CN117344053B - Method for evaluating physiological development process of plant tissue - Google Patents

Method for evaluating physiological development process of plant tissue Download PDF

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CN117344053B
CN117344053B CN202311648157.0A CN202311648157A CN117344053B CN 117344053 B CN117344053 B CN 117344053B CN 202311648157 A CN202311648157 A CN 202311648157A CN 117344053 B CN117344053 B CN 117344053B
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申思
陈先敏
周顺利
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Abstract

The invention relates to the technical field of plant physiological development, in particular to a method for evaluating the physiological development process of plant tissues. The invention firstly utilizes a transcription time map to screen development related genes, namely, the gene expression level is respectively linearly increased or decreased along with the growth time of (sub) tissues; and then the expression level of the genes related to the integrated development is used for detecting the physiological development process of the target plant (sub) tissue, thereby providing an important method for quantitatively evaluating and comparing the physiological development process of the plant tissue or the inside of the tissue. The method provided by the invention is simple to operate, and the physiological development degree of plant tissues can be quantitatively calculated by statistically integrating the expression of thousands of genes related to the development process.

Description

Method for evaluating physiological development process of plant tissue
Technical Field
The invention relates to the technical field of plant physiological development, in particular to a method for evaluating the physiological development process of plant tissues.
Background
Plant tissue often develops and ages at different rates from differentiation to maturation in the same tissue or in different regions of tissue, which makes it difficult to assess the extent of development of different regions within the same tissue or tissue using actual growth times, and quantification of the extent of biological development remains difficult, and lack of methodology limits understanding of physiological functions associated with plant tissue development.
In plants, the extent of physiological development of plant tissue is typically assessed using cellular structural features of the developmental stage, markers of apoptosis or time span/stage of senescence-associated phenotypes (e.g., leaf color changes), and the like. However, these methods can only make ambiguous comparisons between tissues with distinct developmental differences. To date, there is no method by which the degree of physiological development of plants can be quantitatively assessed. Physiological development of organisms involves complex changes in gene expression levels, and medical physiology has demonstrated that the expression levels of some genes of an organism are linearly related with development. In recent years, in plants, arabidopsis leaves were found to have a series of genes that enhance expression with senescence, and in addition to this, the expression levels of some specific genes are closely related to the relevant physiological developmental processes. There is growing evidence that the use of transcriptomics can reflect the stage or extent of plant tissue physiology.
Transcriptomes are the sum of all RNAs transcribed by a living cell and are an important tool for studying cell phenotype and function. In the existing research method, since the physiological development of plant tissues is controlled by highly complex gene functions, quantitative assessment of the physiological development degree of plant tissues by using transcriptome has not been found yet.
Disclosure of Invention
In order to solve the problems, the invention provides a method for evaluating the physiological development process of plant tissues. The method provided by the invention can relatively quantitatively evaluate and compare the physiological development process of plant tissues or inside the tissues.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for evaluating physiological development process of plant tissues, which comprises the following steps:
obtaining a time transcriptome map of plant tissues to be detected from differentiation to maturity;
obtaining positive related genes and negative related genes related to the development of the plant tissue to be detected;
respectively carrying out normalization treatment on the expression levels of the positive correlation gene and the negative correlation gene by using a formula I;
normalization of A gene expression level= (X-min)/(max-min) formula I;
wherein X is the expression level of the A gene at the time point to be detected, min is the minimum expression level of the A gene in the time transcriptome map, max is the maximum expression level of the A gene in the time transcriptome map, and the A gene is any one gene of the positive related genes or any one gene of the negative related genes;
calculating the physiological development index of the plant tissue to be detected by using the formula II, wherein the higher the physiological development index is, the higher the physiological development degree of the plant tissue is;
physiological developmental index = Σnormalized result of positive correlation gene expression level + Σ (normalized result of 1-negative correlation gene expression level) formula ii.
Preferably, the method for obtaining the positive-related gene and the negative-related gene comprises the following steps:
carrying out spearman correlation analysis between the actual growth time in the time transcriptome map and the plant tissue gene expression level at the actual growth time, and screening to obtain a positive correlation gene for plant tissue development and a negative correlation gene for plant tissue development according to a correlation coefficient R value, a gene expression level and a significance p value; the p value of the positive correlation gene of plant tissue development is less than 0.005, the gene expression level is more than or equal to 1, and the correlation coefficient R is more than 0; the p value of the negative correlation gene for plant tissue development is less than 0.005, the gene expression level is more than or equal to 1, and the correlation coefficient R is less than 0.
Preferably, the screening criteria for positive and negative genes further include: at least one time point has an expression level of 1 or more.
