CN117210582B - Specific molecular marker for early sex identification of litchi rough shin cui chi moth and detection method - Google Patents
Specific molecular marker for early sex identification of litchi rough shin cui chi moth and detection method Download PDFInfo
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Abstract
The invention discloses a specific molecular marker for early sex identification of litchi rough shin cui chi moth and a detection method, and relates to the technical field of molecular biology. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the invention also provides a primer group for amplifying the molecular marker, and the nucleotide sequence of the primer group is shown as SEQ ID NO.2 and SEQ ID NO. 3. Carrying out PCR amplification on the cDNA of the sample by using the molecular marker primer group, and when the sequence shown as SEQ ID NO.4 can be obtained through specific amplification, indicating that the measured sample is male worm of litchi rough shank and delphinium acuminatum; when the sequence cannot be amplified, the tested sample is the litchi rough shank cuichondria after female worm. The molecular marker for early sex identification of the litchi rough shank and delphinidia chinensis and the detection method thereof provided by the invention can realize portable, rapid and accurate sex identification of the litchi rough shank and delphinidia chinensis, and provide reliable reference for various researches of the litchi rough shank and delphinidia chinensis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a specific molecular marker for early sex identification of litchi rough shin cui chi moth and a detection method.
Background
Litchi and longan are native in China, namely first and fourth most subtropical fruits in China. The planting area of litchi is approximately 800 ten thousand mu in 2022 nationwide, and the planting area of longan is approximately 410 ten thousand mu, which is the biggest litchi and longan production country in the world. Litchi and longan grow in tropical and subtropical areas, and the characteristics of warm and moist climatic environment and no leaf fall throughout the year lead the litchi and longan to be easily suffered from various diseases and insect pests.
The geometrics pest of the ulnaraceae is one of the important prevention and control objects of litchi and longan leaf eating pests. The pests use litchi rough shin and delphinidia chinensis as raw materialsThalassodes immissariaWalker) is the largest, the insect pest takes the larva (inchworm) to eat the leaves or the flowers and ears as a hazard, the middle-and-high-age larva has binge eating property, and can cause the defect of young leaves, the shedding of young fruits and even the bald branch, thereby seriously affecting the nutrition accumulation of the mother branch of litchi and longan results; the adult flying capability is strong, the distribution range is wide, the spreading is rapid, the yield is reduced for the current year, and the flower and fruit setting in the next year can be influenced. In the litchi garden, the leaf loss caused by the harmful insect of the rough shank cui chi moth is more than 60 percent of the leaf loss caused by all insect diseases, thereby threatening the high and stable yield of litchi, and being the primary leaf feeding pest in the litchi industry. The rough shank cuichuan moth belongs to lepidoptera and ulnara, and takes 7-8 generations in one year, overlaps in orchard generation, and has the characteristics of long larva period, uneven pupa period, long adult period, unobvious peak period and the like, and the prevention and control difficulty is high. At present, only 7 researches on crude shin and delphinidia are reported in China, the literature data is limited, and researches on related morphological identification, biological characteristics, ecology, prevention and treatment technology and the like are urgently needed to be carried out.
The life habit, food intake, host preference, physiological metabolism level and stress resistance of insects are all closely related to sex. Currently, sex determination of insects is largely judged by their morphological differences. However, the litchi rough shank and ulnar moth captured in the field is mostly the pupa of the litchi, and the male and female are difficult to distinguish; even if a small amount of adults are trapped by the lamp, the scale feathers are easy to drop due to the activity of the adults, which makes sex identification difficult. Therefore, there is a need to develop a molecular marker for early sex identification of litchi rough-shin cuisine, and by means of molecular biology, the male and female identification of litchi rough-shin cuisine is realized efficiently and accurately, and reliable reference is provided for various researches of the litchi.
Disclosure of Invention
The invention aims to solve one of the technical problems existing in the prior art. Therefore, the invention provides a molecular marker for early sex identification of litchi rough shank cui chi moth.
