CN110564884B - Method for excavating salix matsudana salt-tolerant pivot gene - Google Patents
Method for excavating salix matsudana salt-tolerant pivot gene Download PDFInfo
- Publication number
- CN110564884B CN110564884B CN201910893406.XA CN201910893406A CN110564884B CN 110564884 B CN110564884 B CN 110564884B CN 201910893406 A CN201910893406 A CN 201910893406A CN 110564884 B CN110564884 B CN 110564884B
- Authority
- CN
- China
- Prior art keywords
- salt
- tolerant
- gene
- progeny
- genes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Botany (AREA)
- Bioinformatics & Computational Biology (AREA)
- Evolutionary Biology (AREA)
- Medical Informatics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for exploring salt-tolerant pivot genes of salix matsudana, which is characterized in that filial generation F is obtained by hybridizing salt-tolerant salix matsudana and salix matsudana1Dividing the population into salt-tolerant progeny and salt-sensitive progeny,and then performing transcriptome sequencing on root systems of salt-tolerant and salt-sensitive offspring respectively to obtain a differential expression gene sequence, constructing a high-density genetic map, positioning salt-tolerant QTLs (quantitative trait loci) and calculating to obtain corresponding QTL (quantitative trait loci) sections, comparing the QTL sections and the differential expression gene sequence with the willow whole genome, analyzing to obtain a plurality of salt-tolerant candidate genes, determining the expression quantity of the plurality of salt-tolerant candidate genes, establishing a different-speed growth model, constructing a normal differential equation set, forming a regulation network by the plurality of salt-tolerant candidate genes, and determining salt-tolerant pivot genes in the regulation network. The invention solves the problem that the salt-tolerant gene can not be accurately found in the traditional salt-tolerant willow breeding.
Description
Technical Field
The invention relates to the technical field of forest breeding, in particular to a method for excavating salix matsudana salt-tolerant pivot genes.
Background
The salt-tolerant gene regulates the salt-tolerant phenotype of the forest through the action of regulatory protein.
The traditional salt-tolerant gene discovery is realized by methods such as homologous gene sequence comparison or transcriptome sequencing comparison, and the like, and the methods cannot accurately find the salt-tolerant pivot gene of the forest. Meanwhile, considering the characteristics of long growth period, complex genetic background, high heterozygosity, weak research foundation, high omics data noise and sparsity and the like of the forest, the traditional method has large limitation and cannot meet the requirement of the development of salt-tolerant genes of the forest.
Salix matsudana is an important tree species of Salix genus of Salicaceae family, originally produced in northeast China, also called as Chinese willow, plays an important role in soil heavy metal remediation, saline-alkali soil greening, biofuel and other aspects, and is widely used for coastal forestation in east China. The different-speed theory is a science for researching the relationship between the structure, the process and the size of a biological organism, and the salt-tolerant pivot genes of the salix matsudana can be accurately discovered based on the different-speed model, so that a new salix matsudana variety with higher salt tolerance is cultivated, and the method contributes to ecological construction of coastal beaches. And the salt-tolerant gene cannot be accurately found by adopting the traditional salt-tolerant willow breeding.
Disclosure of Invention
In order to overcome the defects of the prior art, the method for discovering the salix matsudana salt-tolerant pivot gene is provided so as to solve the problem that the salt-tolerant gene cannot be accurately found in the traditional salt-tolerant willow breeding.
In order to realize the purpose, the method for excavating the salix matsudana salt-tolerant pivot gene comprises the following steps:
hybridizing the salt-tolerant salix matsudana with the salt-tolerant salix matsudana to obtain a filial generation F1A population;
the filial generation F is used1Dividing the population into salt-tolerant filial generations and salt-tolerant filial generations;
under the salt stress of different durations, carrying out transcriptome sequencing on root systems of the salt-tolerant progeny and the salt-sensitive progeny respectively to obtain differential expression gene sequences;
constructing a high-density genetic map based on the salt-tolerant progeny, positioning salt-tolerant QTLs and calculating to obtain corresponding QTL sections;
comparing the QTL sections and the differential expression gene sequences with a willow whole genome, and analyzing to obtain a plurality of salt-tolerant candidate genes;
after the salt-tolerant progeny is stressed by salt for different durations, determining multidimensional data of the salt-tolerant candidate gene, wherein the multidimensional data comprise the expression level of the salt-tolerant candidate gene, the salt-tolerant phenotype index and the salt-tolerant physiological index of the salt-tolerant progeny;
establishing a different-speed growth model based on the multidimensional data;
constructing an ordinary differential equation set based on the different-speed growth model;
constructing a gene regulation network of the salt-tolerant gene based on the ordinary differential equation set;
and calculating the interaction degree among a plurality of salt-tolerant candidate genes by combining the ordinary differential equation set with variable selection and based on the centrality characteristics of the gene regulation network so as to determine the salt-tolerant pivot genes in the gene regulation network.
