CN114958875B - Screening and application of reference gene metG of citrus yellow-long pathogen - Google Patents
Screening and application of reference gene metG of citrus yellow-long pathogen Download PDFInfo
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Abstract
The invention discloses screening and application of a reference gene metG of citrus yellow-dragon bacteria, according to sequencing analysis results of a diaphorina citri and a shaddock transcriptome infected with yellow-dragon disease, carrying out primer specificity screening, relative expression quantity analysis and expression stability evaluation and verification on 15 preliminary screened CLas candidate reference genes with smaller variation coefficients, such as rpoC, con1, con2, clpX, ubiG, rplU, rpsA, metG, omp, ftnA, carD, groL, B I23-RS 01640, ffh, omp/bamA and the like, and finally determining the reference gene metG with strong specificity and stable expression in different hosts, thereby being applicable to gene expression research of CLas in the diaphorina citri and citrus hosts. The reference genes can be stably expressed in CLas insect hosts and plant hosts, the reliability and stability of the yellow-long pathogen gene expression research are improved, the method can be widely used for evaluating the analysis of the expression quantity of the yellow-long pathogen genes in different hosts, and the screened 9 pairs of specific reference primers can also be used for qPCR detection and identification of citrus yellow-long diseases, so that the method has important application value.
Description
Technical Field
The invention relates to the technical field of molecular plant pathology, in particular to screening and application of a reference gene metG of yellow-long citrus germ.
Background
Citrus yellow longdisease (Citrus Huanglongbing, HLB) is a quarantine disease caused by infection with the gram negative bacterium, bacillus phloem ('Candidatus Liberibacter'), which can multiply in the body of citrus and disease-transmitting vector insect diaphorina citri (Diaphorina citri Kuwayama) and more rapidly in diaphorina citri. Based on the pathogen's thermal sensitivity, distribution region, type of transmission medium, and 16SrDNA sequence information, the citrus yellow dragon bacteria are classified into the phloem bacillus asian species (' Candidatus Liberibacter asiaticus ', CLas), african species (' Candidatus Liberibacter africanum ', CLaf), and american species (' Candidatus Liberibacter americanum ', CLam). Currently, the main accepted Chinese species are the Asian species of the Bacillus phloem. HLB is transmitted mainly through sick seedlings and diaphorina citri, can infect almost all citrus plants, has destructive effect on the world citrus industry, and the destructive power of the HLB is far superior to any other citrus diseases, and recent research results show that the citrus yellow dragon disease is an immune disease triggered by phloem bacillus.
Reverse transcription real-time fluorescent quantitative PCR (RT-qPCR) is a common method for researching gene expression, and has extremely high detection sensitivity and specificity. The analysis of the expression level of the yellow dragon germ effector gene in the citrus host and the psyllid host by RT-qPCR has been studied, and the result shows that the significant difference exists in different hosts. For example, the cliband a 00460 effector gene is only expressed in large quantities in citrus, whereas the calasssd 115 effector gene is expressed in substantially higher quantities in psyllids than citrus and the like. These differentially expressed genes are predictive of different important functions in different hosts, respectively. In the analysis of gene expression, the quality of reference gene selection directly affects the accuracy of the result. The internal reference gene with strong specificity and stable expression can effectively correct deviation caused by the processes of RNA extraction and cDNA synthesis through standardization/normalization, thereby ensuring the accuracy of RT-qPCR experimental results and enabling the comparison of target genes among different samples to be possible. And the pathogenic bacteria content of CLas in different yellow dragon disease-causing citrus samples and psyllium samples is different, the reference genes of host plants/insects cannot correct deviation generated by different pathogenic bacteria content, and the comparison between samples can be carried out only by carrying out normalization on the suitable yellow dragon pathogenic bacteria reference genes, otherwise, completely wrong gene expression information can be obtained. The ideal pathogenic bacteria reference genes can be stably expressed in different hosts and different tissues and cells under various experimental conditions, however, most of the reference genes related to the yellow dragon bacteria reported in the literature in recent years have defects, and many of the reference genes are not suitable for cross-host analysis and are only suitable for being used in host plants or host insects. In addition, most of genes are not highly expressed in the yellow dragon germ, and if the common multicopy and super-expressed 16SrDNA, rplJ or mitochondrial cytochrome oxidase COX genes of the yellow dragon disease are used as internal references, larger errors can be introduced in calibrating the low-expression genes in the yellow dragon germ. Therefore, in the study of the expression of the RT-qPCR gene of CLas, the selection of the reference gene which has a Cq or Ct value closer to that of the target gene is preferred. Furthermore, the omp/bamA gene, which is commonly used for molecular or serological detection targets, is also often used as an internal reference, but its expression stability in different hosts has not been evaluated. Therefore, it is important to screen the reference genes which are stably expressed in both plant and insect hosts and are not expressed in the ultra-high level.