The nucleotide sequence of the sex-specific molecular marker provided by the invention is shown as SEQ ID NO.1 and ATGCCGGGGCCAGATGTGAAAGCTAAGGACAATGTTTTTGAAATGTCGGCGGAGGCTACTGCGGCCGTACACAGCGGCGCGGAGTCGAAAGCAGAAAAAAAACTCACTGTACTCGAAACCCACGGATATATTCTCGGTAGAACTATCGGGTCTGGCTCATACGCTACCGTTAAGGTCGCTACAAGTGATAGACACAACTGTCAAGTTGCAATAAAAATAATAAGCAAGTTCCAAGCCCCCGGAGATTATTTGAAAAAGTTTCTACCGAGAGAAATAGAAGTGGTCAAAGGACTGAAACATGAGAACCTTATACGATTCTTGCAAGCTATTGAAACAACACACAGGGTATACATAGTAATGGAATATGCACAAAACGGTAGCCTCTTGGATATAATACGTAAAGACCAACATATAGACGAGGTTCGCGGCCGACGATGGTTTCGACAACTGGTCGAGGCTGTTGAATATTGTCATGAACGAGGAGTCGTTCACAGGGATATAAAATGCGAAAACCTTCTAATGGACCACGGCTTAAACATAAAACTGTCGGATTTCGGTTTCGCTCGCGGTCACATGAAGCCAAAGAACGGAATGTATGTGTTGAGTGAAACATTTTGTGGGAGTTACGCATACGCTTCCCCTGAAATACTAAAGGGGGTTCCGTATAAGCCTCAGGACTCTGATGTATGGAGTATGGGCGTGGTTCTGTACGCTATAGTGTACGGTCGGCTGCCGTTTGATGATACCAATTATACGCAACTACTGAAGCAAGTCCAAAACAAAGTATCATTTCCACGCGAACCGAAAGTAACAGCTGATTGTCGCAAGCTGGTCACCAAGATATTGGCCCCACTCAAGTTACGCGTCAAAATACCTCAAATCCTCGCTGACCCTTGGCTCAATCCTAGCCAAACACCTTCTAAAGACGATGACGAAACAAAGGAAAGTAGCAATGAAATCAAGAGCGCGAATATTGGCACTGCATTCGACAGAAACATGGAACACAAAATGGAAGAAGTCAAAATTTAA.
The invention also provides a primer group for amplifying the molecular marker, and the nucleotide sequence of the primer group is shown as SEQ ID NO.2 and SEQ ID NO. 3. The cDNA library after reverse transcription of the RNA of the litchi rough shank and the ulnara stigmata is amplified by the molecular marker primer group, and the sex of the litchi rough shank and the ulnara stigmata can be identified conveniently, rapidly and accurately according to the amplified products.
In another aspect, the invention provides a detection method for early sex identification of litchi rough shin cui chi moth, comprising the following steps:
s1: extracting RNA of a litchi rough shank and ulnar moth sample;
s2: reverse transcription of the RNA extracted in S1 into cDNA library by reverse transcription PCR reaction;
s3: performing PCR amplification by using the cDNA library obtained in the step S2 as a template and using primer groups with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3 to respectively obtain PCR products corresponding to different cDNA libraries;
s4: and (3) taking 8 mu L of the PCR product in the step (S3) to carry out electrophoresis in agarose gel, analyzing the electrophoresis result, and judging male and female worms of the litchi rough shank cuisine, wherein the primer set shown in SEQ ID NO.2 and SEQ ID NO.3 can amplify the male worms of the litchi rough shank cuisine, and the female worms of the litchi rough shank cuisine are the non-amplified tapes.
In some embodiments of the invention, the primer set SEQ ID NO.2 and SEQ ID NO.3 are androstane specific primers for detecting a fragment of the androstane specific expression molecular marker SEQ ID NO.1 sequence.
