CN115992253A - Primer and method for detecting LGMN gene transcription level of gecko elegans - Google Patents
Primer and method for detecting LGMN gene transcription level of gecko elegans Download PDFInfo
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Abstract
The invention provides a primer and a method for detecting the LGMN gene transcription level of the gecko, wherein the primer sequence is shown as SEQ ID NO. 1-2.
Description
Technical Field
The invention relates to a primer and a method for detecting the LGMN gene transcription level of gecko, belonging to the technical field of biology.
Background
The Papaw gecko belongs to the genus gecko of the order Hemiptera and the family Lepidoptera. The host range is wide, and the pest is an important potential economic pest with strong diffusivity, vitality and fertility. The insect is generally considered native to mexico or central america and the first specimen of gecko was collected in mexico in 1955. Papaya xiu mealybugs live in hidden parts such as veins of leaves, growing points of stems and leaves, concave-convex or overlapping parts of flowers and leaves, concave or groove seams on the surfaces of fruits and the like. The fruit juice of the stems, leaves, flowers, fruits and other organ tissues of the host plants is mainly sucked by nymphs and female adults, so that leaf rolling deformity, rose, greenness, yellowing, withering, falling and the like are caused; can also lead to branches to dry, plants to be short or diapause, fruits to be weak or malformed, flowers to fall off and fruits to fall off, and even lead to death of the whole plant when the damage is serious. Since 1995, it was reported by san Jose et al that papaya gecko hazard has spread to at least 40 countries or regions in central america, north america, pacific, africa, asia, etc., with serious losses to the papaya industry. Papaya gecko now also invades into China, and brings great potential threat to papaya and flowers in China.
The research on the papaya gecko mainly focuses on morphological characteristics, biochemical mechanism, medicament prevention and treatment and the like. Chen Qing et al (2018) determined the difference in enzyme activity in the mealybugs under different temperature incubation conditions and found that both high and low temperatures induced an increase in the protective enzyme activity of mealybugs. Wang Yaru et al (2018) found that the activity of protective enzymes in the body of the papaya gecko eating cassava was significantly reduced and the feeding capacity was also reduced. Chen Qian et al (2020) found that the feeding of some cassava varieties by Gecko was able to inhibit the antioxidant enzyme activity in vivo. The research on LGMN gene expression characteristics of the gecko LGMN fed by different hosts is not reported at home and abroad.
LGMN encodes an aspartic endopeptidase (Asparaginyl Endopeptidase, AEP) with cysteine protease activity, capable of specifically binding asparagine, and is closely associated with part of neurological disorders. LGMN has high expression levels in antigen presenting cells, dendritic cells and B cells. LGMN is a gene related to stress and immunity, and has a direct effect on proliferation and differentiation of cells. After rats were exposed to high oxygen for a period of time, hinkelben et al (2015) found down-regulation of LGMN protein expression. Anders et al (2011) found that LGMN gene was able to specifically inhibit cleavage inactivation of autophagy-related enzyme BHMT. Morito et al (2007) found that LGMN was highly expressed in mouse tubular cells, and further found that LGMN protein could directly degrade fibronectin, one of the major components of extracellular matrix in vitro. Shirahama-Noda et al (2003) found that LGMN plays a key role in the degradation system of lysosomes. Degradation of fibronectin occurs on lysosomes in the involvement of LGMN proteins, and it is speculated that LGMN may play an important role in the reconstitution of extracellular matrix by degradation of proximal tubule extracellular fibronectin. Numerous studies have demonstrated that LGMN is associated with proliferation and differentiation of cells, inhibits cleavage inactivation of autophagy-related enzymes, and is more capable of directly degrading fibronectin in vitro, demonstrating that LGMN can promote autophagy activity of cells.
The research on the expression of the LGMN gene of the gecko in papaya is not reported at home and abroad, so that the real-time fluorescence quantitative PCR is adopted to research the expression characteristics of the LGMN gene of the gecko in papaya under different host treatments, and the result shows that the expression quantity of the LGMN gene is different from different hosts and consistent with the variation trend of the expression quantity of a transcriptome, thereby indicating that the conversion of the hosts can influence the expression regulation of the LGMN gene. If some target genes are used as targets for pest control, a new field of pest molecular regulation can be opened up, and a new method and thinking are provided for controlling agricultural pests.
