CN115094070B - Discovery method and application of corn salt-tolerant gene Zm00001d033878 - Google Patents
Discovery method and application of corn salt-tolerant gene Zm00001d033878 Download PDFInfo
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Abstract
The application relates to the technical field of plant genetics, in particular to a method for discovering a salt-tolerant gene Zm00001d033878 of corn and application thereof. The corn salt-tolerant gene Zm00001d033878 genome DNA obtained by the application has a nucleotide sequence shown in SEQ ID NO. 1; the gene can be used for molecular marker assisted selection of salt-tolerant maize varieties.
Description
Technical Field
The application relates to the technical field of plant genetics, in particular to a method for discovering a corn salt-tolerant gene Zm00001d033878 and application thereof.
Background
Corn is an important grain crop and an important feed source, and is also the grain crop with the highest total yield worldwide. Soil salinization is a worldwide resource problem and ecological problem, and is an abiotic stress which seriously affects the growth and development of plants; the high salt environment greatly jeopardizes the growth and development of plants and can directly lead to a great reduction in yield of crops. Corn is a plant that is more susceptible to salt stress; salt stress is used as an important stress factor affecting the corn yield, and not only affects the corn growth, but also affects the corn quality and yield. At present, the grain problems faced by human beings are increasingly aggravated, and if the standby land resource of the saline-alkali soil is fully utilized, the salt-tolerant corn is cultivated and planted in the saline-alkali soil, so that the method plays an important role in guaranteeing the grain safety. For this reason, there is a need to develop salt tolerance genes and understand their regulatory mechanisms further, so as to develop maize salt tolerance molecular breeding using these genes.
Disclosure of Invention
The application aims to solve the defects in the prior art, and provides a discovery method and application of a corn salt tolerance gene Zm00001d033878, wherein the salt stress response regulation mode of the gene is predicted by carrying out functional analysis on the corn salt tolerance gene.
In order to achieve the above purpose, the present application adopts the following technical scheme:
a maize salt tolerance gene Zm00001d033878, wherein the maize salt tolerance gene Zm00001d033878 genome DNA has a nucleotide sequence shown as SEQ ID NO. 1.
A method for discovering a corn salt tolerance gene Zm00001d033878, which comprises the following steps:
step 1, strand specific transcriptome sequencing and analysis
Selecting and culturing LH196 seeds with tidy and consistent shape and full grains; dividing endosperm-removed seedlings with consistent growth vigor into two groups, wherein one group is blank control, the other group is 250mM NaCl nutrient solution culture group, and continuously treating for 10 days; culturing corn inbred line LH196 under the same conditions, respectively treating with 250mmol/L salt and blank control for 12 hours, taking corn leaves, freezing in liquid nitrogen, and storing at-80 ℃ for later use; extracting RNA in 6 sample leaves by using the kit, and respectively carrying out strand-specific transcriptome sequencing;
performing lncRNA sequencing by using a strand-specific library construction mode; compared with the common transcription banking mode, the strand-specific sequencing can determine the transcription directionality of two strands, and reduce errors in the alignment process; secondly, the lncRNA library construction sequencing can obtain more abundant information; the library establishment of the lncRNA belongs to mRNA+lncRNA, and the data of the mRNA and the data of the lncRNA can be obtained simultaneously only by once library establishment;
identification of differentially expressed genes: screening differential expression genes; clustering analysis of differential expression genes; functional annotation of differentially expressed genes; GO and KEGG enrichment analysis of differentially expressed genes; differentially expressed gene protein interaction networks;
lncRNA analysis: quantitatively analyzing lncRNA expression; analysis of lncRNA differential expression; analysis of lncRNA correlation between samples; annotation enrichment analysis of Cis action difference lncRNA target genes; PPI analysis of Cis action difference lncRNA target genes; annotation enrichment analysis of Trans-action difference lncRNA target genes; performing PPI analysis on a Trans-action difference lncRNA target gene;
combining the differential expression gene and the lncRNA target gene prediction, and exploring a common gene as a corn salt tolerance candidate gene;
step 2, verification of the expression pattern of salt-tolerant genes
Step 2.1, designing and synthesizing a Primer by utilizing a Primer5 for the salt-tolerant candidate gene, reversely transcribing RNA into cDNA, and performing expression pattern verification by utilizing real-time quantitative PCR;
and 2.2, carrying out overexpression verification of candidate genes in arabidopsis thaliana, preparing agrobacterium competence and an overexpression vector, constructing a recombinant plasmid, introducing agrobacterium into arabidopsis thaliana, culturing, screening arabidopsis thaliana transgenic homozygous salt treatment, and carrying out phenotype identification.