In some embodiments of the invention, in the step S1, RNA of the litchi rough shank and ulnar moth sample to be tested is extracted by a kit extraction method.
In some embodiments of the invention, the reverse transcription PCR reaction system is:
5×EvoM-MLVRT Master Mix 2μL
RNA 1μg
RNase free water was made up to 10. Mu.L.
In some embodiments of the invention, in the step S2, the reverse transcription PCR reaction is performed by the following procedure: denaturation at 37 ℃ for 15 min; 85℃reaction 5s,4℃termination reaction.
In some embodiments of the invention, in the step S3, the PCR amplification reaction system is:
2×Master Mix 12.5μL
1 mu L of SEQ ID NO.2 primer
1 mu L of SEQ ID NO.3 primer
cDNA template 1. Mu.L
ddH2O was made up to 25. Mu.L.
In some embodiments of the invention, in the step S3, the PCR amplification is performed by the following procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 1 min, denaturation-annealing-extension for 30 cycles; and finally, the temperature of 72 ℃ is extended for 5 minutes.
In some embodiments of the invention, in the step S4, the agarose gel electrophoresis is performed, and the concentration of the agarose gel is 2%.
In some embodiments of the invention, in the step S4, the agarose gel electrophoresis is performed at a voltage of 100-120V, and the electrophoresis time is 30-40 minutes.
In some embodiments of the invention, in the step S4, the agarose gel electrophoresis is performed according to the method of analysis: if the electrophoresis results of the primer groups SEQ ID NO.2 and SEQ ID NO.3 show a band with the size of 212bp, judging that the litchi rough shank and ulna to be detected is male, and if the band is not amplified, judging that the litchi rough shank and ulna to be detected is female.
The detection method provided by the invention can be used for early male and female identification of the litchi rough shank and ulnar moth.
The molecular marker for early sex identification of the litchi rough shank and delphinidia chinensis and the detection method thereof provided by the invention fill the blank of the litchi rough shank and delphinidia chinensis sex molecular detection technology, and have the following advantages:
because of the lack of effective and reliable sex identification means, most of the field captured litchi rough shin and ulnara are pupa, the morphology is similar, and the male and female are difficult to distinguish; even if a small amount of adults are trapped by the lamp, the scales and feathers are easy to drop due to the activity of the adults, so that the sex identification is difficult; the litchi rough shank delphinidia chinensis nucleic acid sample which is not subjected to sex identification extraction is more difficult to be separated into male and female. The invention carries out PCR amplification on the cDNA library of the litchi rough shank and ulna to be detected through the provided primers, can judge the sex of the litchi rough shank and ulna to be detected according to the amplification result, and can judge the result through the presence or absence of the specific amplification product of the male litchi rough shank and ulna.
The molecular marker is simple and convenient to operate, can carry out sex identification efficiently, quickly and accurately on the litchi rough shank and ulna moth pupae, the imago and the litchi rough shank and ulna moth nucleic acid sample which is not subjected to sex identification and provides reliable reference for the litchi rough shank and ulna moth research work.
Drawings
FIG. 1 is a schematic representation of a litchi rough shin, delphinidia chinensis and pupa;
FIG. 2 is a schematic diagram showing sex identification of different litchi rough shank delphinidia chrysalis by using the sex identification primer provided in the embodiment of the invention (M: DL2000DNA Marker;1: pupa 1;2: pupa 2;3: pupa 3;4: pupa 4);
FIG. 3 is a schematic diagram showing sex identification of different litchi rough-shank cuisine adults by using the sex identification primer provided by the embodiment of the invention (M: DL2000DNA Marker;1: adult 1;2: adult 2;3: adult 3;4: adult 4;5: adult 5;6: adult 6).
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The reagents, methods and apparatus employed in the examples which follow are all conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer.
