Method for discovering cotton fiber strength-related gene and application thereof

Abstract

The present invention belongs to the field of plant genetic technology, and discloses a method for discovering genes related to cotton fiber strength and its application. The present invention reveals a method for discovering genes related to cotton fiber strength based on lncRNA analysis by using near-isogenic lines with different fiber qualities, and specifically applies this method to obtain three cotton fiber strength genes, namely Gomus.D05G015100, Gomus.A05G281300, and Gomus.A10G226800; these three genes can be used to improve the fiber quality of cotton or for molecular marker-assisted breeding of high-strength fiber cotton varieties. The technical solution of the present invention provides a new approach for molecular marker-assisted improvement breeding of cotton fiber quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetics and relates to a method for discovering genes related to cotton fiber strength and its applications. Background Art

[0002] Cotton is an important cash crop in the world and also an important oil crop; cotton fiber is an important resource for the textile industry and also an excellent model for cell biology research. The main cotton varieties promoted in China lack particularly high-quality upland cotton varieties with coordinated fiber length, fiber strength, and fiber fineness. Therefore, while maintaining high cotton yields, it is of utmost importance to further improve the quality of cotton fibers. Identifying candidate genes with excellent fiber quality and cultivating cotton varieties with excellent fiber quality are extremely important. The application of near-isogenic lines with significantly different fiber qualities and high-throughput sequencing technology helps to identify key genes related to fiber development and their regulatory mechanisms. Long non-coding RNA (lncRNA) is an important component of genomic transcription and plays a role in a wide range of biological processes such as plant development and stress responses. Previous studies have shown that lncRNA has an important impact on cotton fiber development. Therefore, in-depth research on it is expected to discover key genes for cotton fiber development and apply them to molecular breeding of cotton fiber quality. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for discovering genes related to cotton fiber strength and its applications.

[0004] The technical solution adopted by the present invention is as follows:

[0005] A method for discovering genes related to cotton fiber strength, comprising: selecting a pair of near-isogenic lines composed of the Gossypium mustelinum introgression line IL9 and its upland cotton recurrent parent PD94042 as materials, selecting cotton fibers at 17 and 21 days after flowering, extracting RNA, reverse-transcribing it into cDNA, constructing a library for lncRNA sequencing analysis, and then predicting and screening target genes to obtain genes related to cotton fiber strength;

[0006] The genes related to cotton fiber strength include Gomus.D05G015100, Gomus.A05G281300, and Gomus.A10G226800;

[0007] Among them, the coding sequence of Gomus.D05G015100 is as shown in SEQ ID NO.1;

[0008] The coding sequence of Gomus.A05G281300 is as shown in SEQ ID NO.2;

[0009] The coding sequence of Gomus.A10G226800 is shown in SEQ ID NO.3.

[0010] The present invention also provides an application of a cotton fiber strength-related gene in improving the cotton fiber strength of cotton, and the cotton fiber strength-related gene is one or more of Gomus.D05G015100, Gomus.A05G281300, and Gomus.A10G226800;

[0011] Among them, the coding sequence of Gomus.D05G015100 is shown in SEQ ID NO.1;

[0012] The coding sequence of Gomus.A05G281300 is shown in SEQ ID NO.2;

[0013] The coding sequence of Gomus.A10G226800 is shown in SEQ ID NO.3.

[0014] The present invention also provides an application of a cotton fiber strength-related gene in molecular marker-assisted breeding of cotton varieties, and the cotton fiber strength-related gene is one or more of Gomus.D05G015100, Gomus.A05G281300, and Gomus.A10G226800;

[0015] Among them, the coding sequence of Gomus.D05G015100 is shown in SEQ ID NO.1;

[0016] The coding sequence of Gomus.A05G281300 is shown in SEQ ID NO.2;

[0017] The coding sequence of Gomus.A10G226800 is shown in SEQ ID NO.3.