Disclosure of Invention
Based on the technical problems existing in the background technology, the invention provides screening and application of a yellow-long-germ reference gene metG, and the gene can be used for simultaneously researching expression analysis of yellow-long germ genes in diaphorina citri and citrus.
The nucleotide sequence of the reference gene metG of the yellow-long orange germ provided by the invention is shown as SEQ ID NO. 1.
The invention provides application of a reference gene metG of citrus yellow-shoot bacteria in researching gene expression analysis of the yellow-shoot bacteria in insect hosts such as diaphorina citri and plant hosts such as citrus.
The screening method of the reference gene metG of the citrus yellow-long germ provided by the invention comprises the following steps:
s1: screening genes with relatively high expression quantity through multi-sample transcriptome data analysis, and screening genes with small variation coefficients as candidate genes;
s2: performing primer specificity screening to remove candidate genes with poor specificity;
s3: sequentially adopting different single internal references to normalize data of all candidate internal reference genes according to Cq or Ct values obtained from RT-qPCR results of different yellow dragon germ internal reference genes, and then comprehensively analyzing the stability of the candidate internal reference genes of pathogenic bacteria in yellow dragon disease-causing citrus psyllium and shaddock by using an online tool RefFinder and combining geNorm, normfinder, bestKeeper and Delta CT four analysis methods;
s4: and (3) evaluating the relative expression quantity and the expression stability of the candidate genes, performing optimal internal reference verification, and finally screening to obtain the optimal internal reference genes.
Preferably, the relative expression level of the reference gene is 2 -ΔCq And (5) calculating by a method.
Preferably, the primer of the internal reference gene metG is shown as SEQ ID NO. 4-5.
The invention provides application of a reference gene metG of citrus yellow-long bacteria in a yellow-long disease qPCR or RT-qPCR detection kit.
The invention provides an application of a primer of an internal reference gene metG in preparation of a yellow dragon disease qPCR or RT-qPCR detection kit.
The invention provides a yellow dragon disease qPCR or RT-qPCR detection kit, which comprises an internal reference gene metG with a nucleotide sequence shown as SEQ ID NO.1 and corresponding primers shown as SEQ ID NO. 4-5.
The beneficial technical effects of the invention are as follows:
(1) The metG internal reference gene is suitable for researching the gene expression of the citrus yellow shoot germ in the insect host citrus psyllid and the plant host citrus, so that the comparison of the target genes of the citrus yellow shoot germ in the plant host and the insect host is possible; makes up the defect that most reported documents are only suitable for the reference genes of the yellow dragon bacteria of plant hosts.
(2) The invention optimizes the internal reference stability evaluation flow, and uses candidate genes as internal references to normalize Cq or Ct values before evaluation, and then carries out 2 times of calculation -ΔCq And comprehensively evaluating the stability of the internal reference, and maximally reducing deviation caused by the difference of bacteria content in each plant sample and insect sample.
(3) Compared with the existing common outer membrane protein gene omp/bamA gene, the metG reference gene has higher expression quantity in a host, and reduces error generation.
Drawings
FIG. 1 is a PCR detection electrophoresis chart of infected diaphorina citri and shaddock yellow dragon disease samples for screening reference genes of citrus yellow dragon bacteria; marker: DL2000 dnastar; YZ1-YZ5: a disease-sensitive grapefruit sample; psy1-Psy5: a psyllid sample; positive: yellow dragon disease positive control; negative: healthy citrus; water: no template water control.