The following test examples were carried out using a kit for rapid RNA extraction by the magnetic bead method provided by Shenzhen Michaelis Biotechnology Co., ltdM-MLVThe reverse transcription kit is provided by Ai Kerui Biotechnology Co., ltd, the 2X Tag PCR MasterMix kit is provided by Tiangen Biotechnology (Beijing) Co., ltd, and the TS-Gelred nucleic acid gel dye is provided by Beijing Optimago Co., ltd.
Example 1 identification of sex of pupae of litchi Philips trichlanicus
Primer design and specificity detection
(1) Primer design
The gene sequence of testis-specific serine (threonine kinase 3) is found in transcriptome data of litchi rough shank and ulnara xylostella by using DESeq2 software, primer 6.0 software is used for Primer design, and 1 pair of specific Primer design is obtained through screening and synthesized by Guangzhou Ai Ji biotechnology company.
Wherein, the forward primer is TGGAGTATGGGCGTGGTTCTGT (SEQ ID NO. 2) and the reverse primer is AGCCAAGGGTCAGCGAGGATT (SEQ ID NO. 3);
the nucleotide sequences of the sequences (212 bp) amplified by SEQ ID NO.2 and SEQ ID NO.3 are: 5'-TGGAGTATGGGCGTGGTTCTGTACGCTATAGTGTACGGTCGGCTGCCGTTTGATGATACCAATTATACGCAACTACTGAAGCAAGTCCAAAACAAAGTATCATTTCCACGCGAACCGAAAGTAACAGCTGATTGTCGCAAGCTGGTCACCAAGATATTGGCCCCACTCAAGTTACGCGTCAAAATACCTCAAATCCTCGCTGACCCTTGGCT-3' (SEQ ID NO. 4).
(2) Extraction of sample RNA
Collecting litchi rough shank and delphinidia chinensis pupa from a litchi garden, placing the litchi rough shank and delphinidia chinensis pupa into a sealing bag, taking the litchi rough shank and delphinidia chinensis pupa back to a laboratory, cleaning the litchi rough shank and delphinidia chinensis pupa with sterile water for 2 times, placing the litchi rough shank and delphinidia chinensis pupa into a 1.5mL centrifuge tube, and extracting sample RNA by using a magnetic bead method RNA rapid extraction kit single head. The integrity of the extracted RNA was checked with a 2% agarose gel. The purity and concentration of the extracted RNA are detected by an enzyme-labeled instrument, and the RNA is stored at-80 ℃ for standby.
(3) cDNA library construction
Taking the sample RNA which is qualified by extraction in the step (2) as a reverse transcription nucleic acid template, operating on ice, and preparing a reverse transcription reaction system in a 0.2ml PCR tube, wherein the reverse transcription reaction system comprises the following steps:
5×EvoM-MLVRT Master Mix 2μL
pupa Bombycis RNA 1 μg
RNase free water was made up to 10. Mu.L;
placing the prepared reverse transcription reaction liquid into a PCR instrument, and performing denaturation at a temperature of 37 ℃ for 15 minutes; 85℃reaction 5s,4℃termination reaction. And (3) after the reaction is finished, obtaining a cDNA library of a corresponding sample, and storing the cDNA library at-20 ℃ for later use.
(4) PCR amplification
And (3) carrying out PCR amplification reaction by taking cDNA of different samples obtained in the step (3) as a detection object according to the following reaction system and amplification procedure. The reaction system of the primer set of SEQ ID NO.2 and SEQ ID NO.3 is as follows:
2×Master Mix 12.5μL
1 mu L of SEQ ID NO.2 primer
1 mu L of SEQ ID NO.3 primer
Pupa of insect cDNA template 1 mu L
ddH2O is complemented to 25 mu L
The procedure for the PCR amplification reaction was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 1 min, denaturation-annealing-extension for 30 cycles; and finally, the temperature of 72 ℃ is extended for 5 minutes.
