| The cucurbitane-type triterpenoid glycosides, mogrosides, are the main active components of Siraitia grosvenorii fruit. Previously the promoter of the key gene SgCS which directly catalyzes the formation of the backbone of mogrosides was cloned in our lab, and E-box capable of binding with bHLH protein was found in the SgCS promoter sequence, which indicates the possible presence of bHLH transcription factors (TFs) affecting the biosynthesis of mogrosides by regulating the expression of SgCS gene. It is recognized that MeJA can increase the accumulation of triterpenoids in plants. In the present work, the effects of MeJA on the content of mogroside and expression of key genes were investigated after the application of MeJA towards S. grosvenorii young fruits in order to increase the content of mogrosides and explore the possible molecular mechanisms of mogrosdies synthesis. Meanwhile, preliminary screening of bHLH TFs which might modulate the biosynthesis of mogrosides and bind with cis-acting elements in the promoter region of SgCS gene was conducted by analyzing S. grosvenorii fruit transcriptome and performing the experiment of yeast one-hybrid. The main content was summarized as follows:1、The effects of MeJA on the accumulations of mogrosides Ⅱ E and intermediates squalene and cucurbitadienol as well as the expression of 13 genes related to the biosynthesis of mogrosides in the fruit of S. grosvenorii was tested by treating fruits with different concentrations of MeJA. The results showed that the application of 500μmol/1 MeJA to the fruits at 30 DAF can indeed promote the accumulation of metabolites. The content of mogroside II E (mass percentage%) increased by 15% over the untreated group and the concentrations of squalene and cucurbitadienol were 19%,22% greater than that of the control, respectively. The transcriptional levels of all the 11 genes were up-regulated following the treatment described above according to the results of qRT PCR determination. Additionally, their responses in the presence of MeJA was related to the concentration and timing of MeJA treatment.2、The transcriptome and gene expression profile of S. grosvenorii fruits at 3,50 and 70 DAF were analyzed. The results showed that there are 468 unigenes annotated as transcription factor, including 79 bHLH,57 WRKY and 50 MYB TFs family which enjoy the largest number of TFs.119 transcription factor unigenes which were differentially expressed in any of the 70 DAF/3 DAF、50 DAF/3 DAF and 70 DAF/50 DAF groups were discovered. What’s more,6 bHLH TFs (bHLH014、bHLH025、 bHLH061、bHLH071、bHLH093、bHLH096) were found with significant expression changes in 50 DAF/3 DAF and 70 DAF/3 DAF groups. The S. grosvenorii fruits where the synthetic pathway of mogrosides was up-regulated after the MeJA inducement was used as materials to determine the expression of the 6 bHLH TFs through qRT-PCR. And results showed that there are 4 bHLH TFs (bHLH014、bHLH025、bHLH093、bHLH096) whose transcriptional level was up-regulated and 2 (bHLH093、bHLH096) increased about 10-fold.3、The 5 E-box motifs were connected as bait and the cDNA library of S. grosvenorii fruit was built in yeast one-hybrid system to screen the bHLH TFs which are able to combine with the promoter of SgCS gene.52 positive clones were obtained, however, the sequencing results showed that no bHLH TFs were found.This study indicates that exogenous application of MeJA can increase the accumulation of mogroside content. The constructed cDNA library of S. grosvenorii fruit as well as the established method will lay a foundation for further experimental design improvement and analyzing the function of bHLH TFs find in the future, and will open up a new way to study the molecular mechanisms in regulation of cucurbitane-type triterpenoid glycosides synthetic pathway. |
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