

[0023] Transcriptome sequencing (as shown in Table 3) and scanning electron microscopy (SEM) were performed on the boll stalk abscission regions of two high-abscission materials H18 and H35 and two low-abscission materials L32 and L50. Figure 3 ).

[0024] Differential expression analysis was performed on 94 genes within the candidate region, and genes containing the region were preliminarily screened. HC04_ D06001880 Sixteen genes, including those with significant differential expression.

[0025] Table 3 Selection of Extreme Materials

[0026] Example 3: In materials with extreme phenotypes GhPDAT Gene expression level detection

[0027] Based on the reference genome annotation information, specific primers were designed: Forward primer F: 5'-GACTTGTGACTGCTGATGGC-3' (SEQ ID NO.4) Reverse primer R: 5'-CTGGTCTCGAACCTGTGTGTT-3' (SEQ ID NO.5) Total RNA was extracted from the stalk abscission zone (AZ) of high-abscission materials Xinluzhong 18 and Xinluzao 35, and low-abscission materials Xinluzao 32 and Xinluzhong 50. After DNase I digestion to remove genomic DNA, cDNA first strand was synthesized using the Novizan 1st Strand CDNASynthesis Kit (+gDNA Wiper) reverse transcription kit (Nanjing, China, catalog number: R212). The qRT-PCR reaction system (20 μL) consisted of: 10 μL of 2×Phanta Max Master Mix (Dye Plus), 0.5 μL each of forward and reverse primers, 2 μL of cDNA template, and ddH2O to a final volume of 20 μL. The reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56℃ annealing for 15 sec, and 72℃ extension for 30 sec, for 28 cycles. Each sample was tested in triplicate, and the relative expression level was calculated using the 2-ΔΔCt method.

[0028] Experimental results show that ( Figure 4 ), HC04_D06001880 The expression level of this gene in the abscission zone tissues of low-abscission materials Xinluzao 32 and Xinluzhong 50 was significantly higher than that in high-abscission materials Xinluzhong 18 and Xinluzao 35 (p<0.01), indicating that this gene may act as a negative regulator of cotton boll shedding. Functional annotation revealed that HC04_D06001880 encodes a diacylglycerol acyltransferase, which is homologous to AT5G13640 in Arabidopsis thaliana, and therefore named it. GhPDAT Gene.

[0029] GhPDAT The genomic DNA sequence of the gene in upland cotton TM-1 is shown in SEQ ID NO.1, the DNA sequence of the coding region is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.3.

[0030] Example 4

[0031] Based on the reference genome annotation information, in GhPDAT Select an appropriate length for the gene CDS region, design specific primers, and add universal adapter sequences to both ends of the primers. Add Kpn to the 5' end of the forward primer. Enzyme cleavage site, BamH added to the 5' end of the reverse primer The enzyme cleavage sites were determined, and the resulting primers are as follows: Forward primer F: 5'-GCGTGAGCTCGGTACCTCCAAGTGGTCGTGTTTGGA-3' (SEQ ID NO.6); Reverse primer R: 5'-GCCTCCATGGGGATCCCATCACCAAAAGTGCCACCC-3' (SEQ ID NO.7).

[0032] cDNA from upland cotton TM-1 was used as a template for amplification to obtain the target fragment. The pCLCrVA vector was prepared using Kpn. and BamH Enzyme digestion was performed to obtain a linearized vector. The PCR product was then ligated into the linearized pCLCrVA vector using In-Fusion enzyme to construct... GhPDAT The VIGS-inhibiting expression vector pCLCrVA::GhPDAT was constructed. After plasmid extraction, the constructed vector was electroporated into Agrobacterium GV3101 strain.