FIG. 2 shows qPCR dissolution curves of 9 specific reference genes of yellow-long citrus bacteria; a: metG; b: rpsA; c: omp31; d: ftnA; e: a carD; f: groL; g: b2i23_rs01640; h: ffh; i: omp/bamA.
FIG. 3 shows qPCR amplification curves of 9 specific reference genes of the yellow-tailed citrus bacteria; a: metG; b: rpsA; c: omp31; d: ftnA; e: a carD; f: groL; g: b2i23_rs01640; h: ffh; i: omp/bamA.
FIG. 4 is a graph showing comparison of the relative expression levels and the expression stability of 9 candidate reference genes according to the present invention; a: metG; b: rpsA; c: omp31; d: ftnA; e: a carD; f: groL; g: b2i23_rs01640; h: ffh; i: omp/bamA.
FIG. 5 shows the analysis of the expression levels of B.flavescens B2I23_RS04900, B.2I23_RS 05695 and lysozyme gene Lyso in grapefruit and psyllium hosts, respectively, using the metG, rpsA and omp/bamA genes as internal references, according to the present invention.
Detailed Description
All internal reference gene primers in the embodiment of the invention are consigned to be synthesized by the Kirsrui biotechnology company.
Examples
1. Pre-screening of candidate reference genes
According to the result of transcriptome sequencing analysis of the yellow-sensitive grapefruit and the psyllids, 273 genes with the expression levels higher than the omp/bamA of the outer membrane protein genes in the yellow-sensitive grapefruit and the psyllids are selected from 1016 common CLas genes, and 15 genes with smaller variation coefficients are selected: rpoC, con1, con2, clpX, ubiG, rplU, rpsA, metG, omp31, ftnA, carD, groL, B2.2I23_RS01640, ffh, omp/bamA were used as candidate CLas reference genes.
2. Design of internal reference gene primer
qPCR primer design was performed on the screened 15 candidate reference genes using NCBI Primer Blast on-line tool. The annealing temperature was set at 60℃as shown in Table 1.
TABLE 1 internal reference Gene primer sequence information
3. Preparation of RT-qPCR templates
1) Sample preparation
Collecting branches of field-sensitive yellow dragon disease grapefruits, grabbing diaphorina citri on yellow dragon disease trees, feeding the diaphorina citri in a laboratory net cage for 1-2 weeks, and detecting the disease rate (the spot check disease rate is the lowest)>60%). The host plant is kriman Ding Gongju, and the raising environment temperature is 25℃:23 ℃ (day: right), photoperiod 16:8 (L: D) with an illumination intensity of 159. Mu. Mol/m 2 s。
2) RNA extraction of disease-sensitive shaddock vein and diaphorina citri sample
Referring to the instruction of the bang-bang organism KK ultrafast plant total RNA extraction kit (KK Fast Plant Total RNA Kit, centrifugal column type), 5 biological replicates of the shaddock and the psyllium RNA samples are respectively extracted from the samples in the step 1), and each psyllium biological replicate sample comprises 60 psyllium.
3) cDNA Synthesis
Referring to the instructions of the TaKaRa PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) kit, the cDNA was synthesized by reverse transcription using the RNA of grapefruit and psyllium in step 2) as templates.
4) Detection of diaphorina citri and shaddock sample yellow dragon disease
PCR yellow dragon disease detection and 1% agarose gel electrophoresis were performed on the psyllium and grapefruit cDNA samples from step 3) by yellow dragon disease detection primers OI1/OI2c (GCGCGTATGC AATACGAGCG GCA/GCCTCGCGAC TTCGCAACCCAT), and the results were observed using a Bio-Rad gel imaging system and photographed at 150V for 30 min. As shown in fig. 1, 5 diaphorina citri samples: CLas-Psy-1, CLas-Psy-2, CLas-Psy-3, CLas-Psy-4, CLas-Psy-5, and 5 grapefruit samples: the bands of CLas-YZ-1, CLas-YZ-2, CLas-YZ-3, CLas-YZ-4 and CLas-YZ-5 are clear, and the size is 1160bp, which shows that 10 samples all confirm the infection of citrus yellow dragon disease.
