CN114277180A - Primer pair, kit, method and application for detecting citrus SOD gene family - Google Patents
Primer pair, kit, method and application for detecting citrus SOD gene family Download PDFInfo
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Abstract
The invention discloses a primer pair, a kit, a method and application for detecting citrus SOD gene family, wherein the primer pair for detecting the citrus SOD gene family comprises 13 primer pairs, namely CSSOD1-CSSOD13, the sequences of which are shown as SEQ ID NO: 1-SEQ ID NO: 26; the 13 pairs of primers provided by the invention can quickly, specifically and accurately detect the expression characteristics of the gene family of the citrus superoxide dismutase by a fluorescent quantitative PCR method, the detection sensitivity is high, the detection result is accurate and reliable, and a foundation is laid for the research of citrus stress resistance.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer pair, a kit, a method and application for detecting an SOD gene family of citrus.
Background
Superoxide dismutase (SOD), a metal antioxidant enzyme, is the first line of defense in the immune system of plants by catalyzing the decomposition of Superoxide anions to hydrogen peroxide (H)2O2) And O2To maintain the dynamic balance of plant Reactive Oxygen Species (ROS). Plant SOD enzymes can be classified into three categories according to the difference of metal cofactors: copper zinc superoxide dismutase (Cu/ZnSOD), iron superoxide dismutase (FeSOD), and manganese superoxide dismutase (MnSOD). Cu/ZnSOD is mainly present in cytosol, chloroplasts, peroxisomes and extracellular fluid; while FeSODs are mainly present in chloroplasts and may also be present in cytosol; MnSODs are present in mitochondria; no nickel superoxide dismutase NiSOD has been found in plants.
Plants can induce secondary oxidative stress when they respond to stress, producing oxidative stress reactions that result in the accumulation of large amounts of ROS in the body. At the cellular and molecular level, ROS can damage biological membranes, cause membrane lipid peroxidation, and change the spatial structure of protein nucleic acid; at the individual level, the physiological balance in the plant body is broken, and the normal growth and development are difficult to maintain. SOD plays an important role as active enzyme for eliminating ROS, and is cooperated with other antioxidant enzyme to relieve the damage of ROS to plants. When the plants are stressed by adversity such as high temperature, low temperature, water, salt and alkali, heavy metal, ozone, ultraviolet rays, nutrient deficiency and the like, the activity of the antioxidant enzyme can be regulated to a certain degree by applying exogenous substances, and the stress resistance of the plants is enhanced. When facing pathogenic bacteria invasion, the pathogenic bacteria infect a host, release toxin in vivo and destroy a plant cell structure. During host defense against toxins, large excess of normal levels of ROS are produced, which breaks the dynamic balance between ROS production and elimination in plants, so changes in SOD levels also play an important role in plant-pathogen interactions.
Superoxide dismutase gene families have been researched in a plurality of plants such as arabidopsis thaliana, plantain angustifolia, sorghum, populus tremuloides and the like, SOD genes play different roles in the growth and development process of the plants, and researches have proved that the SOD genes are related to stress response to adverse conditions. Plant SOD genes that are overexpressed or knocked out have been studied to confirm their role in improving stress resistance: in transgenic rice plants, MnSOD in peas participates in an ROS scavenging system of chloroplasts, so that the drought tolerance of rice is improved; the FeSOD gene of arabidopsis thaliana can improve the tolerance of a transgenic line to in vitro oxidative stress; in the Abscisic acid (ABA) treated AtSOD transgenic plants, the expression of desiccation response transcription factor is strongly up-regulated, and the signal pathway is through elimination.O2-Enhance cold resistance. In summary, the SOD gene family is widely involved in plant responses to abiotic and biotic stresses.
The resistance from stress has a great influence on the development of the citrus industry, such as biological stress including citrus canker and huanglongbing, and abiotic stress including low temperature and drought. The research on SOD gene families in citrus is less, so that the detection of SOD content in citrus is beneficial to the research on the function of oxidative stress reaction of citrus under adverse conditions, and the research on the mechanism of SOD genes for improving the tolerance of citrus to various environmental stresses is necessary. The research on the change of physiology and biochemistry of plants infected by pathogenic bacteria plays an important role in disclosing the disease resistance mechanism of plants. Meanwhile, hormones such as ABA, Salicylic Acid (SA), Jasmonic Acid (JA) and the like are important signal molecules in the response process of growth and development, biotic and abiotic stresses of plants, the response of SOD families to the hormones is researched, and candidate genes related to the growth and development, the biotic and abiotic stresses can also be identified.
