| 【Objective】The study was carried out to provide technical support for the researches on gene function
of wax gourd by constructing a wax gourd yeast two-hybrid cDNA library.【Method】The roots, stems, leaves, male
flowers and tender fruits of the wax gourd inbred line B227（wax gourd cultivar used for genome sequencing ）were used
as materials to extract RNA by using the CTAB method. Then, the extracted RNA was used to isolate and purify mRNA,
and double-stranded cDNA synthesis and DSN homogenization treatment were carried out. Later, cDNA was ligated with
pDONR222 vector through BP recombination and transformed into E.coli DH10B competent cells to construct a primary library. LR recombination reaction was performed with plasmid extracted from primary library and pGADT7-DEST vector,
and the products were transformed into E.coli DH10B competent cells to construct a secondary yeast two-hybrid cDNA
library. The library capacity was calculated by the multiple dilution method, and the recombination rate and the sizes of
the inserted fragments were calculated by colony PCR.【Result】The library quality test showed that the primary library
capacity was 8.24×106
CFU, and the homogenized secondary yeast two-hybrid cDNA library capacity of wax gourd was
1.03×107
CFU. The insert fragments mainly concentrated between 850 bp and 3 000 bp with good polymorphism and the
recombination rate was 100%.【Conclusion】A homogenized yeast two-hybrid cDNA library in wax gourd was successfully
constructed. The library is highly integrated and of good quality, which meets the requirements of yeast two-hybrid
experiments. The library can be used in related experiments such as screening of interaction proteins, laying a foundation for
the study on gene function of wax gourd. |
