Abstract:
Objective Paeonia lactiflora has high ornamental value, but the double dormancy of the P. lactiflora seeds is difficult to release, and severely hinders the breeding process. We used gateway technique to construct the yeast hybrid cDNA library of the P. lactiflora seeds, which are in critical dormancy release periods. It can be used to screen transcription factors and interact proteins that regulates seed dormancy release of P. lactiflora. The construction of this library not only provides scientific support for enriching the P. lactiflora seed dormancy release regulation research, but also establish the foundation of molecular theory for P. lactiflora cultivation breeding and the protection and utilization of wild resources.
Method The P. lactiflora seeds in five critical dormancy release stages were used as samples to extract total RNA and isolate mRNA. The gateway method was used to construct cDNA primary library. Then, the primary library was recombined into pGADT7-DEST, a secondary library vector, by LR recombination to build a secondary library. Finally, the secondary library plasmid was transformed into yeast Y187 strains by transformation test, the construction of a yeast hybrid cDNA library during seed dormancy release of the P. lactiflora was completed.
Result After identification, the cDNA primary library capacity was 1.28 × 107 CFU, the average insert fragment length was more than 1 000 bp, and the recombination rate was 100%; the cDNA secondary library capacity was 1.12 × 107 CFU, the average insert fragment length was more than 1 000 bp, and the recombination rate was 100%; the yeast library titer was 7.0 × 107 CFU/mL, the average insert fragment length was more than 1 000 bp, and the recombination rate was 96%. Using NCBI to compare the sequencing results of 23 positive clones, we found 20 known protein sequences and divided them into 8 categories according to their functions. 7 sequences had been reported to be involved in seed dormancy and germination in these categories.
Conclusion We successfully construct a yeast hybrid cDNA library during seed dormancy release of P. lactiflora. This library has a high quality and a complete genetic information, which conforms to the yeast library screen test standard. It provides basis for revealing molecular regulatory networks of P. lactiflora seeds in the process of dormancy release.
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