Therefore, it is highly desirable to design a kit capable of detecting the SOD content in citrus, and to detect the expression patterns under various stress conditions to obtain stress-related SOD genes for breeding stress-resistant molecules.
Disclosure of Invention
The invention aims to provide a primer pair, a kit and a method for detecting an SOD gene family of citrus, which are used for detecting the SOD content in citrus so as to identify the response of the SOD gene family of the citrus to stress.
The invention is realized by the following technical scheme:
the primer pair for detecting the citrus SOD gene family comprises 13 primer pairs, namely CsSOD 01-CsSOD 13, and the sequences are shown as SEQ ID NO:1 to SEQ ID NO: shown at 26.
The invention designs primers aiming at 13 superoxide dismutase genes of citrus, and obtains the primers through reasonable screening, so that the detection of the superoxide dismutase gene family of the citrus can be realized.
The kit for detecting the citrus SOD gene family comprises the primer pair.
Further, the kit also comprises a reagent for a reaction system for PCR detection.
Further, the reagent for the reaction system includes 2 × SYBRG fluorescent dye.
Further, the reagent for the reaction system further comprises H2O and cDNA.
Further comprises an internal reference primer pair, and the sequences are shown as SEQ ID NO. 27-SEQ ID NO. 28.
The detection method of the citrus SOD gene family comprises the steps of extracting total RNA of a sample to be detected, and carrying out fluorescent quantitative PCR detection by using the kit.
Further, the reaction system for the fluorescent quantitative PCR detection is a 12 μ L system: 4.4uL H2O, 0.3. mu.L of 10mM upstream and downstream primers, 6. mu.L of 2 XSSYBRG fluorescent dye, 1. mu.L of cDNA.
Further, the reaction conditions of the fluorescent quantitative PCR detection are as follows: 60S at 50 ℃ and 2min at 95 ℃ before circulation; 5S at 95 ℃ and 15S at 60 ℃ were cycled for 40 cycles.
The primer pair or the kit is applied to the detection of the expression level of SOD (superoxide dismutase) of a defense mechanism generated when the citrus is subjected to stress (such as pathogenic bacteria invasion and exogenous hormone induction).
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention comprehensively identifies the citrus SOD gene family, compares the whole genome design primers of citrus, screens out multiple pairs of primers to ensure the specificity of the primers, designs 13 kinds of primers of superoxide dismutase genes in total, performs PCR reaction, detects the specificity of the primers through agarose gel electrophoresis, detects two varieties (citrus canker resistant variety kumquat and sensitive variety orange), confirms the universality of the citrus varieties, and detects the expression quantity of SOD in the citrus to identify the response of the citrus superoxide dismutase gene family to adversity and hormone signals.
2. The invention determines the optimal PCR reaction system in the application after carrying out a plurality of experiments by specific primers, respectively identifies the expression characteristics of 13 superoxide dismutase genes of various oranges under the induction of various hormones and the induction of canker bacteria, and is beneficial to researching the stress resistance characteristics of the oranges.
3. The 13 pairs of primers provided by the invention can quickly, specifically and accurately detect the expression characteristics of the gene family of the citrus superoxide dismutase by a fluorescent quantitative PCR method. A large number of early-stage test results show that the detection method is high in sensitivity, accurate and reliable in detection result and lays a foundation for mining candidates related to stress resistance and growth development.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a diagram showing the results of agarose gel electrophoresis after PCR of 13 primer pairs;
FIG. 2 shows the specific positions of 13 pairs of primer pairs on the CDS sequence of the CsSODs gene, wherein the long bar corresponding to each pair of primer pairs represents the CsSOD gene, and the two short bars on the long bar represent the upstream primer and the downstream primer, respectively;
FIG. 3 is a chromosome mapping of the citrus SOD family;
FIG. 4 is a diagram of the structure of the genes of the citrus SOD family;
FIG. 5 is a schematic representation of the protein domains of the citrus SOD family;
FIG. 6 is a graph of the conserved motifs of the citrus SOD family of proteins;
FIG. 7 is a graph showing the induced expression of citrus SOD family by canker pathogen; the different upper and lower case letters on the bars indicate significant differences (P < 0.05) among the different varieties.