Disclosure of Invention
The invention aims to provide a primer and a method for detecting the transcription level of the LGMN gene of the gecko, and different host treatments designed for comprehensively knowing the transcription level of the LGMN gene of the gecko.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
papaya geckos feeding papaya and potato two different hosts are respectively picked into an Eppendorf tube for 20, and are divided into 3 biological replicates, and 6 groups of samples are obtained.
Designing a pair of primers for detecting the LGMN gene transcription level of the gecko, wherein the primers comprise an upstream primer and a downstream primer which accord with the fluorescent quantitative PCR reaction characteristics:
LGMN-F：5`- GCTACCATATCCTCCACCAC-3`，
LGMN-R：5`- TTTTCTCTTCAGCATTCAAC-3`；
upstream and downstream primers of reference gene Tubulin:
Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`
Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`。
the fluorescent quantitative PCR method for detecting the LGMN gene transcription level of the gecko with papaya disclosed by the invention is carried out according to the following steps:
(1) First strand cDNA Synthesis: extracting and purifying total RNA of a sample, and obtaining cDNA (complementary deoxyribonucleic acid) by reverse transcription by using the extracted RNA as a template by adopting a reverse transcription kit;
(2) Conventional PCR assays were performed on the following primers:
fluorescent quantitative upstream and downstream primers of the LGMN gene of gecko:
LGMN-F：5`- GCTACCATATCCTCCACCAC-3`，
LGMN-R：5`- TTTTCTCTTCAGCATTCAAC-3`；
upstream and downstream primers of reference gene Tubulin:
Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`，
Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`；
the total volume of a conventional PCR amplification system is 20 [ mu ] L:2X Taq Plus Master Mix 10 [ mu ] L, an upstream primer 0.8 [ mu ] L (5 uM), a downstream primer 0.8 [ mu ] L (5 uM), 100 ng/[ mu ] L cDNA template 1.0 [ mu ] L and water 7.4 [ mu ] L;
the conventional PCR reaction procedure is: pre-denaturing at 94 ℃ for 5 min, then denaturing at 95 ℃ for 30 s, annealing at 50 ℃ for 30 s, extending at 72 ℃ for 1 min, 35 cycles under this condition, and finally extending at 72 ℃ for 10min;
(3) Real-time fluorescent quantitative PCR reaction:
taking the cDNA obtained in the step (1) as a template, adding the primer in the step (2), performing fluorescent quantitative PCR reaction, setting 3 repetitions for each sample, and taking an average value after amplification.
The real-time fluorescent quantitative PCR amplification system comprises: 2X ChamQ SYBR Color qPCR Master Mix 10 [ mu ] L, an upstream primer 0.8 [ mu ] L (5 uM), a downstream primer 0.8 [ mu ] L (5 uM), 50X ROX Reference Dye 2 0.4 [ mu ] L,100 ng/[ mu ] L cDNA 2.0 [ mu ] L and water to 20 [ mu ] L are supplemented;
the real-time fluorescence quantitative PCR reaction procedure is: pre-denaturation at 95℃for 5 min, then denaturation at 95℃for 5 s, annealing at 50℃for 30 s, extension at 72℃for 40 s,40 cycles.
The more detailed test method of the invention is as follows:
the real-time fluorescence quantitative PCR detection method of the LGMN gene of the gecko of the Chinese flowering quince can be realized by the following steps:
(1) Primer design: according to the sequence of the LGMN gene of the gecko obtained by the transcriptome sequencing result, a DNAMAN software is used for designing a specific primer suitable for fluorescent quantitative PCR detection, and the primer sequence is as follows:
LGMN-F：5`- GCTACCATATCCTCCACCAC-3`，
LGMN-R：5`- TTTTCTCTTCAGCATTCAAC-3`。
meanwhile, according to the sequence of the papaya gecko tubulin gene obtained by a transcriptome sequencing result, a primer for a fluorescent quantitative PCR internal control is designed, wherein the primer sequence is as follows:
Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`
Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`
(2) Papaya gecko treatment test: respectively culturing papaya seedlings and potato seedlings in an artificial climate box, and feeding papaya geckos, wherein 20 papaya geckos are respectively collected in Eppendorf tubes, and the papaya geckos are divided into 3 biological repetition groups, and 6 groups of samples are obtained. After the insect sample is treated, the insect sample is quickly placed into liquid nitrogen for fixation and is preserved at the temperature of minus 80 ℃ for standby.
(3) First strand cDNA Synthesis: with reference to the whole gold companyTransZol TM Up Plus RNA Kit protocol Total RNA was extracted and cDNA first strand was synthesized according to the HiScript Q RT SuperMix for qPCR (+gDNA wind) Kit protocol from Novain.
