Wild wheat blue grain character major gene as well as molecular marker and application thereof
A major gene and molecular marker technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of unclear major gene of wild single wheat blue grain, and achieve the effect of increasing the content
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Embodiment 1
[0038] Example 1 Transcription group sequencing analysis
[0039] Sequencing samples for red grains crocus and blue gemmate wheat - wild one wheat 4A b (4b) Replacement Z18-1244 paste powder layer ( figure 1 .
[0040] 1) Sequencing and sequence assembly
[0041] Transcription group sequencing can efficiently and quickly obtain all transcripts of biological organizations. Clean Data of each sample has reached 5.97 GB, and the q30 base percentage is 93.18% and above. The Clean Reads of each sample with the specified reference genome, respectively, and the ratio of 84.79% to 87.82%, respectively. Based on the comparison result, variable splicing prediction analysis, gene structure optimization analysis, and new genes were discovered, and 105,787 new genes were discovered, 92,072 function comments were obtained. Gene expression quantity analysis is performed based on comparison results. Differential expression genes are identified according to the expression quantity of the gene in d...
Embodiment 2
[0063] Embodiment 2TBMYC4A molecular characteristics
[0064] 1) Preparation for total RNA and CDNA
[0065] Total RNA was extracted from about 0.5 g of paste powder using the Tiangen Rnaprep Pure Plant Kit (TIANGEN, China). CDNA was obtained from total RNA using the Thermo Revertaid First Strand cDNA kit (Thermo-Fisherscientific, Shanghai).
[0066] 2) Design primer separation TBMYC4A transcription
[0067] TBMYC4ACDS-F: atgcgggaaacagctcag;
[0068] TBMYC4ACDS-R: CTATATAGCTTTCTGAAGCGCTTCA.
[0069] Primers TBMYC4ACDS-F, TBMYC4ACDs-R was used to amplify cDNA to separate wild-granulated wheat grain gene in Z18-1244. PCR amplification was performed using Veriti96 ABI Life 2720 (Hunan Innovagene Biotechnology Limited) using Veriti96 ABI Life 2720.
[0070] The PCR amplification program was 95 ° C degeneration for 5 minutes, 35 cycles: 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute 40 seconds, and then extension at 72 ° C for 5 minutes. PCR products were purified fr...
Embodiment 3
[0074] Example 3TBMYC4A instantaneous expression can induce anthocyanin synthesis
[0075]The instantaneous expression vector PLGY-02: TBMYC4A was constructed using homologous recombination. The use of TBMYC4F2 and TBMYC4R2 from the TBMYC4A connected to the base sequence of the PGEM-TEASY vector, and the purpose fragment was carried out by high quality kits, and the electrophoresis was detected without miscellaneous tape. The PLGY-02 linearized carrier is mixed with the TBMYC4A recovery fragment 1: 2 concentration, and the link 37 ° C by the CloneXpress II One Step Cloning Kit kit is 30 min after 30 min, it is placed on ice, and E. coli DH5α is converted to E. coli DH5α, overnight culture, Picking the single spot on the recombinant reaction conversion plate for PCR colony detection, positive single spots sent to Shanghai's generic organism for sequencing. pBRACT214: TaMYC1 and pBRACT214: TaMYB7D interaction has been validated as a white coleoptile capable of inducing a large numbe...
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