Method for developing SSR primers of Blumea balsamifera based on transcriptome sequencing

A technology of transcriptome sequencing and Ainaxiang, which is applied in the fields of molecular biology and bioinformatics, can solve the problems of insufficient yield, low content of medicinal ingredients, and a small number of Ainaxiang SSR primers, etc., achieving convenient and accurate methods Effect

Active Publication Date: 2018-06-22
TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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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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  • Method for developing SSR primers of Blumea balsamifera based on transcriptome sequencing
  • Method for developing SSR primers of Blumea balsamifera based on transcriptome sequencing
  • Method for developing SSR primers of Blumea balsamifera 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 the DNA molecular weight standard, and use 8% non-denaturing polyacrylamide gel for electrophoresis, the electrophoresis bu...

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Abstract

The invention provides a method for developing SSR primers of Blumea balsamifera based on transcriptome sequencing. The method comprises: obtaining a Blumea balsamifera genome transcription set, and forming a sequence database; splicing the sequenced sequences by using Trinity to form a transcriptome, and taking the longest transcripts in each gene as Unigene; carrying out Unigene sequence bioinformatics analysis; carrying out SSR detection on the Unigene by using MISA1.0; and carrying out SSR primer design by using Primer3, and carrying out SSR primer polymorphism identification. According tothe present invention, with the method, 17979 pairs of the SSR primers are successfully designed, the 30 pairs of the primers related to the metabolic pathway of the active ingredient are screened, the DNA of 9 Blumea balsamifera with different sources are verified, 27 pairs of the polymorphic primers are determined, and the plant materials Blumea balsamifera with different geographical sources can be distinguished with the 27 pairs of the SSR primers; and the method provides the new idea for the development of the SSR primers of Blumea balsamifera.

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 Applications(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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