Preferably, the time transcriptome map is obtained by: the time transcriptome map data of the plant tissue to be tested grown under suitable environmental conditions or of the disclosed plant tissue to be tested.
Preferably, the time transcriptome map is obtained by: in the process from differentiation to maturation of plant tissues to be tested, transcriptome composition time transcriptome maps at different time points are obtained.
Preferably, the plant tissues to be tested are the same batch of plant tissues.
Preferably, the units of gene expression level include FPKM, RPKM or TPM.
Preferably, the plant comprises maize.
The beneficial effects are that:
the invention firstly utilizes a transcription time map to screen development related genes, namely, the gene expression level is respectively linearly increased or decreased along with the growth time of (sub) tissues; and then the expression level of the genes related to the integrated development is used for detecting the physiological development process of the target plant (sub) tissue, thereby providing an important method for quantitatively evaluating and comparing the physiological development process of the plant tissue or the inside of the tissue. The method provided by the invention is simple to operate, and the physiological development degree of plant tissues can be quantitatively calculated by statistically integrating the expression of thousands of genes related to the development process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the screening results of maize endosperm development related genes; wherein DAP is the number of days post pollination;
FIG. 2 shows the results of development-related gene expression under drought treatment and 8 days after pollination of normally growing kernels;
FIG. 3 shows the results of gene expression associated with development of high temperature and normal growth kernels at the filling stage 30 days after pollination;
FIG. 4 is a spatiotemporal pattern of genes associated with development of the endosperm of maize in the filling stage.
Detailed Description
The invention provides a method for evaluating physiological development process of plant tissues, which comprises the following steps:
obtaining a time transcriptome map of plant tissues to be detected from differentiation to maturity;
obtaining positive related genes and negative related genes related to the development of the plant tissue to be detected;
respectively carrying out normalization treatment on the expression levels of the positive correlation gene and the negative correlation gene by using a formula I;
normalization of A gene expression level= (X-min)/(max-min) formula I;
wherein X is the expression level of the A gene at the time point to be detected, min is the minimum expression level of the A gene in the time transcriptome map, max is the maximum expression level of the A gene in the time transcriptome map, and the A gene is any one gene of the positive related genes or any one gene of the negative related genes;
calculating the physiological development index of the plant tissue to be detected by using the formula II, wherein the higher the physiological development index is, the higher (old) the physiological development degree of the plant tissue is;
physiological developmental index = Σnormalized result of positive correlation gene expression level + Σ (normalized result of 1-negative correlation gene expression level) formula ii.
The invention obtains the time transcriptome map of the plant tissue to be detected from differentiation to maturation. In the present invention, the plant preferably comprises maize; the time transcriptome map is preferably obtained by: time transcriptome map data of plant tissue to be tested grown under suitable environmental conditions or of published plant tissue to be tested; the method for obtaining the time transcriptome map is preferably as follows: obtaining transcriptome composition time transcriptome maps of different continuous time points in the process of differentiating plant tissues to be detected to maturity; transcriptomes are preferably measured every 2 days; the plant tissues to be tested are preferably the same batch of plant tissues. The present invention is not particularly limited to the methods of sequencing the transcriptome, and sequencing methods well known to those skilled in the art may be employed.
After obtaining a temporal transcriptome map of a plant tissue to be tested from differentiation to maturation, the present invention preferably obtains positive-and negative-related genes related to development of the plant tissue to be tested according to the temporal transcriptome map. In the present invention, the method for obtaining the positive-and negative-associated genes preferably comprises: carrying out spearman correlation analysis between the actual growth time in the time transcriptome map and the plant tissue gene expression level at the actual growth time, and screening to obtain a positive correlation gene for plant tissue development and a negative correlation gene for plant tissue development according to a correlation coefficient R value, a gene expression level and a significance p value; the p value of the positive correlation gene of plant tissue development is less than 0.005, the gene expression level of at least one time point is more than or equal to 1, and the correlation coefficient R is more than 0; the p value of the negative correlation gene of plant tissue development is less than 0.005, the gene expression level of at least one time point is more than or equal to 1, and the correlation coefficient R is less than 0; the means of spearman correlation analysis preferably comprises software with statistical analysis, more preferably Excel or R language.
After obtaining a time transcriptome map, a positive correlation gene and a negative correlation gene, respectively carrying out normalization treatment on the expression levels of the positive correlation gene and the negative correlation gene by using a formula I;
normalization of A gene expression level= (X-min)/(max-min) formula I;
wherein X is the expression level of the A gene at the time point to be detected, min is the minimum expression level of the A gene in the time transcriptome map, max is the maximum expression level of the A gene in the time transcriptome map, and the A gene is any one gene of the positive related genes or any one gene of the negative related genes.