And (3) after the reaction is finished, obtaining a PCR amplification product of the corresponding sample, and storing the PCR amplification product at the temperature of-20 ℃ for standby.
(5) Specific detection
8. Mu.L of the reaction product obtained by the PCR reaction in the step (4) was taken and subjected to electrophoresis in 2% agarose gel at a constant pressure of 110V for 35 minutes. After electrophoresis, the agarose gel is soaked in diluted TS-Gelred nucleic acid gel dye for 30 minutes, and the dyed agarose gel is observed under an ultraviolet lamp.
The sex identification results of the primer set of SEQ ID NO.2 and SEQ ID NO.3 on different litchi rough shank trichla and pupae are shown in figure 2; the sex identification results of the primer set of SEQ ID NO.2 and SEQ ID NO.3 on different litchi rough shank ulnara adults are shown in figure 3.
As can be seen from FIG. 2, the specific band of 212bp can be amplified from the pupae sample 1-3, so that the pupae sample 1-3 is male worms of litchi rough shank delphinium; the non-amplified strip of the pupa sample 4 is the female worm of the litchi rough shank cui chi moth. Which is consistent with the actual situation.
Example 2 identification of known sex of adults of litchi Philips trichlrabi
Primer design and specificity detection
(1) Primer design
The gene sequence of testis-specific serine (threonine kinase 3) is found in transcriptome data of litchi rough shank and ulnara xylostella by using DESeq2 software, primer 6.0 software is used for Primer design, and 1 pair of specific Primer design is obtained through screening and synthesized by Guangzhou Ai Ji biotechnology company.
Wherein, the forward primer is TGGAGTATGGGCGTGGTTCTGT (SEQ ID NO. 2) and the reverse primer is AGCCAAGGGTCAGCGAGGATT (SEQ ID NO. 3);
the nucleotide sequences of the sequences (212 bp) amplified by SEQ ID NO.2 and SEQ ID NO.3 are: 5'-TGGAGTATGGGCGTGGTTCTGTACGCTATAGTGTACGGTCGGCTGCCGTTTGATGATACCAATTATACGCAACTACTGAAGCAAGTCCAAAACAAAGTATCATTTCCACGCGAACCGAAAGTAACAGCTGATTGTCGCAAGCTGGTCACCAAGATATTGGCCCCACTCAAGTTACGCGTCAAAATACCTCAAATCCTCGCTGACCCTTGGCT-3' (SEQ ID NO. 4).
(2) Extraction of sample RNA
In the full-growing period of the adults, 6 heads of the litchi rough shank and delphinidia chinensis adults are trapped by sugar-vinegar liquid, the adults are randomly taken out and identified by a tail device, the adults are numbered as female 1, male 2, female 3, male 4, male 5 and male 6, the adults are placed into a sealing bag in a split manner and brought back to a laboratory, the adults are washed 3 times by using sterile water, and then the adults are placed into a 1.5mL centrifuge tube, and sample RNA is extracted by a single head of a magnetic bead method RNA rapid extraction kit, and the specific steps are carried out according to the specification. The integrity of the extracted RNA was checked with a 2% agarose gel. The purity and concentration of the extracted RNA are detected by an enzyme-labeled instrument, and the RNA is stored at-80 ℃ for standby.
(3) cDNA library construction
Taking the sample RNA which is qualified by extraction in the step (2) as a reverse transcription nucleic acid template, operating on ice, and preparing a reverse transcription reaction system in a 0.2ml PCR tube, wherein the reverse transcription reaction system comprises the following steps:
5×EvoM-MLVRT Master Mix 2μL
adult RNA 1. Mu.g
RNase free water was made up to 10. Mu.L;
placing the prepared reverse transcription reaction liquid into a PCR instrument, and performing denaturation at a temperature of 37 ℃ for 15 minutes; 85℃reaction 5s,4℃termination reaction. And (3) after the reaction is finished, obtaining a cDNA library of a corresponding sample, and storing the cDNA library at-20 ℃ for later use.