FIG. 8A is a graph of SA (salicylic acid) -induced expression analysis of the citrus SOD family, with the lower case letters on the column indicating significant differences in the same variety (P < 0.05).
FIG. 8B is a heat map of SA (salicylic acid) -induced expression of the citrus SOD family, in which J represents Poncirus trifoliata and W represents Citrus aurantium.
FIG. 9A is a graph of MeJA (jasmonic acid) -induced expression analysis of the citrus SOD family, and the lower case letters on the column indicate significant differences in the same variety (P < 0.05).
FIG. 9B is a heat map of MeJA (jasmonic acid) -induced expression of the citrus SOD family, wherein J represents kumquat and W represents late orange.
FIG. 10A is a graph of ABA (abscisic acid) -induced expression analysis of the citrus SOD family, with lower case letters on the bars indicating significant differences in the same variety (P < 0.05).
FIG. 10B is a heat map of ABA (abscisic acid) -induced expression of the SOD family of citrus, wherein J represents kumquat and W represents Citrus aurantiaca.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1:
the first, material and method:
the experimental species were late orange and kumquat, and the leaves were obtained from the modified center nursery of citrus institute of the southwest university/Chinese academy of agricultural sciences. The plant RNA extraction kit is purchased from Beijing Edgeli, the primer is synthesized by Beijing Optimalaceae biotechnology, Inc., and the reverse transcription kit and the real-time fluorescent dye are purchased from TAKARA.
Bioinformatics analysis of citrus SOD family
Taking the SOD family protein sequence of arabidopsis thaliana as input, carrying out BLAST comparison in a third edition of a sweet orange genome database (http:// citrus. hzau.edu.cn /), obtaining the protein sequence of the citrus SOD family, and searching to obtain a corresponding coding framework sequence; predicting the molecular weight and the theoretical isoelectric point (pI) of the amino acid sequence by using online software ProtParam (https:// web. expasy. org/protpa ram /); analyzing the number of introns, exons and the like of the gene on line (http:// citrus. hzau. edu. cn/cgi-bin/orange/blast); carrying out comparison and phylogenetic tree construction by using MEGAX and an Arabidopsis SOD family; protein domain and gene structure analysis was performed using MEME and GSDS, and sequence analysis of the citrus SOD family is shown in table 1:
TABLE 1 sequence information for Citrus SOD family
Hormone and ulcer germ induction treatment of citrus leaves
The treatment of late orange and kumquat leaves was as follows: mature leaves (about 3 months old) were collected, sterilized with 75% ethanol, washed with sterile water, placed in a sterile petri dish, injected with an inoculum of canker bacteria (OD600 ═ 0.5) and incubated at 28 ℃ under light, and the inoculated sites were excised at 0, 6, 12 and 24 Hours (Hours, H), respectively. Soaking leaf disks (diameter 7mm) in jasmonic acid (100 μmol. L)-1) Abscisic acid (100. mu. mol. L)-1) Salicylic acid (10. mu. mol. L)-1) The solution and sterile water (control) were incubated at 28 ℃ with light and samples were taken at 0, 6, 12, 24, 36 and 48H.
Fourth, RNA extraction and reverse transcription
The above samples were extracted with total RNA (edley, beijing) using an RNA extraction kit, reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, japan), and the relative expression amount of the gene in each sample was detected by real-time fluorescent quantitative PCR (qRT-PCR).
Fifthly, primer design and real-time fluorescent quantitative PCR analysis
The Primerblast in NCBI is utilized to design the quantitative PCR primers of the citrus SOD family, the specificity in the citrus genome is fully considered, and the intron regions are crossed as much as possible. The primers of 13 superoxide dismutase genes are designed in total, PCR reaction is carried out, the specificity of the superoxide dismutase genes is detected by agarose gel electrophoresis, and the detection in two varieties (citrus canker resistant variety kumquat and susceptible variety mallow) proves the universality of the citrus varieties (figure 1). The CsActin gene (SEQ ID NO: 27-SEQ ID NO: 28) is used as an internal reference, the hormone of citrus leaf and cDNA extracted after the induction of canker pathogen are used as templates, specific primers shown in Table 2 are used for real-time reverse transcription polymerase chain reaction (RT-PCR), the specific positions of 13 pairs of primer pairs on the CDS sequence of the CsSODs gene are shown in figure 2, and the induced expression characteristics of the 13 genes are analyzed. The specific reaction system and parameters are as follows: for a 12 μ L system: 4.4mL of H2O, 0.3. mu.L of 10mM upstream and downstream primers, 6. mu.L of 2 XSSYBRG fluorescent dye, 1. mu.L of cDNA. Reaction conditions are as follows: 60S at 50 ℃ and 2min at 95 ℃ before circulation; 5S at 95 ℃ and 15S at 60 ℃ were cycled for 40 cycles. Three biological replicates and three replicate-like replicates were performed per treatment. Relative expression amount 2－△CtAnd (4) carrying out calculation by using Excel software to count data.