In the present invention, the units of the gene expression level preferably include FPKM (reads per million maps of transcripts per kilobase), RPKM (reads from a gene per kilobase length per million reads) or TPM (the number of specific genes or transcripts per million transcripts in average in a sample).
After normalization treatment, the invention calculates the physiological development index of the plant tissue to be measured by using the formula II, and the higher the physiological development index is, the higher the physiological development degree of the plant tissue is;
physiological developmental index = Σnormalized result of positive correlation gene expression level + Σ (normalized result of 1-negative correlation gene expression level) formula ii.
According to the invention, the expression levels of the positive correlation genes and the negative correlation genes are respectively normalized, and then the normalization results of each positive correlation gene and each negative correlation gene at the time point to be measured are obtained by integrating the expression levels of the positive correlation genes and the negative correlation genes in the formula II, so that the physiological development index of the plant tissue is obtained, the development degree of the plant girth can be evaluated relatively quantitatively according to the physiological development index, and the physiological development processes of different tissues or different positions in the tissues can be evaluated.
For further explanation of the present invention, a method for assessing the physiological development of plant tissue provided by the present invention is described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Taking corn endosperm as an example, the invention screens development related genes:
a High resolution temporal transcription profile disclosed using corn endosperm, comprising the entire period of fertility (material inbred line B37, 6 to 38 days after pollination, transcriptome measured every two days), see [ Yi F, gu W, chen J, song N, gao X, zhang X, zhou Y, ma X, song W, zhao H et al 2019 High temporal-resolution transcriptome landscape of early maize seed development Plant Cell 31, 974-992 ]. The level correlation coefficient is used to describe the degree and direction of the linear correlation of the expression level of genes (FPKM value) with time according to the Spearman rank correlation, wherein p <0.005 is satisfied while genes whose expression level FPKM.gtoreq.1 at least at one time point are defined as "development-related genes".
According to the correlation coefficient R, when R > 0 is a gene positively correlated with physiological development; r <0 > is a gene negatively related to physiological development. By the above method, 669 genes whose expression level increased with development (positive correlation) and 832 genes whose expression level decreased with development (negative correlation) were screened together, as shown in Table 1.
TABLE 1 genes associated with development
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Note that: the gene ID prefixes in Table 1 are Zm00001, d009907 is exemplified by the complete gene ID Zm00001d009907, from Zm-B73-REFERENCE-GRAMENE-4.0 (also knownas B73v4, https:// maizegdb.org.); e is the base number of the power of 10, taking the gene ID d009907 as an example, the R value of 9.95E-01 is 9.95×10 -1 =0.995, and the other genes correspond to E in R or P values.
The results of the hemmap clustering and box plot using log2 (FPKM+1) showed that, as shown in FIG. 1, the positive-correlated gene expression level gradually increased with time and the negative-correlated gene expression level gradually decreased with time as endosperm developed (FIG. 1).
On the basis, the physiological development degree of corn endosperm grown in different environments is quantitatively evaluated.
Example 2
Using transcriptome data disclosed in literature [ Shen S, liang XG, zhang L, zhao X, liu YP, lin S, gao Z, wang P, wang ZM, zhou SL. 2020, intervening in sibling competition for assimilates by controlled pollination prevents seed abortion under postpollination drought in mail ], plant, cell & Environment 43, 903-919 ], corn grown under drought and normal moisture conditions, respectively, was selected, wherein Drought (WD) treatment was initiated one day (-1 day) prior to pollination to control watering, and soil relative moisture content was maintained at 45% ± 5% until rehydration at 16 days; the Control (CK) ensures a sufficient supply of moisture to maintain its soil relative moisture content above 85% and up to maturity. Corn endosperm was taken from 8 days after pollination under the above environmental conditions, respectively, and the transcriptome data disclosed therein was analyzed;
comparing the expression level of WD vs CK grain development related genes (i.e. genes in table 1), the positive genes in corn grain development which are grown under drought conditions are respectively up-regulated by 83.6% and the negative genes in corn grain development are respectively down-regulated by 83.5% compared with normal grains (figure 2).
