Abstract:

Aiming at the defects of yeast growth complementation method，a new method for detecting the transport activity of candidate SWEET（sugars will eventually be exported transporter）genes was established by using pH indicator to indicate yeast extracellular acidification. In this study，using 0.006% bromocresol violet（BCP）as the pH indicator，the photoabsorption values A₅₈₉ and A₄₃₂ under the wavelength of 589 nm and 432 nm at different pH values were measured，and the correlation function equation between pH value and A₅₈₉/A₄₃₂ ratio（R_589/432）was established. HpSWEET1a and HpSWEET4a genes were isolated from pitaya（Hylocereus polyrhizus）fruit，which encoded amino acid sequences sharing identities of 66.67% and 55.36% with Arabidopsis thaliana AtSWEET1 and AtSWEET4，and belonged to the SWEET Ⅰ and Ⅱ clade，respectively. When expressed in the EBY.VW4000 yeast strain with impaired sugar uptake activity，and subjected to sugar starvation treatment followed by addition of glucose or fructose，the overexpression of HpSWEET1a，HpSWEET4a and AtSWEET1（At1g21460，positive control）caused in the extracellular BCP color from purple to yellow in 3-16 h. This change indicated the occurrence of extracellular acidification. Further calculation revealed that HpSWEET1a，HpSWEET4a and AtSWEET1 caused a decline of 1.00 in the extracellular pH value of yeast cells within 3-16 h，while the vector control only showed a decrease of 0.10. The observed extracellular acidification of yeast cells and the significant decrease in pH value demonstrated that HpSWEET1a and HpSWEET4a，similar as AtSWEET1，exhibited the glucose and fructose transport activity. To streamline the procedure and reduce time，the colorimetric method were employed based on ultra-micro spectrophotometer to measure the R_589/432 value. Following the addition of glucose for 0.5 h，the R_589/432 value reduction with HpSWEET1a was significantly higher compared to that of the vector control，indicating the method’s efficacy. The method developed in this study is superior to the commonly used yeast growth complementary detection in terms of time-consuming，accuracy，operation and flux. It also has a wide range of applications for studying the cell membrane-localized SWEET transporters.

Key words: Hylocereus polyrhizus, sugar transporter, pH indicator, yeast expression, colorimetric method