Further, said converting said offspring F1The method for dividing the population into salt-tolerant filial generations and salt-tolerant filial generations comprises the following steps:
the filial generation F is used1Placing the first stem segment of the population in a NaCl solution with the concentration of 50mM for rooting culture;
after rooting culture for two weeks, determining the salt-tolerant phenotype index of the first stem segment;
(ii) based on the indicator of salt tolerance phenotype, combining said progeny F1The population is divided into salt-tolerant filial generation and salt-tolerant filial generation.
Further, the salt tolerance phenotype indexes comprise the root number, the total root length, the longest root length, the thickest root diameter, the fresh root weight and the dry root weight.
Further, the step of performing transcriptome sequencing on root systems of the salt-tolerant progeny and the salt-sensitive progeny respectively to obtain a differential expression gene sequence comprises:
placing the second stem sections of the salt-tolerant progeny and the salt-sensitive progeny in clear water for rooting culture;
after rooting culture for two weeks, placing the second stem segment in a NaCl solution with the concentration of 200mM for salt stress;
and respectively carrying out transcriptome sequencing on the root system of the second stem segment to obtain a differential expression gene sequence when the second stem segment is subjected to salt stress for 4 hours, 8 hours and 12 hours.
Further, the different-speed growth model is as follows:
in the formula, n is the number of salt-tolerant candidate genes;
m is the number of salt-tolerant filial generations;
alpha is an equilibrium constant term;
beta is a growth index;
x is the sum of the expression amount of all salt-tolerant candidate genes in the same salt-tolerant progeny at a time point;
y is the sum of the expression of the same salt-tolerant candidate gene in all the salt-tolerant progeny at a time point.
Further, the ordinary differential equation system is:
wherein M is the expression level of the salt-tolerant candidate gene.
Further, the method also comprises the following steps:
introducing the salt-tolerant hub gene into willow seedlings, and detecting the salt-tolerant physiological indexes of the willow seedlings;
and determining a salt-tolerant key hub gene in the salt-tolerant hub genes based on the salt-tolerant physiological indexes of the willow seedlings.
Further, the steps of introducing the salt-tolerant hub gene into willow seedlings and detecting the salt-tolerant physiological indexes of the willow seedlings comprise:
constructing a plant expression vector of the salt-tolerant pivot gene;
introducing the plant expression vector into willow seedlings;
and when the willow seedling is stressed by salt for different periods of time, determining the salt tolerance physiological index of the willow seedling.
The invention has the beneficial effects that the method for excavating the salix matsudana salt-tolerant pivot gene can excavate the salix matsudana salt-tolerant pivot gene in a large amount, accurately and quickly to analyze the salt-tolerant mechanism of salix matsudana, so that the breeding efficiency of the salt-tolerant salix matsudana is improved, and meanwhile, the method for excavating the salix matsudana salt-tolerant pivot gene can be applied to other salt-tolerant forests to better breed new species of the salt-tolerant forests.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The invention provides a method for excavating salix matsudana salt-tolerant pivot genes, which comprises the following steps:
s1: hybridizing the salt-tolerant salix matsudana with the salt-tolerant salix matsudana to obtain a filial generation F1And (4) a group.
In the embodiment, the salix matsudana is salix matsudana which can only normally grow in an environment with a salt content of less than 2 per mill. The salt-tolerant salix matsudana is salix matsudana which can grow in an environment with the salt content of more than 4 per mill.
Specifically, the salt-tolerant salix matsudana is taken as a female parent, and the salt-tolerant salix matsudana is taken as a male parent.
In this embodiment, the salt-tolerant salix matsudana is salix matsudana, and the salt-tolerant salix matsudana is salix 9901.
S2: based on willow salt-tolerant phenotype indexes, offspring F1The population is judged as salt-tolerant filial generation and salt-tolerant filial generation.
Specifically, step S2 includes:
step S21, the child F1The first stem section of the population was placed in a NaCl solution at a concentration of 50mM for rooting culture.