Example 3
Corn grown under high and normal temperature environmental conditions, respectively, was selected using transcriptome data as disclosed in literature [ Boehlein SK, liu P, webster A, ribeiro C, suzuki M, wu S, guan JC, stewart JD, tracy WF, settles AM et al 2019. Effects of Long-term exposure to elevated temperature on Zea mays endosperm development during grain fill. Plant Journal 99, 23-40 ], wherein high temperature (HH) treatment: the high temperature (38 ℃ in the daytime and 28 ℃ at night) is continuously carried out on the plants 12 days after the pollination of the corn until the plants are mature; and Control (CK): maize plants were grown consistently in a suitable temperature environment (28 ℃ C. Day, 17 ℃ C. Night). Corn endosperm was taken from samples 30 days after pollination under the above environmental conditions, respectively, and the transcriptome data disclosed therein was analyzed;
comparing the expression pattern of hhvs CK endosperm development related genes (i.e., genes of table 1), genes positively correlated with development were each up-regulated by 86.2% and genes negatively correlated with development were each down-regulated by 67.6% (fig. 3).
The high temperature and drought can promote the development of corn endosperm, and the results of examples 2 and 3 show that the expression of the development related genes obtained by screening according to the invention increases or decreases linearly with the development of endosperm respectively. In addition, among other published transcriptome data of grains or endosperm aged or accelerated in external stimulus, the genes positively correlated with development screened by the present invention are mostly promoted, while the genes negatively correlated with development are mostly suppressed. In summary, the development related genes screened by the invention can be used for evaluating the physiological development process of plant tissues.
Example 4
Corn hybrid Zhengdan 958 is selected as a test material and planted in the university of agriculture Wu Qiao laboratory at Hebei province (37 degrees 36 'N, 116 degrees 21' E, altitude 19.7 m) in 2020. Sowing for 5 months and 20 days, sowing 3 seeds in each pot barrel (height 30 cm, radius 17 cm), wherein the barrel contains dry sand (sandy loam) 10 kg rich in mineral substance, and applying 11 g compound fertilizer (N15%, P) on each pot edge 2 O 5 15%，K 2 O15%) sowingAnd then a layer of general nutrient soil (rich in peat, organic matters, vermiculite and the like) purchased from He agricultural technology development limited company in Huaian city is covered. And (3) setting seedlings in the three-leaf period (V3) to ensure that a seedling with consistent growth vigor is reserved in each pot. 5 g urea (N46%) was applied during the large flare period (V12) for normal growth in the later stages of the plant. Plants are grown in natural environment, and are watered manually and quantitatively to keep the water content of the potting soil sufficient and consistent. Throughout the growth process, the plants are protected from insect pests or abiotic stresses. All the spikes were bagged prior to laying to prevent natural pollination. When all filaments were shed (about day 5 post-shed), fresh pollen was collected for artificial synchronous pollination. The middle 5 rounds of kernels were harvested at 15, 20, 25, 35 Days (DAP). Immediately after each grain sample, the embryo and seed coat were discarded, and split evenly according to endosperm length into upper (AE), middle (ME) and lower (BE) endosperm sections, which were immediately frozen for storage at-80 ℃ for transcriptome sequencing.
The preserved endosperm was used to extract total RNA using TRizol reagent (Invitrogen, USA) and RN40-EASYspin kit (Aidlab, china), respectively. cDNA synthesis is carried out on RNA with the A260/A280 ratio more than or equal to 1.9. cDNA libraries were prepared using NEBNext Ultra II RNA Library Prep Kit and sequenced on the Illumina Hiseq2500 platform from BioMarkerTechnologies Illumina company (Beijing, china). Reads were mapped to maize B73 ReferenceGenome version 3 (V3, http:// www.maizegdb.org/assambly /) after removing the linker sequences using Topht 2 and filtering low quality sequences. Expression of each gene was normalized to Fragments (FPKM) read per million maps per kilobase of transcription.
After transcriptome sequencing, each sample was screened for expression levels of all development-related genes (i.e., the genes of table 1), and the results are shown in fig. 4.
FIG. 4 shows that the expression of development related genes is different at different times and different regions of endosperm. Most genes positively correlated with development are highly expressed in AE or ME for 35 days, and BE is lowly expressed. Genes that are inversely related to development show the opposite pattern, their expression is higher in BE and gradually decreases in all endosperm over time. Whereas the degree of development of endosperm from bottom to top at the same time point is known to increase gradually [ Young TE, galie DR, and DeMason DA. Ethylene-mediated programmed cell death during maize endosperm development of wild-type and shrunken2 genetypes 1997 Plant Physiology 115, 737-751 ], it is seen that the time transcription profile of 1501 development related genes screened in example 1 of the present invention can provide a basis for further quantification of AE, ME and BE development processes.
In order to further quantitatively compare the physiological development among AE, ME and BE, the FPKM values of the development-related genes were mathematically integrated, thereby calculating the relative degree of physiological development. Wherein the FPKM values of all selected development related genes are normalized by 'maximum-minimum values' in all samples, respectively, and the normalization range is 0-1. Subsequently, the relative degree of physiological development of each sample was calculated as follows:
physiological developmental index = Σnormalized FPKM (developmental positive-related gene) +Σ [ 1-normalized FPKM (developmental negative-related gene) ]; the results are shown in Table 2.