[0018] G.mustelinum v1.1|Gomus.D05G015100.1 CDS

[0019]

[0020] G.mustelinum v1.1|Gomus.A05G281300.1 CDS

[0021]

[0022] G.mustelinum v1.1|Gomus.A10G226800.1 CDS

[0023]

[0024] Compared with the prior art, the present invention selects a pair of near-isogenic lines composed of the Gossypium mustelinum introgression line IL9 and its Gossypium hirsutum recurrent parent PD94042 as materials, selects cotton fibers at 17 and 21 days after flowering, performs lncRNA sequencing analysis, then predicts and screens target genes, obtains genes related to cotton fiber strength, and both expression verification and functional verification show that the method for discovering fiber strength functional genes provided by the present invention is accurate and efficient. These genes can be used to improve the fiber quality of cotton and can also be used as molecular markers for seed selection and breeding. Description of the Drawings

[0025] Figure 1 It is a lncRNA differential expression map;

[0026] Figure 2 It is the expression analysis of lncRNA target genes related to fiber strength; Note: The asterisks above the bar graphs represent statistically significant differences (***P < 0.001, ****P < 0.0001);

[0027] Figure 3 It is the phenotypic map of the leaf color of each plant at the seedling stage in the VIGS functional verification experiment of target genes related to fiber strength in cotton; among them, the first column in the figure is the negative control (empty vector pCLCrV::00), the second column is the positive control (plant pCLCrV::GhPDS with phytoene desaturase gene silenced), and the third column is the experimental group (pCLCrV:Target gene); the target genes in groups A-C are Gomus.A05G281300, Gomus.D05G015100, and Gomus.A10G226800 respectively; pCLCrV:Target gene represents the experimental groups with 3 target genes silenced respectively;

[0028] Figure 4 It is the gene expression verification of VIGS-silenced plants; A: Leaves; B: 17dpa - cotton fibers; C: 21dpa - cotton fibers. The asterisks above the bar graphs indicate statistically significant differences (***P < 0.01, ***P < 0.001, ****P < 0.0001). Detailed Embodiments

[0029] The present invention provides a method for discovering genes related to cotton fiber strength, including:

[0030] Step 1, lncRNA sequencing analysis

[0031] Using a pair of near-isogenic lines composed of the Gossypium arboreum introgression line IL9 with outstanding comprehensive fiber quality performance and its upland cotton recurrent parent PD94042 as materials, cotton fibers at 17 and 21 days post anthesis (dpa) were selected, RNA was extracted, reverse transcribed into cDNA, and a library was constructed for lncRNA sequencing analysis. lncRNA is a class of long non-coding RNAs, with a length usually >200bp. According to the different positional relationships with coding sequences, it can be divided into intergenic lncRNA (abbreviated as lincRNA), intronic lncRNA, anti-sense lncRNA, sense lncRNA, bidirectional lncRNA and other types. Among them, lincRNA accounts for the highest proportion. This experiment mainly screened the transcripts of these three types: lincRNA, intronic lncRNA, and anti-sense lncRNA.

[0032] Differential expression analysis was performed on the identified lncRNAs. A total of 203 differential lncRNAs were obtained, showing the same trend as the results in mRNA. The largest number of differences was between IL9-17dpa and IL9-21dpa, with a total of 120. The smallest number of differences was between PD94042-17dpa and IL9-17dpa, only 8. The statistical results are as Figure 1 shown.

[0033] Step 2: Prediction of lncRNA target genes

[0034] Due to the characteristic that lncRNA itself does not have coding function, its principle of action is usually achieved in two ways, that is, by acting on protein-coding target genes in cis and trans manners; another is based on the base complementary pairing between lncRNA and mRNA to produce an effect. Based on this principle, the target gene prediction tool LncTar was used to predict the target genes of our lncRNAs. The basic principle of cis-acting target gene prediction believes that the function of lncRNA is related to the protein-coding gene adjacent to its coordinates. Therefore, the protein-coding genes located about 100kb upstream and downstream of lncRNA were selected as its target genes. However, the prediction of trans-acting target genes is different from that of cis-acting. The basic principle of its prediction is that the function of lncRNA is not related to the positional relationship between itself and the coding gene, but related to the co-expressed protein-coding gene. The target genes can be predicted by analyzing the correlation or co-expression of the expression levels of lncRNA and protein-coding genes between samples. Then, according to the relevant annotation information (source), the target genes related to fiber strength were screened.

[0035] According to the relevant annotation information, three target genes related to fiber strength were screened in this study, and their functional annotations are shown in Table 1.