4. Fluorescent quantitative PCR amplification
Diluting 10 cDNA samples in the step 3 by 5 times, and taking the 10 cDNA samples as templates for fluorescence quantitative PCR, wherein each sample is subjected to 3 technical repetitions; the fluorescent dye was LightCycler 480SYBR Green I Master (Roche). The reaction system is shown in Table 2.
TABLE 2 fluorescent quantitative PCR reaction System
The reaction procedure: pre-denaturation at 95 ℃ for 5min; (denaturation at 95℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s,45 cycles (fluorescence signal collection)); (95 ℃,5s,4.8 ℃/s;65 ℃,60s;97 ℃,1 s); 37℃for 30s.
5. Candidate reference gene specificity verification
Through qPCR primer evaluation verification in the step 4, amplification curves of rpoC, con1, con2 and clpX, ubiG, rplU in 15 reference genes are subjected to nonspecific amplification, and are not suitable for serving as the reference genes; rpsA, metG, omp31, ftnA, carD, groL, B2I 23-RS 01640, ffh, omp/bamA, which have smooth 9 gene amplification curves, single peak dissolution curve and good specificity (figures 2 and 3); taking each specific candidate gene as an internal reference gene in sequence, normalizing the relative expression quantity of other internal reference genes in different hosts, and passing through 2 -Δct The relative expression of the genes is calculated by the method, meanwhile, the One-wayANOVA is used for carrying out difference significance analysis, and the result shows that the relative expression of 9 genes is higher (figure 4), so that a new gene can be enteredAnd (5) screening the expression stability of the reference genes.
6. Analysis of expression stability of candidate reference genes of yellow-long bacteria in different hosts
The stability of the expression of the 9 screened specific CLas candidate reference genes in the body infected with the yellow dragon disease grapefruit and the psyllium respectively is evaluated according to a comprehensive evaluation method of four different algorithms geNorm, normfinder, bestKeeper and Delta CT in RefFinder (http:// bloom. Cn/. Comprehensive evaluation results show (FIG. 4, tables 3-20) that when metG, carD, ftnA, omp and ffh are used as reference genes, the expression stability of rpsA in two types of hosts is the best; B2I23_RS01640, groL, rpsA as reference gene showed the best expression stability of metG in both hosts.
The comprehensive evaluation stability numerical results are summarized and summed, and the numerical values are sorted from small to large, the smaller the numerical values are, the more stable the numerical values are, the better the expression stability of metG (metanine-tRNA (tRNA) gene is found, the next is rpsA (30S ribosomal protein S1), the expression quantity is relatively high (FIG. 4, table 21), and the method can be used as a CLas preferential reference gene in diaphorina citri and citrus for carrying out yellow-corydalina bacteria gene screening analysis verification.
TABLE 3 ranking of the expression stability of the genes with CarD as an internal reference
TABLE 4 comprehensive ranking of stability by different evaluation methods using CarD as a reference gene
TABLE 5 ranking of the expression stability of the genes with ftnA as an internal reference
TABLE 6 comprehensive ranking of stability by different evaluation methods using ftnA as reference gene
TABLE 7 ranking of the expression stability of genes with omp31 as a reference gene
TABLE 8 comprehensive ranking of stability by different evaluation methods using omp31 as reference gene
TABLE 9 ranking of the expression stability of the genes with ffh as reference gene
| Method | rpsA | metG | omp | ftnA | groL | B2I23_RS01640 | carD | omp31 |
| DeltaCT | 3.466 | 3.675 | 3.941 | 3.561 | 3.651 | 3.691 | 3.89 | 9.698 |
| BestKeeper | 0.802 | 0.399 | 0.305 | 2.395 | 3.226 | 2.715 | 3.334 | 8.609 |
| Normfinder | 2.403 | 2.839 | 3.369 | 1.106 | 1.022 | 1.112 | 1.74 | 9.577 |
| Genorm | 0.709 | 0.665 | 0.665 | 1.785 | 2.56 | 2.285 | 2.696 | 4.447 |
| RefFinder | 2.59 | 2.632 | 2.646 | 2.826 | 3.224 | 4.401 | 5.856 | 8 |
Table 10 comprehensive stability ranking of different evaluation methods using ffh as reference gene
TABLE 11 ranking of expression stability of genes with groL as an internal reference
TABLE 12 comprehensive ranking of stability by different evaluation methods using groL as reference gene
TABLE 13 ranking of the expression stability of the genes with B2I23_RS01640 as reference genes
Table 14 comprehensive stability ranking of different evaluation methods using B2I23_RS01640 as reference gene
TABLE 15 ranking of the expression stability of the genes with rpsA as reference gene
TABLE 16 comprehensive ranking of stability by different evaluation methods with rpsA as reference gene
TABLE 17 stability of expression of genes with metG as an internal Gene
TABLE 18 comprehensive ranking of stability by different evaluation methods of metG as reference gene
TABLE 19 stability of expression of genes with omp as an internal Gene
TABLE 20 comprehensive ranking of stability by different evaluation methods using omp as reference gene
Table 21 summarizing and ranking the results of mutual evaluation of stability by taking different genes as internal parameters
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7. Comparing the expression level analysis of the MetG, rpsA and omp/bamA as the internal reference to the yellow dragon germ B2I23_RS04900, B2I23_RS05695 gene and lysozyme gene Lyso in the diaphorina citri and shaddock, respectively.