[0033] VIGS vector infection of cotton: After delinting L32 seeds, selected seeds were planted in an artificial climate chamber suitable for cotton growth (light / dark, 16 h, 28℃ / 8 h, 22℃). After 10 days, when the cotyledons of the seedlings were fully expanded, VIGS bacterial suspension was injected. Agrobacterium was activated and propagated with Agrobacterium containing pCLCrVA::GhPDAT, empty vector pCLCrVA, positive control vector pCLCrVA::GhPDS, and helper vector pCLCrVB, respectively. The suspension was shaken in a 28℃ shaking incubator until the OD600 value was 1.5~2.0. The bacterial suspension was centrifuged at 4000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were resuspended in a resuspension solution (100 mL resuspension solution: 1 mL MES-KOH (1M), pH 5.7; 1 mL MgCl2 (1M); 200 µL acetylsylgenone (20 µmol / L); sterile water to a final volume of 100 mL) and the OD600 value was adjusted to approximately 1.2. The cells were then incubated in the dark at room temperature for 3 h. The resuspensions of the empty vector, positive control, and gene vector and helper vector pCLCrVB were mixed separately at a 1:1 ratio. A small incision was made on the underside of the cotyledons to inject the bacterial solution. The cotton seedlings injected with the infection solution were then incubated in the dark for 24 h, followed by normal light incubation.

[0034] Identification of Silent Plants: When cotton plants reach the three-leaf or four-leaf stage, the leaves of the positive control PDS plants begin to turn white, indicating that the virus has successfully infected the cotton plants. Tender leaves were taken from cotton plants injected with pCLCrVA::GhPDAT and those without the vector, RNA was extracted, reverse transcribed, and then subjected to quantitative real-time PCR to detect the target gene. GhPDAT The expression level changes were measured to determine the silencing efficiency (method as in Example 3). The silencing efficiency was detected by qRT-PCR 15 days after inoculation.

[0035] The results showed that ( Figure 5 ), VIGS plants GhPDAT The expression level of pCLCrVA was reduced by more than 75% compared to the control. Phenotypic statistics showed that pCLCrVA:: GhPDAT The bud and boll shedding rate of the silent plants was 26.8%, which was 2.37 times higher than that of the empty vector control (11.3%), confirming... GhPDAT It is a negative regulator of cotton boll shedding.

[0036]

[0037]

[0038] SEQ ID NO.3: MSLIRRRKHPFNESFRKPEKEHENGDFSYESKRKKIPYEHEEERPLSKWSCLDTWCWLIGCVCVTWWFLLFLYNAMPASFPKYVTEVITGPLPDPPGVKLRKLGLTAKHPVVFVPAAVTGGLELWEGRKCAQGLFRKRLWGGTFGDVYKRPQCWVEHMSLDNET GMDPCGIRVRPISGLVTADGLGSGYFVWTTLVSNLARIGYEEKTMFMAAYDWRLSFQNTQVRDQTLSRIKNNIELMVATRGGQKAVIIPHSAGALFFLHFMKWVEAPPPMGGGGGPYWCSKHIKAVVNIAAPFLGVPKAIPLFLSAESKDVSLVRAIAPGFLENDIFQPQ ILQHVMRLSHSWDSTVSMIPRGGDTIWGSLDWSPEEGYSCDQKREMKNSTRVTNPARAKSAISLTRSANFGRIVSFGKDVAEAPSNDIDRIDFKGAVKGHRAENRTCRDLWAGYHGMGFEGTKAVADYKTYTAESIVELLHFVAPKMMARSTAHFSHGIADNMDDPKYK HYKYWSNPLETTLPIAPEMEIFSFYGTGLPTERSYVYKLSPTAECHIPFQIDSSANDEETCLKGGAYSVDGDGTVPVLSAGFMCAKAWHGKTKFNPSGIRTYVREYRHSPPTTLLEGCGTLNCAHVDIMRSFALIEDVIRIAAGASGEELGGNRVHSNIFKWSEKINLQL

[0039] 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.

Claims

1. The application of the cotton GhPDAT gene in regulating cotton boll shedding, characterized by, The GhPDAT gene negatively regulates cotton boll shedding; the nucleotide sequence of the coding region of the GhPDAT gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the GhPDAT gene is shown in SEQ ID NO.1.

Images