In this embodiment, child F is randomly selected1400 progeny F in the population1Intercepting 400 filial generation F1And 400 progeny F were added1The first stem section of (2) was placed in a NaCl solution of 50mM concentration for rooting culture.
And step S22, determining the salt-tolerant phenotype index of the first stem segment after rooting culture for two weeks.
In 400 progeny F1After two weeks of rooting culture of the first stem segment, the salt tolerance phenotype index of the root system of the first stem segment is determined.
Indices of salt tolerant phenotype of willows include, but are not limited to: root number, total root length, longest root length, thickest root diameter, fresh root weight, and dry root weight.
Specifically, the number of roots is the number of roots generated after stem segments are cultured.
The total root length is the total length of all the resulting roots of the stem segment.
The longest root length is the length of the longest root generated after stem section culture.
The diameter of the thickest root is the maximum diameter of the thickest root produced after stem section culture.
The fresh weight of the root is the total weight of all the fresh roots produced after stem section culture.
The dry root weight is the total weight of all roots generated after stem section culture after drying.
Step S23, based on the salt tolerance standard and the salt sensitivity standard, the filial generation F is processed1Progeny F in the population1Respectively judging to be salt-tolerant filial generation or salt-sensitive filial generation.
Specifically, the salt tolerance standard is the average value of all salt tolerance phenotype index values which are larger than the measured salt tolerance phenotype index.
The salt sensitivity standard is the average value of the plant death or salt tolerance phenotype indexes smaller than the measured salt tolerance phenotype indexes.
Respectively adding 400 filial generation F according to salt tolerance standard and salt sensitivity standard1And judging to be salt-tolerant progeny or salt-sensitive progeny.
S3: under the salt stress of different durations, carrying out transcriptome sequencing on root systems of the salt-tolerant progeny and the salt-sensitive progeny respectively to obtain a differential expression gene sequence.
In this example, transcriptome sequencing analysis was performed on RNA extracted from the root system of the first stem segment determined as salt-tolerant progeny and salt-sensitive progeny, respectively, to obtain Differentially Expressed Gene Sequences (DEGs).
Specifically, step S3 includes:
and step S31, randomly selecting 30 salt-tolerant progenies and 30 salt-sensitive progenies. And intercepting the second stem segment of the salt-tolerant filial generation and the salt-sensitive filial generation. And placing the second stem segment in clear water for rooting culture.
And step S32, after the second stem segment is subjected to rooting culture for two weeks, placing the second stem segment in a NaCl solution with the concentration of 200mM for salt stress.
And S33, respectively extracting RNA from the root system of the second stem segment under the salt stress of different durations, and performing transcriptome sequencing to obtain the differentially expressed gene sequence.
Specifically, under salt stress of different durations, namely 4 hours, 8 hours and 12 hours of salt stress of the second stem segment, RNA is extracted from the root system of the second stem segment and transcriptome sequencing is carried out to obtain a differential expression gene sequence (namely, 30 salt-tolerant progenies and 30 salt-sensitive progenies form three biological repeated mixed samples respectively with each 10 strains, after the salt stress of 4 hours, 8 hours and 12 hours, the salt-tolerant progenies and the salt-sensitive progenies are pairwise compared with DEGs of different salt-tolerant materials with the same duration, and the DEGs with the same duration are the final Differential Expression Gene Sequence (DEGs)).
And comparing the sequence of the differentially expressed gene sequence DEGs obtained by sequencing analysis of the transcriptome with the GO, KEGG, NR, Swiss-Prot, COG and eggNOG gene databases by using BLAST software to obtain the annotation information of the differentially expressed gene sequence DEGs in the 6 gene databases.
S4: and constructing a high-density genetic map based on the salt-tolerant progeny, positioning the salt-tolerant QTLs and calculating to obtain a corresponding QTL section.
Specifically, 400 progeny F were used1The population constructs a high-density genetic map based on SNP markers, locates salt tolerance QTLs related to root system measurement indexes of the salix matsudana, and analyzes the QTL sections with important salt tolerance of the salix matsudana root system by calculating LOD values and contribution rates of the QTLs.
S5: comparing the QTL sections and the differential expression gene sequences with the willow whole genome, and analyzing to obtain a plurality of salt-tolerant candidate genes.