TABLE 2 primers for detecting expression of citrus SOD family
Sixth, result analysis and discussion
1. Informatics analysis of citrus SOD family.
Chromosomal localization of the citrus SOD family is shown in FIG. 3, with the uneven distribution of CsSODs across the 4 chromosomes of citrus. There are six CsSODs genes on chromosome 3, and there is one gene cluster (CsSOD 01-CsSOD 06) according to the definition of Holub for the gene cluster, and in this cluster, four genes are involved in tandem replication events. Although CsSOD1 and CsSOD6 are closely related genes located on the same chromosome, they are not considered tandem repeat genes because of their low similarity in amino acid sequence. Chromosome 5 contains two distant CsSOD genes, CsCOD07 and CsCOD 08. The 4 CsSOD genes (CsSOD 09-CsSOD 12) are located on chromosome 7, wherein CsSOD09 and CsSOD10 are tandem repeats. Chromosome 8 contains only CsSOD13 a CsSOD gene.
The gene structure of citrus SOD family is shown in FIG. 4, the number of exons of CsSOD01 is the highest (14), the number of exons of CsSODs gene family varies from 14 to 5, and the length of introns varies greatly. The CsSOD09 and CsSOD10 genes showed the same intron/exon organization pattern. The protein structure domain of the citrus SOD family is shown in a figure 5 (CsSOD01 in the figure 5 sequentially comprises a protein sequence, copper/zinc superoxide dismutase, a protein sequence, copper/zinc superoxide dismutase and a protein sequence, CsSOD02-CsSOD07 sequentially comprises a protein sequence, copper/zinc superoxide dismutase and a protein sequence, CsSOD08 sequentially comprises a protein sequence, a heavy metal structure domain, a protein sequence, copper/zinc superoxide dismutase and a protein sequence, CsSOD09-CsSOD012 sequentially comprises a protein sequence, copper/manganese superoxide dismutase, an alpha-hairpin structure domain, a protein sequence, copper/manganese superoxide dismutase, a C-terminal structure domain and a protein sequence, and CsSOD013 sequentially comprises a protein sequence, copper/zinc superoxide dismutase and a protein sequence). Protein conserved motifs of the citrus SOD family are shown in figure 6, and among the citrus SOD proteins, MEME identified 11 conserved protein motifs (Motif). Based on the conserved domain prediction of PFAM, two classes of CsSODs were found, both containing completely different motifs. Motif 2 is located in Cu/ZnSODs (CsSOD 01-07 and 13), and the Cu/ZnSODs except CsSOD01 all contain Motif 6. Members of the Fe-MnSODs class include Motif3, Motif4, and Motif 10. In addition to CsSOD12, the Fe/MnSOD contained Motif 5, 7 and 9.
2. The citrus SOD family was analyzed by the induction expression of the ulcer germs, as shown in FIG. 7.
Researches the induction effect of Xcc on CsSOD genes of the mallotus virens and the kumquats, and further determines the function of the CsSODs in the sweet oranges. The expression of CsSODs is detected by qRT-PCR, and the result shows that the CsSOD gene is induced or inhibited in the Xcc infection process. In the kumquat variety resisting canker, the expression quantity of CsSOD 01-CsSOD 08 is the lowest at 6H, except CsSOD04, the expression quantity of CsSOD initially decreases and then reverses to increase at 12H, and finally is moderately inhibited. While the expression level of CsSOD09 decreased after increasing, the expression level of CsSOD13 was opposite. In contrast, in the ulcer-sensitive variety of late orange, the expression of CsSOD 07-13 is obviously inhibited; CsSOD01, 03 and 05 was expressed in the highest amount at 12H. The expression pattern of CsSOD03 was similar in late orange and kumquat, whereas the expression pattern of CsSOD11 was completely opposite in both varieties.