[0036] Table 1 Functional annotation information of target genes

[0037]

[0038] Step 3: Expression verification of target genes related to fiber strength

[0039] Total RNA of plant materials was extracted and purified, and the RNA was reverse transcribed into cDNA for qRT-PCR experiments. The Primer Premier 5 software was used to screen and design appropriate primers through the target gene sequences, and the designed primer sequences were sent to Suzhou Hongxun Biotechnology Co., Ltd. for primer synthesis to carry out the expression verification of target genes. For the relevant target genes, their homologous genes sequences in Gossypium mustelinum were used for qRT-PCR verification. The expression levels of these three target genes were significantly up-regulated or down-regulated to varying degrees ( Figure 2 ), and the expression trends were consistent with the transcriptome data results.

[0040] Step 4: VIGS functional verification of target genes related to fiber strength in cotton

[0041] Gene cloning of candidate genes and functional verification based on virus-induced gene silencing (VIGS) were carried out in cotton. Gene-silenced plants of the target genes Gomus.D05G015100, Gomus.A05G281300, and Gomus.A10G226800 were obtained by VIGS technology, and the silenced plants showed a phenotype of significantly reduced fiber strength; it was considered that these three genes play an important role in cotton fiber strength.

[0042] Figure 3 This is the phenotypic diagram of the leaf color of each plant at the seedling stage in the VIGS functional verification experiment of target genes related to fiber strength in cotton. The first column in the figure is the negative control, the second column is the positive control, and the third column is the experimental group; the target genes in groups A-C are Gomus.A05G281300, Gomus.D05G015100, and Gomus.A10G226800, respectively. pCLCrV::00 is an empty vector, representing the negative control; pCLCrV::GhPDS is a plant in which the phytoene desaturase (PDS) gene is silenced. The silencing of this gene results in the loss of protection, and the synthesis of chlorophyll is affected by photobleaching, resulting in the bleaching of its true leaves; therefore, the plant with the silenced GhPDS gene is used as the positive control. pCLCrV:Target gene represents the experimental groups in which the three target genes are respectively silenced.

[0043] The seedlings of the Gossypium mustelinum introgression line IL9 showed symptoms 15 days after VIGS treatment. As Figure 3 shown in Figure 3 , in the negative control, there was only a slight wilting state; in the positive control, the plants with the phytoene desaturase (PDS) gene silenced showed a lack of protection, and the synthesis of chlorophyll was affected by photo-bleaching, resulting in the appearance of bleaching in their true leaves (

[0044] ), which proved the successful injection of the VIGS bacterial solution. After confirming the successful injection of the bacterial solution, to ensure that the subsequent normal growth and development of cotton were not affected, the plants were transplanted from small pots into large pots with a diameter of 21 cm. Figure 4 )

[0045] Figure 4 Verification of gene expression in VIGS-silenced plants; Note: A: Leaves; B: Cotton fibers at 17 dpa; C: Cotton fibers at 21 dpa. The asterisks above the bar graphs indicate statistically significant differences (***P < 0.01, ***P < 0.001, ****P < 0.0001).

[0046] As Figure 4 shown, the expression levels of the target genes were significantly decreased compared with the negative control, both in leaves and in fibers. This indicated that the VIGS infection was successful and correct, and the target genes were effectively silenced.

[0047] After the VIGS plants grew for 5 - 6 months, mature cotton fibers were collected under natural conditions, and three biological replicates were collected for each candidate gene. The relevant phenotypes were measured in terms of fiber length, fiber strength, and fiber uniformity.

[0048] Table 2 Measured values of fiber phenotypes of VIGS plants

[0049]

[0050] (*All are the significance of differences compared with pCLCrV::00; *P < 0.05, **P < 0.01)

[0051] After the determination of the relevant phenotypes of VIGS plants (Table 2), it was found that the fiber strength values of all three gene-silenced plants were significantly lower than those of the negative control group.

Claims

1. Use of genes related to cotton fiber strength in improving the cotton fiber strength of cotton, wherein the genes related to cotton fiber strength are Gomus.D05G015100 , Gomus.A05G281300 and Gomus.A10G226800 one or more of them ； Among them, Gomus.D05G015100 The coding sequence is shown in SEQ ID NO. 1; Gomus.A05G281300 The coding sequence is shown in SEQ ID NO. 2; Gomus.A10G226800 The coding sequence is shown in SEQ ID NO. 3.

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