The application also provides application of the corn salt-tolerant gene Zm00001d033878 in molecular marker assisted selection of salt-tolerant corn varieties, and a salt stress response regulation mode of the gene is predicted.
By adopting the technical scheme: according to the application, the regulation and control modes of the salt stress response lncRNA and the target gene Zm00001d033878 thereof are estimated through the analysis of the lncRNA and the target gene thereof in the early stage. Under salt stress, lncRNA in corn seedling leaves is differentially expressed, the up-regulated expression of a target gene Zm00001d033878 is regulated, and Zm00001d033878 participates in activating certain salt tolerance paths in plants, so that corn can better adapt to salt stress.
Compared with the prior art, the application has the following beneficial effects:
the application takes a maize salt-tolerant inbred line LH196 as a material, carries out the sequence of a chain-specific transcriptome, the sequence of lncRNA and the prediction of a target gene under salt stress and contrast, preliminarily determines a salt-tolerant candidate gene through the combined analysis of the salt-tolerant candidate gene and the salt-tolerant candidate gene, carries out RT-qPCR expression analysis on the salt-tolerant difference gene, carries out functional verification in arabidopsis at last, and finally obtains the maize salt-tolerant gene Zm00001d033878 genome DNA with the functions of
A nucleotide sequence shown in SEQ ID NO. 1; the salt stress response regulation mode of the gene is predicted; the gene can be transferred to corn materials with excellent agronomic characters through molecular assisted breeding, so as to create corn materials or varieties with salt tolerance and excellent agronomic characters.
Drawings
FIG. 1 is a diagram showing the result of RT-qPCR expression verification of a salt-tolerant gene Zm00001d033878 of corn under salt stress in corn seedling leaves;
FIG. 2 is a graph showing the comparison of the difference in phenotype of two groups of Arabidopsis under salt stress in the present application;
wherein, WT is wild type Arabidopsis, OE is an overexpression gene Zm00001d033878 Arabidopsis strain, CK is a control group, and Salt is a 250mmol saline treatment group;
FIG. 3 is a schematic diagram showing the prediction of the regulation pattern of the salt stress response maize salt tolerance gene Zm00001d 033878.
Detailed Description
The following technical solutions in the embodiments of the present application will be clearly and completely described with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present application, and thus the protection scope of the present application is more clearly defined. The described embodiments of the present application are intended to be only a few, but not all embodiments of the present application, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the present application.
A maize salt tolerance gene Zm00001d033878, wherein the maize salt tolerance gene Zm00001d033878 genome DNA has a nucleotide sequence shown as SEQ ID NO. 1.
Specifically, the application takes a maize salt-tolerant inbred line LH196 as a material, carries out chain-specific transcriptome sequencing, lncRNA sequencing and target gene prediction under salt stress and control, preliminarily determines salt-tolerant candidate genes through combined analysis of the salt-tolerant candidate genes, carries out RT-qPCR expression analysis on salt-tolerant differential genes, and finally carries out functional verification in arabidopsis. The method comprises the following specific steps:
referring to fig. 1-3, a method for discovering a maize salt tolerance gene Zm00001d033878 comprises the following steps:
step 1, strand specific transcriptome sequencing and analysis
Selecting and culturing LH196 seeds with tidy and consistent shape and full grains; dividing endosperm-removed seedlings with consistent growth vigor into two groups, wherein one group is blank control, the other group is 250mM NaCl nutrient solution culture group, and continuously treating for 10 days; culturing corn inbred line LH196 under the same conditions, respectively treating with 250mmol/L salt and blank control for 12 hours, taking corn leaves, freezing in liquid nitrogen, and storing at-80 ℃ for later use; extracting RNA in 6 sample leaves by using the kit, and respectively carrying out strand-specific transcriptome sequencing;
the method has the advantages that the method adopts a chain-specific library construction mode to carry out lncRNA sequencing, and compared with a common transcription library construction mode, the chain-specific sequencing can determine the transcription directionality of two chains, so that errors in the comparison process are reduced; secondly, the lncRNA library construction sequencing can obtain more abundant information; the library establishment of the lncRNA belongs to mRNA+lncRNA, and the data of the mRNA and the data of the lncRNA can be obtained simultaneously only by once library establishment;
identification of differentially expressed genes: screening differential expression genes; clustering analysis of differential expression genes; functional annotation of differentially expressed genes; GO and KEGG enrichment analysis of differentially expressed genes; differentially expressed gene protein interaction networks;
lncRNA analysis: quantitatively analyzing lncRNA expression; analysis of lncRNA differential expression; analysis of lncRNA correlation between samples; annotation enrichment analysis of Cis action difference lncRNA target genes; PPI analysis of Cis action difference lncRNA target genes; annotation enrichment analysis of Trans-action difference lncRNA target genes; performing PPI analysis on a Trans-action difference lncRNA target gene;
and (3) combining the differential expression gene and the lncRNA target gene to predict, and exploring a common gene as a corn salt tolerance candidate gene.