Comparing the carefully positioned important QTL sections of the root systems of the salt-tolerant progeny and the DEGs separated from the root system transcription components of the salt-tolerant progeny to the whole genome of the salix matsudana, and analyzing to obtain a salt-tolerant candidate gene group of the salix matsudana root system, namely a plurality of salt-tolerant candidate genes.
Specifically, a batch of genes are obtained through QTL sections, a batch of genes are obtained through DEGs annotation of transcriptomes, the genes are compared with the whole genome, and the coincident genes of the genes and the whole genome are determined as salt-tolerant candidate genes.
S6: and determining the multidimensional data of the salt-tolerant candidate genes after the salt-tolerant progeny is stressed by salt for different durations. The multidimensional data comprises the relative expression quantity of the salt-tolerant candidate gene, the root salt-tolerant phenotype index of the salt-tolerant progeny and the salt-tolerant physiological index.
Specifically, step S6 includes:
and step S61, intercepting the third stem segment of the randomly selected 30 salt-tolerant progeny plants. Placing the third stem segment in 50mM NaCl solution for rooting, dynamically observing and culturing salt-tolerant phenotype indexes of root systems for 0, 3, 6, 9, 12 and 15 days, and simultaneously determining the expression quantity of salt-tolerant candidate genes.
And step S62, intercepting the fourth stem segment of the randomly selected 30 salt-tolerant progeny plants. And placing the fourth stem segment in clear water for rooting. After rooting in clear water for two weeks, the rooted fourth stem segments were placed in a 200mM NaCl solution for salt stress for 0 hour, 6 hours, 12 hours, 24 hours and 48 hours, wherein the salt stress for 6 hours, 12 hours, 24 hours and 48 hours was set as a test group, and the salt stress for 0 hour was set as a control group.
And respectively measuring the expression quantity of the salt-tolerant candidate gene of the fourth stem segment of the group to be tested and the control group when the third stem segment is stressed by salt for 0 hour, 6 hours, 12 hours, 24 hours and 48 hours, and simultaneously measuring the salt-tolerant physiological index of the fourth stem segment of the salt-tolerant progeny.
Physiological indicators of salt tolerance include conductivity, malondialdehyde, proline, SOD (Superoxide dismutase), POD (Peroxidase), CAT (Catalase), H2O2(hydrogen peroxide) and soluble sugar content.
Specifically, the data of the expression level of the salt-tolerant candidate gene is expressed according to F ═ 2-ΔΔctAnd (4) formula calculation.
In this embodiment, the calculation formula of the relative expression level (F) of the salt-tolerant candidate gene is:
F＝2- [ (average Ct value of target gene in test group-average Ct value of reference in test group) - (pair)Mean Ct value of control group target gene-mean Ct value of control group internal reference gene)]。
In the calculation formula of the relative expression of the salt-tolerant candidate gene, the specific contents of the superscript of 2 are as follows: - [ (mean Ct value of target gene in test group-mean Ct value of reference gene in test group) - (mean Ct value of target gene in control group-mean Ct value of reference gene in control group) ].
The gene expression levels of the test group at 6, 12, 24 and 48 hours of salt stress and the gene expression level of the control group at 0 hour.
S7: based on the multidimensional data, namely the relative expression quantity of the salt-tolerant candidate gene, the salt-tolerant physiological index and the salt-tolerant phenotype index, the multidimensional data are standardized in a certain gene or phenotype index, and based on the sum of all indexes, a different-speed growth model is established.
Taking the list of gene expression levels as an example, the gene expression level at time t can be expressed as:
wherein xmThe total expression quantity of all the salt-tolerant candidate genes of the salt-tolerant progeny salix matsudana individual m at the t-th time point is expressed, so that at a time point, the total expression quantity y of all the salt-tolerant progeny salix matsudana individuals based on one salt-tolerant candidate gene is expressednmWith the total expression level xmI.e. the relationship between parts and the whole, the following different-speed growth model is constructed:
in the formula, n is the number of salt-tolerant candidate genes;
m is the number of salt-tolerant filial generations;
alpha is an equilibrium constant term;
beta is a growth index;
alpha and beta are unknown parameters and can be obtained by nonlinear least square estimation. Wherein, beta is more than 0, the gene expression trend is generally increased, and the gene expression trend is reduced otherwise.
x is the sum of the expression amount of all salt-tolerant candidate genes in the same salt-tolerant progeny at a time point;
y is the sum of the expression of the same salt-tolerant candidate gene in all the salt-tolerant progeny at one time point.
S8: and constructing an ordinary differential equation set based on the different-speed growth model.