(4) Real-time fluorescent quantitative PCR reaction: real-time fluorescent quantitative PCR was performed using the ChamQ SYBR Color qPCR Master Mix (2X) kit from Nanjinopran Biotechnology Co., ltd. Using the first strand of the synthesized cDNA as a template, using the LGMN-F, LGMN-R and Tubulin-F, tubulin-R as specific primers, performing a fluorescent quantitative PCR procedure, setting 3 parallel repeats for each sample, and taking the average of the obtained parallel Ct values after amplification.
The real-time fluorescent quantitative PCR amplification system comprises: 2X ChamQ SYBR Color qPCR Master Mix 10 [ mu ] L, an upstream primer 0.8 [ mu ] L (5 uM), a downstream primer 0.8 [ mu ] L (5 uM), 50X ROX Reference Dye 2 0.4 [ mu ] L,100 ng/[ mu ] L cDNA 2.0 [ mu ] L and water to 20 [ mu ] L are supplemented;
the real-time fluorescence quantitative PCR reaction procedure is: pre-denaturation at 95℃for 5 min, then denaturation at 95℃for 5 s, annealing at 50℃for 30 s, extension at 72℃for 40 s,40 cycles.
(5) The expression quantity of the LGMN gene of the gecko of the papaya relative to the tubulin gene is calculated by the following formula: relative mRNA expression = 2 -ΔΔCt X 100%, where Ct value = target gene Ct value-tubulin Ct value.
(6) FIG. 3 shows the differential expression of LGMN gene from Gecko feeding different hosts. The result shows that the expression quantity of LGMN gene is different due to different hosts and is consistent with the variation trend of the expression quantity of transcriptome, thus providing important basic data for developing the research of LGMN gene of Gecko in depth.
Compared with the prior art, the invention has the following advantages and effects:
(1) The different host treatment measures in the invention are applicable to most of the LGMN genes of insects, and have important significance for comprehensively and deeply researching the gene expression characteristics.
(2) The efficient and rapid fluorescent quantitative PCR method comprises a fluorescent quantitative PCR program and the like, is used for 50 minutes practically, and has the characteristics of being efficient and rapid, and compared with the prior art, the reaction time is greatly shortened.
(3) The fluorescent quantitative PCR method provides a conventional PCR electrophoresis result diagram, and can intuitively and rapidly reflect the specificity of the primer.
Drawings
The attached drawing shows the fluorescent quantitative PCR detection result of the LGMN gene transcription level of the gecko.
Fig. 1: electrophoresis results of conventional PCR products of fluorescent quantitative primers of LGMN gene of gecko: the product length is 142 bp, and the Marker strips are from top to bottom: 2000. 1000, 750, 500, 250, 100bp.
Fig. 2: conventional PCR product electrophoresis result of fluorescent quantitative primers of gecko tubulin genes: the product length is 202 bp, and the Marker strips are sequentially from top to bottom: 2000. 1000, 750, 500, 250, 100bp.
Fig. 3: the LGMN gene differential expression after the papaya gecko feeds different hosts, the columnar and left coordinate axes are the fluorescent quantitative expression quantity, the scattered point and right coordinate axes are the transcriptome expression quantity, MF6 represents the papaya gecko feeding papaya, and TF6 represents the papaya gecko feeding potato.
Detailed description of the preferred embodiments
The present invention is described below with reference to specific embodiments and drawings, but the embodiments are not limited to the invention, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the invention should be made in the equivalent manner, and the modifications, substitutions, and the embodiments are included in the protection scope of the present invention.
Example 1
Treatment of test materials
Respectively culturing papaya seedlings and potato seedlings in an artificial climate box, wherein the papaya seedlings and the potato seedlings are used for raising papaya geckos, 20 papaya geckos are respectively picked in Eppendorf tubes by different hosts, and the papaya geckos are divided into 3 biological repetition groups, and 6 groups of samples are obtained. Collecting insect sample, rapidly fixing in liquid nitrogen, and storing at-80deg.C.
Example 2
Primer design
(1) Primer design: according to the sequence of the LGMN gene of the gecko obtained by transcriptome sequencing, a DNAMAN software is used for designing a specific primer suitable for fluorescent quantitative PCR detection, and the primer sequence is as follows:
LGMN-F：5`- GCTACCATATCCTCCACCAC-3`，
LGMN-R：5`- TTTTCTCTTCAGCATTCAAC-3`。
the length of the fluorescent quantitative PCR product of the LGMN gene of the gecko is 142 bp, the agarose gel electrophoresis result shows that the amplified product is consistent in length and is a single strip, which indicates that the designed primer has strong specificity and is suitable for real-time fluorescent quantitative PCR detection, and the agarose gel electrophoresis result is shown in figure 1.