TABLE 2 physiological developmental index differences in different regions of corn endosperm during filling
Endosperm region For 15 days For 20 days 25 days For 35 days
Endosperm upper section (AE) 732.3 782.6 788.6 1093.1
Endosperm middle part (ME) 591.4 621.9 632.8 748.4
Endosperm lower part (BE) 409.8 418.4 432.9 563.1
In 15-35 days of endosperm, the physiological development indexes of AE, ME and BE are 732.3-1093.1, 591.4-748.4 and 409.8-563.1 respectively, and the incremental amplitudes are 360.8, 157.0 and 153.3 respectively. 15-35 days, the physiological development speed of AE is faster, and the speeds of ME and BE are close to half of AE.
In summary, the physiological development index is used as a parameter reflecting the physiological development degree, and the calculation method thereof synthesizes a series of expression level values of development related genes, overcomes the difficulty of evaluating the physiological development process in tissues by using actual time, and can know the development processes in tissues such as different regions of endosperm.
Animal and plant physiology involves complex gene expression changes, but many of the development-related genes that undergo significant changes are in accordance with a common pattern. In an organism, the developmental process is often not controlled by a single gene or metabolic pathway, but rather by multiple organelles and metabolic pathways in the body. The invention provides a method for quantitatively calculating the physiological development index by counting and integrating the expression of thousands of age-related genes, provides an important method for quantitatively evaluating the physiological processes in tissues such as endosperm and the like, and distinguishes the physiological development degree and the actual development time of plant tissues.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (7)

1. A method of assessing the physiological development of plant tissue comprising:
obtaining a time transcriptome map of plant tissues to be detected from differentiation to maturity; the plant tissue to be detected is corn endosperm;
obtaining positive related genes and negative related genes related to the development of the plant tissue to be detected;
the positive and negative genes are shown in the following table:
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wherein, the prefix of the gene ID in the table is Zm00001, and d009907 is taken as an example, the complete gene ID is Zm00001d009907, and the gene ID is obtained from a Zm-B73-REFERENCE-GRAMENE-4.0 database; e is the base number of the power of 10, taking the gene ID d009907 as an example, the R value of 9.95E-01 is 9.95×10 -1 =0.995, E in R or P values corresponding to other genes is the same;
respectively carrying out normalization treatment on the expression levels of the positive correlation gene and the negative correlation gene by using a formula I;
normalization of A gene expression level= (X-min)/(max-min) formula I;
wherein X is the expression level of the A gene at the time point to be detected, min is the minimum expression level of the A gene in the time transcriptome map, max is the maximum expression level of the A gene in the time transcriptome map, and the A gene is any one gene of the positive related genes or any one gene of the negative related genes;
calculating the physiological development index of the plant tissue to be detected by using the formula II, wherein the higher the physiological development index is, the higher the physiological development degree of the plant tissue is;
physiological developmental index = Σnormalized result of positive correlation gene expression level + Σ (normalized result of 1-negative correlation gene expression level) formula ii.
2. The method according to claim 1, wherein the method for obtaining the positive-and negative-associated genes comprises:
carrying out Szelman correlation analysis between the actual growth time in the time transcriptome map and the plant tissue gene expression level at the actual growth time according to the correlation coefficient R value, the gene expression level and the significancepThe value is screened to obtain a positive related gene of plant tissue development and a negative related gene of plant tissue development; the positive correlation gene for plant tissue developmentpValue of<0.005, the gene expression level is more than or equal to 1, and the correlation coefficient R is more than 0; the negative related gene of plant tissue developmentpValue of<0.005, gene expression level is not less than 1, and relatedThe coefficient R is less than 0.
3. The method of claim 2, wherein the screening criteria for positive and negative genes further comprise: at least one time point has an expression level of 1 or more.
4. The method according to claim 1 or 2, wherein the temporal transcriptome map is obtained as follows: the time transcriptome map data of the plant tissue to be tested grown under suitable environmental conditions or of the disclosed plant tissue to be tested.
5. The method according to claim 1 or 2, wherein the obtaining of the temporal transcriptome map is: in the process from differentiation to maturation of plant tissues to be tested, transcriptome composition time transcriptome maps at different time points are obtained.
6. The method of claim 5, wherein the plant tissue to be tested is the same batch of plant tissue.
7. The method of claim 1 or 2, wherein the units of gene expression level comprise FPKM, RPKM or TPM.
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