Firstly, screening differential genes, and then estimating two key parameters alpha and beta (see formula I) of the differential growth model by a nonlinear least square method aiming at the gene expression quantity of each salt-tolerant candidate differential gene based on the differential growth model. And finally obtaining the continuous expression quantity of the salt-tolerant candidate gene by utilizing the estimated parameters.
The differential gene analysis screening process is completed based on R package MaSigpro.
In particular, as P-value<0.05 and goodness of fit R2>0.8 was used as the screening criterion.
Secondly, clustering analysis of the expression amounts of the differential genes is performed using a k-means clustering algorithm (k-means clustering algorithm), and the optimal clustering number is determined by a method of Gap statistical (Gap statistical).
And finally, constructing an ordinary differential equation system based on the multi-dimensional variables, and then selecting the variables. Considering the interaction between the variable M (M ═ 1.., p) and other variables, the variables can be expressed as factors such as the expression level of the gene, physiological indices of salt tolerance, and the like. And (3) considering the sparsity of a subsequently established regulation and control network of the salt-tolerant candidate genes, and selecting variables by adopting a grouped minimum angle regression algorithm (GroupLasso).
The constructed ordinary differential equation set is as follows:
wherein M is the expression level of the salt-tolerant candidate gene.
S9: and constructing a gene regulation network of the salt-tolerant gene based on the ordinary differential equation group.
Based on the relative change of the gene expression level of each salt-tolerant candidate gene i, the relative change is a connection function containing all salt-tolerant candidate genes, which are respectively expressed as f1,...,fi. And describing the interaction effect of all salt-tolerant candidate genes by using a connection function to form a gene regulation network of the salt-tolerant genes.
S10: and (3) calculating the interaction degree among a plurality of salt-tolerant candidate genes by combining a normal differential equation set with variable selection and based on the centrality characteristics of the gene regulation network so as to determine the salt-tolerant hub genes in the gene regulation network.
Whether interaction exists among the salt-tolerant candidate genes can be obtained through variable selection, then a high-dimensional differential equation is constructed according to a sparse gene interaction network, parameter estimation of the high-dimensional differential equation is completed by utilizing maximum likelihood estimation or a nonlinear least square method in combination with a smoothing method, and further the interaction degree of the salt-tolerant candidate genes is quantized. And finally, completing the screening of the core hub through core-edge characteristic indexes such as connectivity, compactness, betweenness, eccentricity, feature vectors and the like, wherein the specific critical value can be set according to the number of genes and the complexity of the network.
The core-edge characteristics include Connectivity, Closeness (Closeness), Betweenness (Betweenness), Eccentricity (eccentricities), eigenvectors (eigenvectors), and PageRank algorithm.
Specifically, connectivity refers to the number of genes directly linked to one another within a network.
Compactness refers to the closeness of one gene to other genes within the network.
Compactness is quantified by the following formula:
in the formula, V represents the number of salt-tolerant candidate genes in the network, dist (i, j) represents the minimum distance between the salt-tolerant candidate genes i and the salt-tolerant candidate genes j in the network.
The betweenness reflects the importance degree of the connection function of the salt-tolerant candidate gene as a bridge. The betweenness is quantified by the following equation:
③ injkIs the shortest path number g between the salt-tolerant candidate gene j and the salt-tolerant candidate gene kjk(i) Is that the salt-tolerant candidate gene i is positioned on the shortest path gjkThe number of (2).
Eccentricity is used to describe the longest distance from other salt-tolerant candidate genes to one salt-tolerant candidate gene. The eccentricity is quantified as follows:
the feature vectors are used for measuring the importance of the salt tolerance candidate genes in the regulation network. The importance of the current salt-tolerant candidate gene depends on the importance of the neighbor salt-tolerant candidate gene. For the salt-tolerant candidate gene i, the feature vector center fraction is proportional to the total integral of the salt-tolerant candidate genes directly connected with the feature vector center fraction, and the calculation and quantification are carried out by the following formula:
in the formula, lambda is a constant satisfying the equation Ax ═ lambda x, aijAnd (3) representing whether connection exists between the salt-tolerant candidate genes in the regulatory network, wherein N is the number of the total salt-tolerant candidate genes.
For the salt-tolerant candidate gene i, the PageRank center score is defined as:
sixthly, d is a damping factor and is usually set to 0.85.