Step 2, verifying the expression mode of salt tolerance genes and predicting the salt stress response regulation mode thereof
Step 2.1, designing and synthesizing a Primer by utilizing a Primer5 for the salt-tolerant candidate gene, reversely transcribing RNA into cDNA, and performing expression pattern verification by utilizing real-time quantitative PCR;
and 2.2, carrying out overexpression verification of candidate genes in arabidopsis thaliana, preparing agrobacterium competence and an overexpression vector, constructing a recombinant plasmid, introducing agrobacterium into arabidopsis thaliana, culturing, screening arabidopsis thaliana transgenic homozygous salt treatment, and carrying out phenotype identification.
By the above differential gene expression screening, 271 Differential Expression Genes (DEG) were obtained in total, of which 209 genes were up-regulated and 62 genes were down-regulated. To understand the role and involved regulatory pathways of these 271 differentially expressed genes in maize salt stress response, the present application annotated them with GO and KEGG functions. According to the application, DESeq2 differential expression analysis is used, and 28 differential expression lncRNAs are finally obtained through screening. The lncRNA of the present application was subjected to target gene prediction using LncTar target gene prediction tool. Finally, the gene Zm00001d033878 was obtained as both a differentially expressed gene from the transcriptome sequencing results and as a target gene for lncRNA. The gene is subjected to RT-qPCR expression analysis (figure 1) and transgenic Arabidopsis functional verification (figure 2), which shows that the over-expression of the gene can improve salt tolerance and predicts the salt stress response regulation mode (figure 3) of the gene.
In conclusion, the corn salt tolerance gene Zm00001d033878 genome DNA obtained according to the application has the nucleotide sequence shown in SEQ ID NO. 1. The gene can be transferred to corn materials with excellent agronomic characters through molecular assisted breeding, so as to create corn materials or varieties with salt tolerance and excellent agronomic characters.
The description and practice of the application disclosed herein will be readily apparent to those skilled in the art, and may be modified and adapted in several ways without departing from the principles of the application. Accordingly, modifications or improvements may be made without departing from the spirit of the application and are also to be considered within the scope of the application.
SEQUENCE LISTING
<110> university of Nantong
<120> discovery of one maize salt tolerance gene Zm00001d033878 and uses thereof
<130> 2022
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2840
<212> DNA
<213> Gossypium hirsutum L.
<400> 1
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aggctccccg ctcctgcggg agaacgcaag gcaatcgatc tcccgcccgc ctccgatccc 180
tcctccattc tcgctcgtcc acggtctgcc ctatataacc cctcgctccg ttggtttcgt 240
cgcctccgtt cccccaaggg agaagggtac gttggtgata tcccgcgtcc ccagcgagga 300
tgctgtcatt gcgctgccac gccgccgcgc cggcgacccg cggcgtcgca tgtgaacacg 360
gggcgcgttc atcgtgttgt cttctttctt tgccccccgt ctctgtctct ctctctctct 420
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Claims (1)
1. The application of the maize salt-tolerant gene Zm00001d033878 in the molecular marker assisted selection of salt-tolerant maize varieties is characterized in that the genomic DNA of the maize salt-tolerant gene Zm00001d033878 is a nucleotide sequence shown as SEQ ID NO. 1.
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