3. The citrus SOD family is subjected to salicylic acid, jasmonic acid and abscisic acid induced expression analysis, and is respectively shown in FIGS. 8A, 8B, 9A, 8B, 10A and 10B.
All CsSOD genes were expressed in both varieties, and they showed different expression patterns. After SA treatment, the SA-induced expression levels of CsSOD01, 03, 06, 07, 09, 10, 12 and 13 in kumquat and CsSOD02, 04 in late mallow were significantly up-regulated at 6H. After jasmonic acid treatment, the expression levels of CsSOD13, 12, 10, 06, 09 and 01 in kumquat are the highest at 12H; the expression levels of CsSOD03 and 04 in late caragana orange were highest at 48H, the expression level of CsSOD10 showed a tendency to increase, and the expression level was highest at 48H. After ABA treatment, the expression levels of CsSOD13, 12, 10, 05, 06, 07, 03, 02 and 01 in kumquat reached the highest values at 36H, while CsSOD04, 08 and CsSOD11 reached the highest values at 48H; the expression level of CsSOD02, 05, 09, 10 in Citrus aurantium is always down-regulated; the expression levels of CsSOD06 and CsSOD08 were gradually up-regulated in both kumquat and late orange. In general, most of the CsSOD gene expression was altered under hormone induction.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
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Claims (10)
1. The primer pair for detecting the citrus SOD gene family is characterized by comprising 13 primer pairs, and the sequences are shown as SEQ ID NO. 1-SEQ ID NO. 26.
2. Kit for detecting citrus SOD gene family, comprising the primer pair according to claim 1.
3. The kit for detecting citrus SOD gene family according to claim 2, further comprising reagents for reaction system of PCR detection.
4. The kit for detecting citrus SOD gene family according to claim 3, wherein the reagent for reaction system comprises 2 x SYBRG fluorescent dye.
5. The kit for detecting citrus SOD gene family according to claim 4, wherein the reagent for reaction system further comprises H2O and cDNA.
6. The kit for detecting the citrus SOD gene family according to claim 2, further comprising an internal reference primer pair, wherein the sequence is shown as SEQ ID NO. 27-SEQ ID NO. 28.
7. The method for detecting the citrus SOD gene family is characterized in that total RNA of a sample to be detected is extracted, and fluorescent quantitative PCR detection is carried out by using the kit of any one of claims 2 to 6.
8. The detection method according to claim 7, wherein the reaction system of the fluorescence quantitative PCR detection is a 12 μ L system: 4.4uL H2O, 0.3. mu.L 10mM forward primer, 0.3. mu.L 10mM reverse primer, 6. mu.L 2 XSSYBRG fluorescent dye, 1. mu.L cDNA.
9. The detection method according to claim 7, wherein the reaction conditions of the fluorescent quantitative PCR detection are as follows: 60S at 50 ℃ and 2min at 95 ℃ before circulation; 5S at 95 ℃ and 15S at 60 ℃ were cycled for 40 cycles.
10. Use of the primer pair according to claim 1 or the kit according to any one of claims 2 to 6 for detecting the expression level of SOD in the defense mechanism of citrus infected by pathogenic bacteria.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116516053A (en) * | 2023-05-23 | 2023-08-01 | 中国农业科学院郑州果树研究所 | Primer pair, kit and method for detecting watermelon LIM gene family and application |
| CN116814653A (en) * | 2023-07-27 | 2023-09-29 | 西部(重庆)科学城种质创制大科学中心 | Citrus ABI5 gene family and detection method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116516053A (en) * | 2023-05-23 | 2023-08-01 | 中国农业科学院郑州果树研究所 | Primer pair, kit and method for detecting watermelon LIM gene family and application |
| CN116516053B (en) * | 2023-05-23 | 2023-11-10 | 中国农业科学院郑州果树研究所 | Primer pair, kit and method for detecting watermelon LIM gene family and application |
| CN116814653A (en) * | 2023-07-27 | 2023-09-29 | 西部(重庆)科学城种质创制大科学中心 | Citrus ABI5 gene family and detection method and application thereof |
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