S11: the function of the salt-tolerant hub gene is verified through transient transformation, and the salt-tolerant key hub gene is determined based on the function of the salt-tolerant hub gene and the up-down relation of the salt-tolerant hub gene in a gene regulation network.
Specifically, step S11 includes:
s111, introducing the salt-tolerant pivot gene into willow seedlings, and detecting salt-tolerant physiological indexes of the willow seedlings.
Specifically, the method comprises the following steps: and constructing a plant expression vector of the salt-tolerant pivot gene.
The salt-tolerant hub is introduced into willow seedlings through a plant expression vector.
And when the willow seedling is stressed by salt for different periods of time, determining the salt tolerance physiological index of the willow seedling.
The willow seedling introduced into the salt-tolerant hub is placed in a NaCl solution with the concentration of 200mM for salt stress treatment for 48 hours, 72 hours and 96 hours, the salt-tolerant phenotype of the willow seedling is analyzed through methods such as biochemical dyeing, enzyme activity analysis and the like, the salt-tolerant physiological index is determined, and the influence of an exogenous gene on the salt tolerance of the willow seedling is analyzed.
And S112, determining a salt-tolerant key hub gene in the salt-tolerant hub genes based on the salt-tolerant physiological indexes of the willow seedlings.
The specific implementation process comprises the following steps:
constructing a differential equation:
determining salt-tolerant progeny by using a transcriptome sequencing technology (RNA-seq), and obtaining n salt-tolerant candidate genes or functional modules (salt-tolerant physiological indexes) at t time points or data of salt-tolerant candidate gene expression amount of a sample under a salt stress treatment condition. Let G1(t),....,Gi(t) represents the gene expression level of gene i at a time point or in a sample t, where t is t0,., T, i is 1, n. The dynamic changes of the n genes and their interactions can be described using the systematic differential equations (ODE), which are specifically expressed as follows:
the system of equations is a refinement of the above system of ordinary differential equations, the parameter Ω represents an unknown parameter, and the degree of interaction between genes can be quantified by estimating the unknown parameter.
Wherein the relative change of each gene i is a linkage function comprising all genes, respectively denoted as f1,...,fi(ii) a Each connection function depends on a parameter Ω ═ Ωj1,...,ΩjiAnd (4) describing (expressing) the interaction effect of all salt-tolerant candidate genes by utilizing the parameters to form a gene regulation network of the salt-tolerant genes. And completing parameter estimation based on the maximum likelihood estimation of the fourth-order Runge Kutta method and the nonlinear least square method.
Determination of salt-tolerant pivot genes with significant interaction
Based on the sparsity characteristic of a gene regulation network and the biological connotation of a biological system, a high-dimensional variable selection technology Group LASSO and an Adaptive Group LASSO are applied to ODE corresponding to each gene in the ordinary differential equation set to preliminarily determine salt-tolerant pivot genes with obvious interaction.
Screening of salt-tolerant key pivot genes based on gene regulation network characteristics
Nodes in a gene regulatory network are evaluated using centrality features of the gene regulatory network. The centrality features include connectivity, compactness, betweenness, eccentricity, feature vectors, and PageRank. The indexes such as the connection number, the interaction degree and the like of each salt-tolerant candidate gene and other salt-tolerant candidate genes in the gene regulation network are obtained by utilizing the centrality characteristics, the critical value of each index is determined by arrangement detection according to the complexity of network topology, and finally the salt-tolerant key hub gene is screened and determined.
The method for excavating the salix matsudana salt-tolerant pivot gene can be used for excavating a large amount of salix matsudana salt-tolerant pivot genes accurately and quickly to analyze the salt-tolerant mechanism of salix matsudana and improve the breeding efficiency of salt-tolerant willows.
Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present application and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of this type are intended to be covered by the present invention.