(2) Meanwhile, according to the sequence of the palaemon carica papaya tubulin gene obtained by transcriptome sequencing, a primer for fluorescent quantitative PCR internal control is designed, wherein the primer sequence is as follows:
Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`
Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`
the length of the fluorescent quantitative PCR product of the gecko tubulin gene is 202 bp, the agarose gel electrophoresis result shows that the amplified product has consistent length and is a single band, which indicates that the designed primer has strong specificity and is suitable for real-time fluorescent quantitative PCR detection, and the agarose gel electrophoresis result is shown in figure 2.
Example 3
Extraction of Total RNA
With reference to the whole gold companyTransZol TM Total RNA was extracted from the Up Plus RNA Kit instructions, the worms were ground to a powder with liquid nitrogen, and 1ml was addedTransZol TM Up, transferring to a 1.5 ml centrifuge tube, standing at room temperature for 5 min, adding 200 μl chloroform/1 mlTransZol TM Up, vigorously shake 30 s, incubate for 3 min at room temperature. After centrifugation at 10000 g for 15 min at 4℃the upper aqueous phase was removed (generally<80%) in a new centrifuge tube, add 1/3 volume of absolute ethanol and gently mix upside down. And sleeving the RNA centrifugal column in a 2 ml collecting pipe, transferring all the mixture obtained in the previous step into the centrifugal column, centrifuging for 12000 g and 30-60 s, discarding the mobile phase, and reusing the collecting pipe. 500. Mu. LCB9 was added, centrifuged at 12000 g at room temperature for 30 s, the mobile phase was discarded, 500. Mu. LCB9 was added, centrifuged at 12000 g at room temperature for 30 s, and the mobile phase was discarded. Diluted 500 μLWB9, 12000 g was added to centrifuge 30 s, the mobile phase was discarded, diluted 500 μLWB9, 12000 g was added to centrifuge 30 s, and the mobile phase was discarded. 12000 g, centrifuging for 2 min, thoroughly removing residual ethanol, standing for several minutes at room temperature, and thoroughly airing the centrifugal column. The column was put into RNase-free Tube, and 50. Mu. LRNase-free Wa was addedthe ter is positioned at the center of the centrifugal column, and is kept stand at room temperature for 1 min, and 12000 g is centrifuged for 1 min to elute RNA. The RNA obtained was placed at-80℃for further use.
Example 4
First strand cDNA Synthesis: the first strand of cDNA was reverse transcribed using the RNA of example 3 as a template according to the procedure of HiScript Q RT SuperMix for qPCR (+gDNA wind) kit instructions from Norpraise, as follows:
(1) Genomic DNA removal reaction.
(2) Preparing a reverse transcription reaction system.
(3) Carrying out reverse transcription reaction, incubating at 50deg.C for 15 min, heating at 85deg.C for 2 min, and storing at-20deg.C and-80deg.C.
Example 5
Fluorescent quantitative PCR reactions were performed. Real-time fluorescent quantitative PCR was performed using the ChamQ SYBR Color qPCR Master Mix (2X) kit from Nanjinopran Biotechnology Co., ltd. The cDNA in example 4 was used as a template, and the primers in example 2 were used to perform a real-time fluorescent quantitative PCR amplification reaction with 3 replicates per sample, and the average of the parallel Ct values obtained after amplification was taken.
(1) The real-time fluorescent quantitative PCR amplification system is as follows:
the real-time fluorescent quantitative PCR amplification system comprises: 2X ChamQ SYBR Color qPCR Master Mix 10 [ mu ] L, an upstream primer 0.8 [ mu ] L (5 uM), a downstream primer 0.8 [ mu ] L (5 uM), 50X ROX Reference Dye 2 0.4 [ mu ] L,100 ng/[ mu ] L cDNA 2.0 [ mu ] L and water to 20 [ mu ] L are supplemented;
(2) The real-time fluorescence quantitative PCR reaction procedure is: pre-denaturation at 95℃for 5 min, then denaturation at 95℃for 5 s, annealing at 50℃for 30 s, extension at 72℃for 40 s,40 cycles.
(3) After the real-time fluorescent quantitative PCR is completedCalculating the ratio 2 of the relative expression under different processing conditions according to Ct values -ΔΔCt . The expression quantity of the LGMN gene of the gecko of the papaya relative to the tubulin gene is calculated by the following formula: relative mRNA expression = 2 -ΔΔCt X 100%, where Ct value = target gene Ct value-tubulin Ct value.
Table 1: c (T) value, average value, standard deviation, fluorescence quantitative expression quantity and transcriptome expression quantity of LGMN gene after feeding different hosts by Chinese flowering quince mealybugs
Note that: MF6 represents papaya gecko eating papaya, TF6 represents papaya gecko eating potato.
(4) FIG. 3 shows the differential expression of LGMN gene from Gecko feeding different hosts. The results showed that the expression level of LGMN gene was different depending on the host, and was consistent with the trend of the expression level of transcriptome. This provides important basic data for the deep research of the LGMN gene of the gecko of Chinese flowering quince.
Claims (2)
1. A fluorescent quantitative PCR primer for detecting the LGMN gene transcription level of gecko, which is characterized in that: the method comprises the following steps of:
LGMN-F：5`- GCTACCATATCCTCCACCAC-3`，
LGMN-R：5`- TTTTCTCTTCAGCATTCAAC-3`。
2. a fluorescent quantitative PCR method for detecting the LGMN gene transcript level of gecko by using the fluorescent quantitative PCR primer according to claim 1, wherein: the method comprises the following steps:
(1) First strand cDNA Synthesis: extracting and purifying total RNA of a sample, and obtaining cDNA (complementary deoxyribonucleic acid) by reverse transcription by using the extracted RNA as a template by adopting a reverse transcription kit;
(2) Conventional PCR assays were performed with the following primers:
fluorescent quantitative upstream and downstream primers of the LGMN gene of gecko:
LGMN-F：5`- GCTACCATATCCTCCACCAC-3`，
LGMN-R：5`- TTTTCTCTTCAGCATTCAAC-3`；
upstream and downstream primers of reference gene Tubulin:
Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`，
Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`；
the total volume of a conventional PCR amplification system is 20 [ mu ] L:2X Taq Plus Master Mix 10 [ mu ] L,5uM upstream primer 0.8 [ mu ] L,5uM downstream primer 0.8 [ mu ] L,100 ng/[ mu ] L cDNA template 1.0 [ mu ] L and water 7.4 [ mu ] L;
the conventional PCR reaction procedure is: pre-denaturing at 94 ℃ for 5 min, then denaturing at 95 ℃ for 30 s, annealing at 50 ℃ for 30 s, extending at 72 ℃ for 1 min, 35 cycles under this condition, and finally extending at 72 ℃ for 10min;
(3) Real-time fluorescent quantitative PCR reaction:
taking the cDNA obtained in the step (1) as a template, adding the primer in the step (2), performing fluorescent quantitative PCR reaction, setting 3 repetitions for each sample, and taking an average value after amplification;
the real-time fluorescent quantitative PCR amplification system comprises: 2X ChamQ SYBR Color qPCR Master Mix 10 [ mu ] L,5uM upstream primer 0.8 [ mu ] L,5uM downstream primer 0.8 [ mu ] L, 50X ROX Reference Dye 2 0.4 [ mu ] L,100 ng/[ mu ] L cDNA 2.0 [ mu ] L and water is supplemented to 20 [ mu ] L;
the real-time fluorescence quantitative PCR reaction procedure is: pre-denaturation at 95℃for 5 min, then denaturation at 95℃for 5 s, annealing at 50℃for 30 s, elongation at 72℃for 40 s,40 cycles.
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| US20140039013A1 (en) * | 2010-10-25 | 2014-02-06 | Laurence Zwiebel | Composition for Inhibition of Insect Host Sensing |
| CN109609664A (en) * | 2019-02-03 | 2019-04-12 | 福建省农业科学院农业质量标准与检测技术研究所 | For identifying the specific primer and its detection method of pawpaw show mealybug |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20140039013A1 (en) * | 2010-10-25 | 2014-02-06 | Laurence Zwiebel | Composition for Inhibition of Insect Host Sensing |
| CN109609664A (en) * | 2019-02-03 | 2019-04-12 | 福建省农业科学院农业质量标准与检测技术研究所 | For identifying the specific primer and its detection method of pawpaw show mealybug |
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| ZHENG, LIZHEN等: "De Novo Transcription Responses Describe Host-Related Differentiation of Paracoccus marginatus (Hemiptera: Pseudococcidae)", INSECTS, vol. 13, no. 9, 19 September 2022 (2022-09-19), pages 1 - 18 * |
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