In order to further verify the reliability of the reference genes screened by the invention, 2 genes of CLas gene B2I23_RS05695 and lysozyme gene Lyso which are only highly expressed in shaddock and 1 gene of CLas gene B2I23_RS04900 which are only highly expressed in the shaddock are selected from the sequencing analysis results of the transcriptome of the diaphorina citri and the shaddock infected with the yellow-long disease, the expression conditions of the 3 genes in the diaphorina citri and the citrus are respectively analyzed and compared by using metG, rpsA and omp/bamA as the reference genes according to the method of the embodiment 2-step 6 of the invention, the primer sequence information of the three genes is shown in table 22, and the annealing temperature is set to 60 ℃.
TABLE 22_CLas Gene primer sequence information
The template and qPCR system used in the fluorescent quantitative PCR are consistent with those in the step 1; the fluorescent dye used was Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (YEASEN), reaction procedure: pre-denaturation at 95℃for 2min; (denaturation at 95℃for 10s; annealing at 60℃for 20s;40 cycles (fluorescence signal collection)); (95 ℃,5s,4.8 ℃/s;65 ℃,60s;97 ℃,1 s); 40℃for 30s.
Based on Cq values of the 3 genes in RT-qPCR experiments, 2 were performed on the 3 genes with the genes metG, rpsA and omp/bamA as internal references, respectively -ΔCq Data were normalized and differential significance analysis was performed with t-tests. As shown in fig. 5, when metG was used as the reference gene, the expression level of CLas gene b2i23_rs05695 and lysozyme gene Lyso was significantly higher Yu Mushi in grapefruit only and the difference was very significant; the relative expression quantity of the CLas gene B2I23_RS04900 which is only highly expressed in the diaphorina citri is higher than that of the shaddock in the diaphorina citri, and the difference is obvious. When rpsA is used as an internal reference, the lysozyme gene Lyso is highly expressed in the infected grapefruit, and the difference is obvious; the gene B2I23_RS05695 is highly expressed in the affected grapefruit and has extremely obvious difference in two types of affected hosts; while the gene B2I23_RS04900 is not significantly different in the two types of affected hosts. When the outer membrane protein gene omp/bamA is used as an internal reference, the lysozyme gene Lyso is highly expressed in the infected shaddock, and the difference is very obvious; the expression quantity of the gene B2I23_RS05695 in the infected shaddock is higher than that in the infected psyllium, and the difference is obvious; the difference of the gene B2I23_RS04900 in the infected diaphorina citri and the shaddock is not obvious. In addition, as can be seen from fig. 5, the same sample was used to detect the expression analysis results of three genes in two types of hosts, and the concentration of scattered spots in the expression results with the gene metG as an internal reference was higher, indicating that the results were more stable. From the results, the gene metG was the optimal internal reference; genes rpsA and omp/bamA times; the present invention shows that the screened internal reference gene metG is stable and reliable and may be used in yellow dragon germGene expression studies in different hosts.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
<110> Gannan university of teachers and students
<120> screening and application of reference gene metG of yellow-orange-dragon germ
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gaccaacagt agcgtacaac aggagcga 28
<210> 8
<211> 23
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qCLas_ftnAF)
<400> 8
gcttgggctg catctcataa tct 23
<210> 9
<211> 26
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qCLas_ftnAF)
<400> 9
gttatgttga cgggatttgg tgctct 26
<210> 10
<211> 26
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_1640F)
<400> 10