Claims (6)
1. A method for excavating salix matsudana salt-tolerant pivot genes is characterized by comprising the following steps:
hybridizing the salt-tolerant salix matsudana with the salt-tolerant salix matsudana to obtain a filial generation F1A population;
the filial generation F is used1Dividing the population into salt-tolerant filial generations and salt-tolerant filial generations;
under the salt stress of different durations, carrying out transcriptome sequencing on root systems of the salt-tolerant progeny and the salt-sensitive progeny respectively to obtain differential expression gene sequences;
constructing a high-density genetic map based on the salt-tolerant progeny, positioning salt-tolerant QTLs and calculating to obtain corresponding QTL sections;
comparing the QTL sections and the differential expression gene sequences with a willow whole genome, and analyzing to obtain a plurality of salt-tolerant candidate genes;
after the salt-tolerant progeny is stressed by salt for different durations, determining multidimensional data of the salt-tolerant candidate gene, wherein the multidimensional data comprise the expression level of the salt-tolerant candidate gene, the salt-tolerant phenotype index and the salt-tolerant physiological index of the salt-tolerant progeny;
establishing a different-speed growth model based on the multidimensional data;
constructing an ordinary differential equation set based on the different-speed growth model;
constructing a gene regulation network of the salt-tolerant gene based on the ordinary differential equation set;
calculating the interaction degree among a plurality of salt-tolerant candidate genes by combining the ordinary differential equation set with variable selection and based on the centrality characteristics of the gene regulation network so as to determine the salt-tolerant pivot genes in the gene regulation network;
the abnormal growth model is
In the formula, n is the number of salt-tolerant candidate genes;
m is the number of salt-tolerant filial generations;
alpha is an equilibrium constant term;
beta is a growth index;
x is the sum of the expression amount of all salt-tolerant candidate genes in the same salt-tolerant progeny at a time point;
y is the sum of the expression amount of the same salt-tolerant candidate gene in all salt-tolerant progeny at a time point;
the system of ordinary differential equations is
Wherein M is the expression level of the salt-tolerant candidate gene.
2. The method for discovering Salix matsudana salt-tolerant hub gene according to claim 1, wherein the progeny F is obtained1The method for dividing the population into salt-tolerant filial generations and salt-tolerant filial generations comprises the following steps:
the filial generation F is used1Placing the first stem segment of the population in a NaCl solution with the concentration of 50mM for rooting culture;
after rooting culture for two weeks, determining the salt-tolerant phenotype index of the first stem segment;
(ii) based on the indicator of salt tolerance phenotype, combining said progeny F1The population is divided into salt-tolerant filial generation and salt-tolerant filial generation.
3. The method for exploring salix matsudana salt-tolerant hub gene according to claim 2, wherein the salt-tolerant phenotype indexes comprise root number, total root length, longest root length, thickest root diameter, fresh root weight and dry root weight.
4. The method for discovering salix matsudana salt-tolerant hub gene according to claim 2, wherein the step of performing transcriptome sequencing on root systems of the salt-tolerant progeny and the salt-sensitive progeny respectively to obtain differentially expressed gene sequences comprises:
placing the second stem sections of the salt-tolerant progeny and the salt-sensitive progeny in clear water for rooting culture;
after rooting culture for two weeks, placing the second stem segment in a NaCl solution with the concentration of 200mM for salt stress;
and respectively carrying out transcriptome sequencing on the root system of the second stem segment to obtain a differential expression gene sequence when the second stem segment is subjected to salt stress for 4 hours, 8 hours and 12 hours.
5. The method for discovering salix matsudana salt-tolerant hub gene according to claim 1, further comprising:
introducing the salt-tolerant hub gene into willow seedlings, and detecting the salt-tolerant physiological indexes of the willow seedlings;
and determining a salt-tolerant key hub gene in the salt-tolerant hub genes based on the salt-tolerant physiological indexes of the willow seedlings.