(4) PCR amplification
And (3) carrying out PCR amplification reaction by taking cDNA of different samples obtained in the step (3) as a detection object according to the following reaction system and amplification procedure. The reaction system of the primer set of SEQ ID NO.2 and SEQ ID NO.3 is as follows:
2×Master Mix 12.5μL
1 mu L of SEQ ID NO.2 primer
1 mu L of SEQ ID NO.3 primer
Adult cDNA template 1. Mu.L
ddH2O is complemented to 25 mu L
The procedure for the PCR amplification reaction was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 1 min, denaturation-annealing-extension for 30 cycles; and finally, the temperature of 72 ℃ is extended for 5 minutes.
And (3) after the reaction is finished, obtaining a PCR amplification product of the corresponding sample, and storing the PCR amplification product at the temperature of-20 ℃ for standby.
(5) Specific detection
8. Mu.L of the reaction product obtained by the PCR reaction in the step (4) was taken and subjected to electrophoresis in 2% agarose gel at a constant pressure of 110V for 35 minutes. After electrophoresis, the agarose gel is soaked in diluted TS-Gelred nucleic acid gel dye for 30 minutes, and the dyed agarose gel is observed under an ultraviolet lamp.
The sex identification results of the primer set of SEQ ID NO.2 and SEQ ID NO.3 on different litchi rough shank ulnara adults are shown in figure 3.
As can be seen from FIG. 3, the specific bands of 212bp can be amplified by the adult samples 1-2 and 5-6, so that the adult samples 1-2 and 5-6 are male worms of the litchi rough shank, delphinium and the like; the non-amplified strip of the adult sample 3-4 is the female worm of the litchi rough shank cui chi moth. Which is consistent with the actual situation.
The detection method provided by the embodiment has good sensitivity.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (8)
1. The primer for early sex identification of litchi rough shank and delphinidia koidz is characterized by being shown as SEQ ID NO.2 and SEQ ID NO. 3.
2. The use of the primer of claim 1 in the sex early identification of litchi rough shank cuisine.
3. The method for early sex identification of the litchi rough shank and delphinidia chinensis is characterized by comprising the following steps of:
s1: extracting RNA of a litchi rough shank and ulnar moth sample;
s2: reverse transcribing the RNA extracted in S1 into cDNA;
s3: performing PCR amplification by using the cDNA obtained in the step S2 as a template and using a primer group with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO. 3;
s4: and (3) electrophoresis is carried out on the PCR amplified product in the step (S3) in agarose gel, and the male and female worms are judged according to the electrophoresis result, wherein the primer group shown in SEQ ID No.2-3 can amplify the male worm of the litchi rough shank and the female worm of the delphinium when the primer group is not amplified.
4. The method of claim 3, wherein in step S1, the litchi rough shank and ulna moth sample is litchi rough shank and ulna moth pupae.
5. The method of claim 3, wherein in step S2, the reverse transcription reaction system is a 10 μl amplification system: 5 XEvoM-MLVRT Master Mix 2. Mu.L, 1. Mu.g of sample RNA, and RNase free water were added to 10. Mu.L.
6. The method of claim 3, wherein in step S2, the reverse transcription procedure is: denaturation at 37 ℃ for 15 min; 85℃reaction 5s,4℃termination reaction.
7. The method of claim 3, wherein in step S3, the PCR amplification reaction system is 25 μl amplification system: 2 XMaster Mix 12.5. Mu.L, 1. Mu.L each of the 10. Mu.M molecular marker primer set, 1. Mu.L each of the cDNA template, and ddH2O to 25. Mu.L.
8. The method of claim 3, wherein in step S3, the PCR amplification procedure is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 1 min, denaturation-annealing-extension for 30 cycles; and finally, the temperature of 72 ℃ is extended for 5 minutes.
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