gttatgttga cgggatttgg tgctct 26
<210> 11
<211> 26
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qclas_1640f)
<400> 11
aatacaccca aaggcgcgaa tatgaa 26
<210> 12
<211> 22
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_cadf)
<400> 12
ttgtccgtga cttacaccgc ac 22
<210> 13
<211> 28
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qclas_cadf)
<400> 13
attgagagcg gattcatata gctggcgt 28
<210> 14
<211> 22
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_grolf)
<400> 14
taatacgctt gccgaggctg ta 22
<210> 15
<211> 23
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qclas_grolf)
<400> 15
tagtaacgcg aggagcaccg aaa 23
<210> 16
<211> 21
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qCLas_ffhF)
<400> 16
cccggaatgc cctctctcaa a 21
<210> 17
<211> 22
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qCLas_ffhF)
<400> 17
acttgggtta gcacgctctt ct 22
<210> 18
<211> 25
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_ompf)
<400> 18
agggtatagg tccgcgtgtg gataa 25
<210> 19
<211> 25
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qclas_ompf)
<400> 19
aaccagccct ttcaggaaca agagg 25
<210> 20
<211> 26
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_4900F)
<400> 20
gcacgacgtt tacgcgaaaa atcaga 26
<210> 21
<211> 28
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qclas_4900F)
<400> 21
cttctgcaag aatctttgcc ctatgctt 28
<210> 22
<211> 32
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_5695F)
<400> 22
gtttatccga tcatggtaaa gtagcaggag aa 32
<210> 23
<211> 25
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_5695F)
<400> 23
tggttgcggt tctctttttg gctgt 25
<210> 24
<211> 23
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction upstream primer of phloem bacillus (qclas_lysof)
<400> 24
ggaaaaagcc gctgaggaat gta 23
<210> 25
<211> 23
<212> DNA
<213> real-time fluorescent quantitative polymerase chain reaction downstream primer of phloem bacillus (qclas_lysof)
<400> 25
acttaacctc tgcatcacgc ctt 23
Claims (3)
1. Reference gene of citrus yellow-long germmetGApplication of diaphorina citri gene expression analysis in insect host diaphorina citri and plant host citrus, and reference gene of diaphorina citrimetGThe nucleotide sequence of the polypeptide is shown as SEQ ID NO. 1.
2. The reference gene of yellow croaker as set forth in claim 1metGIs characterized in that the method comprises the following steps:
s1: screening genes with relatively high expression quantity through multi-sample transcriptome data analysis, and screening genes with small variation coefficients as candidate genes;
s2: performing primer specificity screening to remove candidate genes with poor specificity to obtain reference genesmetGThe reference genemetGThe primer of (2) is shown as SEQ ID NO. 4-5;
s3: sequentially adopting different single internal references to normalize data of all candidate internal reference genes according to Cq or Ct values obtained from RT-qPCR results of different yellow dragon germ internal reference genes, and then comprehensively analyzing the stability of the candidate internal reference genes of pathogenic bacteria in yellow dragon disease-causing citrus psyllium and shaddock by using an online tool RefFinder and combining geNorm, normfinder, bestKeeper and Delta CT four analysis methods;
s4: and (3) evaluating the relative expression quantity and the expression stability of the candidate genes, performing optimal internal reference verification, and finally screening to obtain the optimal internal reference genes.
3. The reference gene of yellow-tailed citrus bacteria according to claim 2metGCharacterized in that the relative expression level of the reference gene is 2 -ΔCq And (5) calculating by a method.
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| WO2015170325A2 (en) * | 2014-05-04 | 2015-11-12 | Forrest Innovations Ltd. | Compositions and methods for reducing pathogen-induced citrus greening |
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