6. The method for discovering salix matsudana salt-tolerant hub gene according to claim 5, wherein the steps of introducing the salt-tolerant hub gene into willow seedlings and detecting the salt-tolerant physiological indexes of the willow seedlings comprise:
constructing a plant expression vector of the salt-tolerant pivot gene;
introducing the plant expression vector into willow seedlings;
and when the willow seedling is stressed by salt for different periods of time, determining the salt tolerance physiological index of the willow seedling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910893406.XA CN110564884B (en) | 2019-09-20 | 2019-09-20 | Method for excavating salix matsudana salt-tolerant pivot gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910893406.XA CN110564884B (en) | 2019-09-20 | 2019-09-20 | Method for excavating salix matsudana salt-tolerant pivot gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110564884A CN110564884A (en) | 2019-12-13 |
| CN110564884B true CN110564884B (en) | 2020-12-25 |
Family
ID=68781514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910893406.XA Active CN110564884B (en) | 2019-09-20 | 2019-09-20 | Method for excavating salix matsudana salt-tolerant pivot gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110564884B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111567399B (en) * | 2020-06-03 | 2021-02-02 | 南通大学 | Quick breeding method for new flooding-resistant willow variety |
| CN113345520B (en) * | 2021-06-07 | 2021-12-14 | 北京林业大学 | Richards equation-based Quantitative Trait Locus (QTL) positioning frame method for trees |
| CN114634993B (en) * | 2022-04-27 | 2023-03-14 | 南通大学 | Transcriptome and proteome combined analysis-based cotton salt-tolerant gene discovery method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108651273B (en) * | 2018-05-17 | 2021-06-15 | 南通大学 | Method for breeding salt-tolerant lagerstroemia indica leaves by polymerization based on gene detection |
| CN109699489A (en) * | 2019-01-18 | 2019-05-03 | 南通大学 | It polymerize the willow breeding method of salt tolerant, fast-growing character |
-
2019
- 2019-09-20 CN CN201910893406.XA patent/CN110564884B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110564884A (en) | 2019-12-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gali et al. | Genome-wide association mapping for agronomic and seed quality traits of field pea (Pisum sativum L.) | |
| Jia et al. | A haplotype map of genomic variations and genome-wide association studies of agronomic traits in foxtail millet (Setaria italica) | |
| Migicovsky et al. | Morphometrics reveals complex and heritable apple leaf shapes | |
| Flood et al. | Natural genetic variation in plant photosynthesis | |
| Andrew et al. | Adaptation with gene flow across the landscape in a dune sunflower | |
| CN110564884B (en) | Method for excavating salix matsudana salt-tolerant pivot gene | |
| Koopman et al. | AFLP markers as a tool to reconstruct complex relationships: a case study in Rosa (Rosaceae) | |
| Guo et al. | Functional phenomics and genetics of the root economics space in winter wheat using high‐throughput phenotyping of respiration and architecture | |
| Twyford et al. | Multi-level patterns of genetic structure and isolation by distance in the widespread plant Mimulus guttatus | |
| Gosseau et al. | Heliaphen, an outdoor high-throughput phenotyping platform for genetic studies and crop modeling | |
| Hamilton et al. | Fine-scale environmental variation contributes to introgression in a three-species spruce hybrid complex | |
| CN109830261B (en) | Method for screening quantitative trait candidate genes | |
| Helmstetter et al. | Genetic diversity and domestication of hazelnut (Corylus avellana L.) in Turkey | |
| Masalia et al. | Multiple genomic regions influence root morphology and seedling growth in cultivated sunflower (Helianthus annuus L.) under well-watered and water-limited conditions | |
| Tsumura et al. | Effects of the last glacial period on genetic diversity and genetic differentiation in Cryptomeria japonica in East Asia | |
| Panahi et al. | Characterization of Iranian grapevine cultivars using machine learning models | |
| Ogbonna et al. | Comprehensive genotyping of a Brazilian cassava (Manihot esculenta Crantz) germplasm bank: insights into diversification and domestication | |
| Chen et al. | Genetic diversity of Prunus sibirica L. superior accessions based on the SSR markers developed using restriction-site associated DNA sequencing | |
| Hamadeh et al. | Evolution of almond genetic diversity and farmer practices in Lebanon: impacts of the diffusion of a graft-propagated cultivar in a traditional system based on seed-propagation | |
| Roumet et al. | How to escape from crop‐to‐weed gene flow: Phenological variation and isolation‐by‐time within weedy sunflower populations | |
| Dong et al. | VitisGDB: the multifunctional database for grapevine breeding and genetics | |
| Chang et al. | Application of 3D-volumetric analysis and hyperspectral imaging systems for investigation of heterosis and cytoplasmic effects in pepper | |
| Hasan et al. | Genetic and genomic resources and their exploitation for unlocking genetic potential from the wild relatives | |
| Huang et al. | Genetic diversity and geographic distribution of north American Setaria viridis populations | |
| Chen et al. | Genetic architecture behind developmental and seasonal control of tree growth and wood properties in Norway spruce |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Jian Inventor after: Jiang Yuna Inventor after: Gao Junfeng Inventor after: Jiang Libo Inventor after: Liu Guoyuan Inventor after: Yu Chunmei Inventor after: Chen Yanhong Inventor after: Lian Bolin Inventor after: Ke Yongchao Inventor before: Zhang Jian Inventor before: Jiang Libo Inventor before: Yu Chunmei Inventor before: Chen Yanhong Inventor before: Liu Guoyuan Inventor before: Lian Bolin Inventor before: Jiang Yuna Inventor before: Ke Yongchao Inventor before: Gao Junfeng |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |