CN115820820A - Primer and method for detecting LAMAN gene transcription level of caraway elegans - Google Patents

Abstract

The invention provides a primer and a method for detecting the transcriptional level of the LAMAN gene of the Lecanicillium papaya Lecaninum, wherein the sequence of the primer is shown as SEQ ID NO. 1-2.

Description

Primer and method for detecting LAMAN gene transcription level of caracoccus elegans

Technical Field

The invention relates to a primer and a method for detecting the LAMAN gene transcription level of the lecanicillium elegans, belonging to the technical field of biology.

Background

The Carica papaya Lecanicillium lecanii belongs to the hemiptera, the Lecanicillium lecanii superfamily, the Lecanicillium lecanii genus of the Lecanicillium lecanii family. The host range is wide, and the insect is an important potential economic insect with strong diffusivity, strong vitality and strong fecundity. The insect was generally thought to be native to Mexico or Central America, and the first specimen of the papaya Lecanicillium was collected in Mexico in 1955. The pawpaw and mealybug lives in hidden positions such as leaf veins of leaves, growing points of stems and leaves, concave-convex or overlapped parts of flowers and leaves, depressions or furrows on the surfaces of fruits and the like. Nymphs and female adults mainly prick and suck the juice of organs and tissues such as stems, leaves, flowers and fruits of host plants, so that leaf curling and malformation, rosettes, greenness and yellowing, withering and shedding and the like are caused; it can also cause dry branches, short or diapause plants, weak or abnormal fruits, flower and fruit drop, and even death of the whole plant when the damage is serious. Since the report of the hazard of the carica papaya fargecko in santa martensii 1995, it has now spread to at least 40 countries or regions in central america, north america, the pacific, africa, asia, etc., with serious losses to the papaya industry. The papaya mealybugs also invade China, and great potential threats are brought to the papaya and flower industries in China.

The research on the caraway chaenomeles elegans mainly focuses on morphological characteristics, biochemical mechanism, medicament prevention and treatment and the like. Chen Qing and the like (2018) determine the activity difference of enzymes in the bodies of the eleckoes chaenomeles sinensis under different temperature culture conditions, and the results show that the activity of the protective enzymes of the eleckoes chaenomeles sinensis can be improved under the induction of high temperature and low temperature. Wang Yaru et al (2018) found that the activity of protective enzymes in bodies of pawpaw and mealybugs eating cassava is remarkably reduced, and the eating ability is also reduced. Chen Qian et al (2020) found that eating some cassava varieties, papaya, lecanicillium elegans, inhibited antioxidant enzyme activity in vivo. The research on LAMAN gene expression characteristics of the papaya mealybug bred by different hosts is not reported at home and abroad.

LAMAN, also known as Lysosomal α -D-mannosidase (LAMAN, EC 3.2.1.24), is a key enzyme for N-glycan modification of eukaryotic proteins, and its trimming process for mannose residues is an essential step that complicates the N-sugar chain of glycoproteins, and is crucial for protein synthesis and folding of the correct conformation. LAMAN maps to chromosome 19, spanning 21.5 kb genomic DNA (gDNA), containing 24 exons, and cDNA containing 1 reading frame of 2964 bp (Wu Xiaoyun, 2013). The transcription start sites were mainly 3, located upstream of the start codon-309 bp, -196 bp and-191 bp. In the sequence upstream of the initiation site, there is no characterized CAAT or TATA sequence, but the 5' end region contains multiple GC-rich sites to which transcription factors can bind (Riise, 1997).

The alpha-mannosidase encoded by the LAMAN gene mostly has a conserved sequence of glycoside hydrolase 38, 47 family. At present, the enzyme is divided into three categories, namely, category I, category II and category No. (Liu Gongxia, 2006) by the difference of gene conserved sequences.

Class ii α -mannosidases have conserved sequences of glycoside hydrolase 38 family (Cobucci-ponzanno, 2010), are mainly distributed in lysosomes, golgi bodies, endoplasmic reticulum, cytoplasm, and the like, and participate in synthesis and degradation of glycoproteins (Hossain, 2010, shashidhara, uno, 2010. LAMAN is the most common lysosomal acid α -mannosidase, and abnormalities in this enzyme cause anemia of congenital dyserythropoiesis and the like (Wang Shanshan, 2012).

In biochemical metabolic pathways, α -mannosidases are primarily involved in the modification of glycoprotein glycan hydrolysis and in the modification of protein glycosylation. Glycosylation and glycoprotein glycan hydrolysis are closely related to folding, maturation, transport, half-life and biological activity of nascent glycoproteins, and may play a role in cell adhesion, hormonal activity, immune surveillance, etc. (Moremen, 2002). When LAMAN is abnormal, oligosaccharide metabolism is disordered, glycoprotein hydrolysis cannot be carried out, oligosaccharide is accumulated in lysosomes, extensive vacuolar degeneration occurs, and the symptoms are that the development of nerve skeleton is incomplete, the liver and spleen are enlarged, and the like. LAMAN deficiency can lead to accumulation of oligomers in various organs, systems, particularly mannose-rich oligosaccharides, in lysosomes in cells, causing lysosome swelling, which in turn can cause severe functional impairment of the cells.

The research on the expression of the LAMAN gene of the Lecanicillium papaya is not reported at home and abroad, so that the expression characteristics of the LAMAN gene of the Lecanicillium papaya processed by different hosts are researched by adopting real-time fluorescent quantitative PCR (polymerase chain reaction), and the result shows that the expression quantity of the LAMAN gene is different in different hosts and is consistent with the change trend of the expression quantity of a transcriptome, and the host transformation can influence the expression regulation and control of the LAMAN gene. If some target genes are used as targets for pest control, a new field of pest molecule regulation can be developed, and a new method and a new thought are provided for prevention and control of agricultural pests.

Disclosure of Invention

The invention aims to provide primers and a method for detecting the transcriptional level of the LAMAN gene of the Lecanicillium papaya and process different hosts designed for comprehensively understanding the transcriptional level of the LAMAN gene of the Lecanicillium papaya.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

20 pawpaw mealybugs eating two different hosts of pawpaw and potato are respectively collected into Eppendorf tubes and divided into 3 biological replicates, and 6 groups of samples are obtained.

A pair of primers for detecting the transcription level of the LAMAN gene of the Lecanicillium elegans is designed, and the primers comprise an upstream primer and a downstream primer which accord with the characteristics of fluorescent quantitative PCR reaction:

upstream and downstream primers for LAMAN gene:

LAMAN-F：5`- CTTGGTTCGGCAAAGGAGCA-3`，

LAMAN-R：5`- GATTGGATGTGGGGAGGGTA-3`；

upstream and downstream primers of internal reference Tubulin gene:

Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`，

Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`。

the invention discloses a fluorescent quantitative PCR method for detecting the LAMAN gene transcription level of the Carica elegans Lecanicilli, which comprises the following steps:

(1) First strand cDNA Synthesis: extracting and purifying total RNA of a sample, and carrying out reverse transcription by using the extracted RNA as a template by adopting a reverse transcription kit to obtain cDNA;

(2) The following primers are used for carrying out conventional PCR detection on the LAMAN gene of the Carica elegans

Fluorescent quantitative upstream and downstream primers of the gene LAMAN of the Carica elegans Lecanicillium:

LAMAN-F：5`- CTTGGTTCGGCAAAGGAGCA-3`，

LAMAN-R：5`- GATTGGATGTGGGGAGGGTA-3`；

upstream and downstream primers of internal reference Tubulin gene:

Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`，

Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`；

the total volume of the conventional PCR amplification system is 20 muL: 2X Taq Plus Master Mix 10 muL, upstream primer 0.8 muL (5 uM), downstream primer 0.8 muL (5 uM), 100 ng/muL cDNA template 1.0 muL and water 7.4 muL;

the conventional PCR reaction procedure was: pre-denaturation at 94 ℃ for 5 min, denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 1 min, wherein 35 cycles are performed under the condition, and finally extension is performed at 72 ℃ for 10min;

(3) Real-time fluorescent quantitative PCR reaction:

adding the primers obtained in the step (2) into the cDNA obtained in the step (1) as a template to perform fluorescent quantitative PCR reaction, setting 3 times of repetition for each sample, and taking an average value after amplification;

the real-time fluorescent quantitative PCR amplification system is as follows: 2X ChamQ SYBR Color qPCR Master Mix 10 muL, upstream primer 0.8 muL (5 uM), downstream primer 0.8 muL (5 uM), 50X ROX Reference Dye 2.4 muL, 100 ng/muL cDNA 2.0 muL, and water is replenished to 20 muL;

the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min, followed by denaturation at 95 ℃ for 5 s, annealing at 55 ℃ 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 fluorescent quantitative PCR detection method of the LAMAN gene of the Carica elegans can be realized by the following steps:

(1) Designing a primer: according to the sequence of the LAMAN gene of the Lecanicillium lecanii obtained by sequencing the transcriptome, DNAMAN software is used for designing a specific primer suitable for fluorescent quantitative PCR detection, and the primer sequence is as follows:

LAMAN-F：5`- CTTGGTTCGGCAAAGGAGCA-3`，

LAMAN-R：5`- GATTGGATGTGGGGAGGGTA-3`；

meanwhile, according to the sequence of the gene of the Lecanicillium elegans Tubulin obtained by the sequencing result of the transcriptome, a primer for a control Tubulin in the fluorescent quantitative PCR is designed, and the sequence of the primer is as follows:

Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`，

Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`。

(2) Pawpaw and mealybug treatment test: the method comprises the steps of respectively cultivating papaya seedlings and potato seedlings in an artificial climate box for breeding the pawpaw mealybugs, respectively taking 20 pawpaw mealybugs fed with different hosts into Eppendorf tubes, and dividing the pawpaw mealybugs into 3 biological repeated samples, wherein the total number of the biological repeated samples is 6. After being treated, the insect sample is quickly put into liquid nitrogen for fixation and is stored at minus 80 ℃ for standby.

(3) First strand cDNA Synthesis: with reference to the whole formula of goldTransZol ^TM Total RNA was extracted using Up Plus RNA Kit, and first strand cDNA was synthesized according to HiScript Q RT Supermix for qPCR (+ gDNA wiper) Kit of Novomedium.

(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 Biotech Inc., nanjing Novozam. And (3) performing a fluorescent quantitative PCR program by using the synthesized first strand of the cDNA as a template and the LAMAN-F, LAMAN-R and the Tubulin-F, tubulin-R as specific primers, setting 3 parallel repeats for each sample, and taking the average of the parallel Ct values after amplification.

The real-time fluorescent quantitative PCR amplification system is as follows: 2X ChamQ SYBR Color qPCR Master Mix 10 muL, upstream primer 0.8 muL (5 uM), downstream primer 0.8 muL (5 uM), 50X ROX Reference Dye 2.4 muL, 100 ng/muL cDNA 2.0 muL, and water is replenished to 20 muL;

the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min, followed by denaturation at 95 ℃ for 5 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 40 s,40 cycles.

(5) The expression of the diaphora elegans LAMAN gene relative to tubulin gene is calculated as follows: 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 LAMAN gene in Carica papaya and Lecanicillium lecanii eating different hosts. The result shows that the expression level of the LAMAN gene is different due to different hosts and is consistent with the expression level change trend of a transcriptome, which provides important basic data for deeply developing the research of the LAMAN gene of the Carica papaya mealybug.

Compared with the prior art, the invention has the following advantages and effects:

(1) The different host processing measures in the invention are suitable for most of the LAMAN genes of insects, which has important significance for the comprehensive and deep research of gene expression characteristics.

(2) The efficient and fast fluorescence quantitative PCR method comprises a fluorescence quantitative PCR program and the like, is actually used for 50 min, greatly shortens the reaction time compared with the prior art, and has the characteristics of high efficiency and fast speed.

(3) The fluorescence quantitative PCR method provided by the invention provides a conventional PCR electrophoresis result picture, and can intuitively and quickly reflect the specificity of the primer.

Drawings

FIG. 1: the conventional PCR product electrophoresis result of the fluorescent quantitative primer for the LAMAN gene of the Carica elegans: the length of the product is 162 bp, and the marker bands are sequentially from top to bottom: 2000. 1000, 750, 500, 250 and 100bp. The lanes are sequentially Marker, LAMAN gene and Marker from left to right.

FIG. 2: electrophoresis results of conventional PCR products of fluorescent quantitative primers of the chaenomeles elegans tubululin gene are as follows: the length of the product is 202 bp, and the marker bands are sequentially from top to bottom: 2000. 1000, 750, 500, 250 and 100bp. The lanes from left to right are Marker, tubulin gene, marker.

FIG. 3: the differential expression of LAMAN gene of the Carica papaya, namely the Lecanicillium elegans after eating different hosts, is realized by taking the column axis and the left axis as the expression quantity of fluorescence quantification and taking the scatter point axis and the right axis as the expression quantity of transcriptome. MF indicates eating pawpaw, and TF indicates eating potato.

Detailed description of the preferred embodiments

The present invention is described below with reference to the following embodiments and the accompanying drawings, wherein the embodiments are not limited to the invention, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be regarded as equivalent replacements within the scope of the present invention.

Example 1

Treatment of test materials

Pawpaw seedlings and potato seedlings are respectively cultivated in an artificial climate box and used for feeding pawpaw mealybugs, 20 pawpaw mealybugs are respectively picked from different hosts and put into an Eppendorf tube, and the two pawpaw mealybugs are divided into 3 biological replicates, and 6 groups of samples are obtained. The insect sample is collected and then quickly placed into liquid nitrogen for fixation, and is stored at the temperature of minus 80 ℃ for standby.

Example 2

Design of primers

(1) Designing a primer: according to the sequence of the LAMAN gene of the Lecanicillium elegans obtained by transcriptome sequencing, DNAMAN software is used for designing a specific primer suitable for fluorescent quantitative PCR detection, and the primer sequence is as follows:

LAMAN-F：5`- CTTGGTTCGGCAAAGGAGCA-3`，

LAMAN-R：5`- GATTGGATGTGGGGAGGGTA-3`。

the length of the fluorescent quantitative PCR product of the LAMAN gene of the eleicerus elegans is 162 bp, and the agarose gel electrophoresis result shows that the amplified products have consistent length and are single strips, 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 gene of the lecanicillium elegans tubululin obtained by transcriptome sequencing, primers for a fluorescent quantitative PCR internal control are designed, and the sequences of the primers are as follows:

Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`，

Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`。

the length of the fluorescent quantitative PCR product of the gene of the chaenomeles elegans tubululin is 202 bp, and the agarose gel electrophoresis result shows that the amplified products have consistent length and are single strips, 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 body of goldTransZol ^TM Up Plus RNA Kit instructions for total RNA extraction, liquid nitrogen grinding the worm body into powder, adding 1mlTransZol ^TM Up, transferred to a 1.5 ml centrifuge tube, left to stand at room temperature for 5 min, and added with 200. Mu.L chloroform/1 mlTransZol ^TM Up, 30 s were shaken vigorously and incubated at room temperature for 3 min. Centrifuging at 4 deg.C 10000 g for 15 min, and removing the upper aqueous phase (generally<80%) into a new centrifuge tube, add 1/3 volume of absolute ethanol, mix by gentle inversion. Sheathing the RNA centrifugal column in a 2 ml collecting pipe, transferring the mixture into the centrifugal column, centrifuging at 12000 g for 30-60 s, discarding the mobile phase, and reusing the collecting pipe. 500 μ LCB9 was added, room temperature 12000 g centrifuged 30 s was discarded, and 500 μ LCB9 was added, room temperature 12000 g centrifuged 30 s was discarded, and the mobile phase was discarded. Diluted 500 μ LWB9, 12000 g centrifuged 30 s was added and mobile phase discarded, and diluted 500 μ LWB9, 12000 g centrifuged 30 s was added and mobile phase discarded. 12000 g, centrifuging for 2 min, completely removing residual ethanol, standing at room temperature for several minutes, and completely airing the centrifugal column. The column was placed in an RNase-free Tube, 50. Mu.L Nase-free Water was added to the center of the column, and the column was left standing at room temperature for 1 min, centrifuged at 12000 g for 1 min, and RNA was eluted. The RNA obtained was stored at-80 ℃ until use.

Example 4

First strand cDNA Synthesis: reverse transcription was performed to generate the first strand of cDNA using the RNA of example 3 as a template according to the procedures of HiScript Q RT Supermix for qPCR (+ gDNA wiper) kit of Novomedium, and the specific steps were as follows:

(1) Reaction for removing genomic DNA

(2) Preparing a reverse transcription reaction system.

(3) Performing reverse transcription reaction, incubating at 50 deg.C for 15 min, heating at 85 deg.C for 2 min, and storing at-20 deg.C and-80 deg.C.

Example 5

And carrying out fluorescent quantitative PCR reaction. Real-time fluorescent quantitative PCR was performed using the ChamQ SYBR Color qPCR Master Mix (2X) kit from Biotech Inc., nanjing Novozam. Real-time fluorescent quantitative PCR amplification reaction was performed using the cDNA of example 4 as a template and the primers of example 2, 3 replicates were taken for each sample, and the average of parallel Ct values obtained after amplification was taken.

(1) The real-time fluorescent quantitative PCR amplification system comprises the following components:

the real-time fluorescent quantitative PCR amplification system is as follows: 2X ChamQ SYBR Color qPCR Master Mix 10 muL, upstream primer 0.8 muL (5 muM), downstream primer 0.8 muL (5 muM), 50X ROX Reference Dye 2.4 muL, 100 ng/muL cDNA 2.0 muL, and water is replenished to 20 muL;

(2) The real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min, followed by denaturation at 95 ℃ for 5 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 40 s,40 cycles.

(3) After the real-time fluorescent quantitative PCR is finished, calculating the ratio 2 of relative expression amounts under different treatment conditions according to the Ct value ^-ΔΔCt . The expression of the diaphora elegans LAMAN gene relative to tubulin gene is calculated as follows: 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 LAMAN gene of pawpaw and mealybug eating different hosts (MF represents eating pawpaw, TF represents eating potato)

(4) FIG. 3 shows the differential expression of LAMAN gene in Carica papaya Lecanicillium caudatum after feeding on different hosts. The results showed that the expression level of LAMAN gene was different depending on the host, and was consistent with the tendency of change in the expression level of transcriptome. The method provides important basic data for deeply developing the research on the LAMAN gene of the lecanium elegans.

Claims (2)

1. A fluorescent quantitative PCR primer for detecting the LAMAN gene transcription level of the eleicerus elegans is characterized in that: comprises an upstream primer and a downstream primer which accord with the characteristics of fluorescent quantitative PCR reaction:

LAMAN-F：5`- CTTGGTTCGGCAAAGGAGCA-3`，

LAMAN-R：5`- GATTGGATGTGGGGAGGGTA-3`。

2. a fluorescent quantitative PCR method for detecting the transcription level of the LAMAN gene of elecoccus chachii by using the fluorescent quantitative PCR primer of claim 1, characterized in that: the method comprises the following steps:

(1) First strand cDNA Synthesis: extracting and purifying total RNA of a sample, and carrying out reverse transcription by adopting a reverse transcription kit by taking the extracted RNA as a template to obtain cDNA;

(2) The following primers were used for conventional PCR detection of the LAMAN gene of Carica elegans:

fluorescent quantitative upstream and downstream primers of the gene LAMAN of the Carica elegans Lecanicillium:

LAMAN-F：5`- CTTGGTTCGGCAAAGGAGCA-3`，

LAMAN-R：5`- GATTGGATGTGGGGAGGGTA-3`；

upstream and downstream primers of internal reference Tubulin gene:

Tubulin-F: 5`-CTTCACTTCTTCATGCCTGG-3`，

Tubulin-R: 5`-TTTGTTCGTCAACTTCTTTC-3`；

the total volume of the conventional PCR amplification system is 20 muL: 2X Taq Plus Master Mix 10 muL, 5 muM upstream primer 0.8 muL, 5 muM downstream primer 0.8 muL, 100 ng/muL cDNA template 1.0 muL and water 7.4 muL;

the conventional PCR reaction procedure was: pre-denaturing at 94 ℃ for 5 min, then denaturing at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extending at 72 ℃ for 1 min, under the condition, 35 cycles are carried out, and finally extending at 72 ℃ for 10min;

(3) Real-time fluorescent quantitative PCR reaction:

adding the primers obtained in the step (2) into the cDNA obtained in the step (1) as a template to perform fluorescent quantitative PCR reaction, setting 3 times of repetition for each sample, and taking an average value after amplification;

the real-time fluorescent quantitative PCR amplification system is as follows: 2.0 muL of 2X ChamQ SYBR Color qPCR Master Mix 10 muL, 5 muM upstream primer 0.8 muL, 5 muM downstream primer 0.8 muL, 50X ROX Reference Dye 2.4 muL, 100 ng/muL cDNA, and water is replenished to 20 muL;

the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min, followed by denaturation at 95 ℃ for 5 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 40 s,40 cycles.

Images