A method for developing Ainaxiang ssr primers based on transcriptome sequencing

A technology of Ainaxiang and primer pairs, which is applied in the fields of molecular biology and bioinformatics, and can solve the problems of insufficient yield, low content of medicinal ingredients, and a small number of Ainaxiang SSR primers, etc.

Active Publication Date: 2021-06-04
TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low content of medicinal ingredients, such as the yield of moxa powder extracted from fresh leaves is less than 10mg / g
[0005] At present, there is no complete gene sequence information for Ainaxiang, and the number of SSR primers for Ainaxiang is relatively small

Method used

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  • A method for developing Ainaxiang ssr primers based on transcriptome sequencing
  • A method for developing Ainaxiang ssr primers based on transcriptome sequencing
  • A method for developing Ainaxiang ssr primers based on transcriptome sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 RNA-seq analysis and design of SSR primers

[0043] 1. RNA-seq analysis sample processing and sequencing process see figure 1 . The specific method is as follows:

[0044] For the extraction of total RNA of Ainaxiang, eukaryotes build a library with an initial amount of 5ug total RNA;

[0045] After the mRNA was separated by the magnetic bead method, the ion interrupted the mRNA (Truseq TM RNA sample prep Kit);

[0046] Recover 200-700bp fragments, reverse transcribe and synthesize double-stranded cDNA, fill in, add A to the 3' end, and connect the index adapter (Truseq TM RNA sample prep Kit);

[0047] Library enrichment, PCR amplification for 15 cycles;

[0048] 2% agarose gel recovery target band (Certified Low Range Ultra Agarose);

[0049] TBS380 (Picogreen) quantified, mixed according to the data ratio on the machine;

[0050] Perform bridge PCR amplification on cBot to generate clusters;

[0051] Hiseq2000 sequencing platform for 2*100bp sequ...

Embodiment 2

[0094] Example 2 Excavation of high-throughput SSR sites of Ainaxiang

[0095] Identification of SSR loci

[0096] First install the Perl language, download est-trimmer.pl from the http: / / pgrc.Lpk-gatersleben.de / misa website, and remove the short and long sequences in the transcriptome sequence; from http: / / www.bioinformatics , download CD-HIT software from org / cd-hit / to remove redundant sequences.

[0097] Download from http:pgrc.lpk-gatersleben.de / misa website and use MISA software to identify and locate SSR in the sequence, the parameters are set as follows: single nucleotide, dinucleotide, trinucleotide, tetranucleotide, five The number of repeats of nucleotides and hexanucleotides is at least 10, 6, 5, 3, 3, 3.

[0098] Design of SSR primers

[0099] Use Primer3 to batch design SSR primers, website: http: / / sourceforge.net / projects / primer3 / files / primer3 / 1.1.4 / primer3-1.1.4-WINXP.zip / download, primer design parameters are primer length 18-22bp ,T m 55-65°C. The diffe...

Embodiment 3

[0100] Example 3 Using SSR primers to identify polymorphisms in Ainaxiang DNA from different places

[0101] Extract 9 copies of Ainaxiang DNA, use 0.8% agarose gel electrophoresis to check the quality, dilute the DNA concentration to 50ng / ul, and store it at -20°C for future use. The DNA of the materials used for primer development was used to identify the success rate of primer design by PCR. The total volume of PCR reaction is 25μl, 1.5mmol / L MgCl 2, 100 μmol / L each of the four dNTPs, 80ng each of the primers, 1.5U Taqplus DNA polymerase (high fidelity), 50ng DNA. The reaction program was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 51-60°C for 45 s, extension at 72°C for 45 s, 32-35 cycles, and finally extension at 72°C for 5 min. After the reaction, add 2ul of sample buffer to the product, use 100bp DNA ladder as DNA molecular weight standard, use 8% non-denaturing polyacrylamide gel for electrophoresis, electrophoresis buffer level 0...

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Abstract

The present invention provides a method for developing SSR primers of Ainaxiang based on transcriptome sequencing: obtain the collection of Ainaxiang genome transcripts to form a sequence database; use Trinity to splice the sequenced sequences into a transcriptome, and take the longest of each gene Transcripts were used as Unigene; Unigene sequence bioinformatics analysis; Unigene was detected by SSR using MISA1.0; Primer3 was used to design SSR primers and identify polymorphisms of SSR primers. The present invention successfully designed 17,979 pairs of SSR primers, screened 30 pairs of primers related to the metabolic pathway of active ingredients, and verified 9 Ainaxiang DNA from different sources. Among them, there are 27 pairs of polymorphic primers. Using these 27 pairs of SSR primers It is possible to distinguish the plant material of Ainara from different geographical origins. The invention provides a new idea for the development of the SSR primers of Ainaxiang.

Description

technical field [0001] The invention relates to molecular biology and bioinformatics, in particular to a pair of Ainaxiang SSR primers, an Ainaxiang SSR primer set, and a method for developing Ainaxiang SSR primers based on transcriptome sequencing. Background technique [0002] Blumea balsamifera (L.) DC., also known as Dafeng AI, is a perennial herb of Compositae, widely distributed in Hainan, Guizhou, Guangxi, Yunan and other provinces in my country. In addition to containing volatile oil (mainly L-borneol, l-borneol), Ainaxiang leaves are used as a refined natural borneol (Ai tablet), and also contain other various active ingredients, such as flavonoids (mainly Ainaxiang and dioxin). Hydrogen flavonoids, etc.), sesquiterpenes, etc. It has various pharmacological activities such as antibacterial, insecticidal, antioxidative, antityrosinase and antitumor. Aina sesame oil (volatile oil) has been successfully used in "Jinhoujian Spray", "Yanlishuang Dropping Pills" and oth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895C12Q1/6869
CPCC12Q1/6869C12Q1/6895C12Q2600/156C12Q2535/122C12Q2537/165
Inventor 官玲亮庞玉新陈振夏于福来王凯谢小丽江芊
Owner TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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