New Use of S-Adenosyl-L-Homocysteine Hydrolase Gene

Abstract

The present invention belongs to the field of plant genetic technology, and discloses a new use of S-adenosyl-L-homocysteine hydrolase gene. The present invention reveals that the S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400 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 way 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 new use of S-adenosyl-L-homocysteine hydrolase gene, specifically to a new use of S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400. Background Art

[0002] Cotton is an important cash crop in the world. Cotton fiber is an important resource for the textile industry and also an excellent model for cell biology research. While continuing to maintain high cotton yields, it is of utmost importance to further improve the quality of cotton fibers. Therefore, it is extremely important to discover and identify candidate genes for excellent fiber quality and cultivate cotton varieties with excellent fiber quality. 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. S-adenosyl-L-homocysteine hydrolase (SAHH) is a key enzyme widely present in organisms that regulates intracellular methylation reactions. It can reversibly catalyze the decomposition of S-adenosyl-L-homocysteine (SAH) into adenosine (Ado) and homocysteine (Hcy), enabling the smooth progress of SAH methylation transfer. Therefore, SAHH plays a role similar to a switch in gene expression regulation. Previous studies randomly sequenced the cotton fiber mixed cDNA library of high-quality fiber upland cotton line 7235 and cloned a full-length GhSAHH gene, which is expressed in various tissues and organs of upland cotton TM-1 and is predominantly expressed in roots and hypocotyls, and may be related to the formation of fiber quality. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a new use of cotton S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400.

[0004] The present invention provides 1. The application of S-adenosyl-L-homocysteine hydrolase gene in molecular marker-assisted breeding of cotton varieties, and the coding sequence of S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400 is shown as SEQ ID NO.1.

[0005] G.mustelinum v1.1|Gomus.A12G023400.1CDS1809

[0006]

[0007] The present invention also provides an application of the S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400 shown in SEQ ID NO.1 in improving the strength of cotton fibers.

[0008] Compared with the prior art, the present invention provides a new use of the cotton S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400, which is an important gene affecting the quality of cotton fiber strength and can be applied to the improvement of cotton fiber strength or the molecular marker-assisted breeding of cotton varieties based on fiber strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 For the expression analysis of lncRNA target genes related to fiber development; the asterisks above the bar graphs represent statistically significant differences (***P < 0.001);

[0010] Figure 2 For the phenotypic map of the leaf color of VIGS plants at the seedling stage; 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; pCLCrV::00 is the empty vector, representing the negative control; pCLCrV::GhPDS is the 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, leading to 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 group of the target gene;

[0011] Figure 3 For the verification of gene expression in VIGS-silenced plants: A: leaves; B: 17 dpa - cotton fibers; C: 21 dpa - cotton fibers. The asterisks above the bar graphs indicate statistically significant differences (***P < 0.001, ****P < 0.0001). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus more clearly define the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0013] Example 1 lncRNA Sequencing Analysis and Target Gene Prediction

[0014] Using a pair of near-isogenic lines consisting of the Gossypium barbadense introgressed 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, usually with a length > 200bp. According to their positional relationship with coding sequences, they can be classified 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. In this experiment, transcripts of these three types, namely lincRNA, intronic lncRNA, and anti-sense lncRNA, were mainly screened.

[0015] Due to the characteristic that lncRNA itself does not have coding function, the principle by which it exerts its function is usually through two ways, namely cis and trans ways, to act on protein-coding target genes; 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 lncRNA. 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, protein-coding genes located about 100kb upstream and downstream of lncRNA were selected as its target genes. However, the prediction of trans-acting related 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 is related to the co-expressed protein-coding gene. Its target genes can be predicted by analyzing the expression correlation or co-expression between lncRNA and protein-coding genes among samples.

[0016] According to relevant annotation information, the present invention analyzed and obtained 1 target gene related to fiber development: S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400, and its function annotation is shown in Table 1.

[0017] Table 1 Function annotation information of target genes

[0018]

[0019] Example 2: Expression verification of target genes

[0020] Total RNA of plant materials was extracted and purified, and RNA was reverse transcribed into cDNA for qRT-PCR experiments. PrimerPremier5 software was used to screen and design suitable primers through the target gene sequence, and the designed primer sequences were sent to Suzhou Hongxun Biotechnology Co., Ltd. for primer synthesis. For the target gene Gomus.A12G023400, qRT-PCR verification was performed using its yellow-brown cotton homologous gene sequence. The results are as follows Figure 1 As shown, the expression level of the target gene is consistent with the expression trend of the transcriptome data results.

[0021] Example 3: VIGS Function Verification of Target Genes in Cotton

[0022] Gene cloning of candidate genes and functional verification based on virus-induced genesilencing (VIGS) were carried out in cotton.

[0023] Negative control: empty vector pCLCrV::00;

[0024] Positive control: plant pCLCrV::GhPDS in which the phytoene desaturase (PDS) gene was silenced;

[0025] Experimental group: gene silenced plant pCLCrV:Gomus.A12G023400 of the target gene Gomus.A12G023400 obtained by VIGS technology.

[0026] The yellow-brown cotton introgression line IL9 seedlings became diseased 15 days after VIGS treatment, while the negative control showed only slight wilting ( Figure 2 Left); In the positive control, the plant whose phytoene desaturase (PDS) gene was silenced resulted in the loss of protection, and the synthesis of chlorophyll was affected by photobleaching, resulting in bleaching of its true leaves ( Figure 2 The VIGS bacterial solution injection was successful. Figure 2 right.

[0027] After the albino phenotype appeared in pCLCrV::GhPDS, leaves and fibers at 17 dpa and 21 dpa of the pCLCrV::Gomus.A12G023400 silenced plants were taken to extract total RNA, which was then reverse transcribed and subjected to qRT-PCR experiments. The results are shown in Figure 2. Figure 3 shown. Figure 3Verification of gene expression in VIGS-silenced plants. Note: A: Leaf; B: 17 dpa - cotton fiber; C: 21 dpa - cotton fiber. Asterisks above the bar graphs indicate statistically significant differences (***P < 0.001, ****P < 0.0001).

[0028] It can be seen from Figure 3 that the expression level of the target gene showed a significant decrease compared to the negative control, whether in leaves or in fibers. This indicates that the VIGS infection was successful and correct, and the target gene was effectively silenced.

[0029] After the VIGS plants (experimental group) grew for 5 - 6 months, mature cotton fibers in the natural state were collected, and three biological replicates were taken for measurement of relevant phenotypes in terms of fiber length, fiber strength, and fiber regularity.

[0030] Table 2 Measured values of fiber phenotypes of VIGS plants

[0031]

[0032] (*All are the significance of differences compared with pCLCrV::00; *P < 0.05, **P < 0.01)

[0033] After measuring the relevant phenotypes of the VIGS plants (Table 2), it was found that the fiber strength value of the plants with the Gomus.A12G023400 gene silenced was significantly lower than that of the negative control group.

Claims

1. Application of S-adenosyl-L-homocysteine hydrolase gene in molecular marker-assisted breeding of cotton varieties based on fiber strength, S-adenosyl-L-homocysteine hydrolase gene Gomus.A12G023400 The coding sequence thereof is as shown in SEQ ID NO.

1.

2. Application of S-adenosyl-L-homocysteine hydrolase gene shown in SEQ ID NO.1 in improving the fiber strength of cotton Gomus.A12G023400 ​

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