| Cucumber(Cucumis sativus L.)uses stachyose as the main assimilate for transport,and stachyose synthase is a key enzyme to catalyze the synthesis of stachyose.There are three different CsSTS transcripts in cucumber leaves which are encoded by the same gene and generated by alternative polyadenylation(APA).However,the exact function of thees three CsSTS transcripts and their regulatory roles in the assimilate distribution of cucumber are still unclear.In this study,we have investigated the stability,translation efficiency,and expression levels of the three CsSTS transcripts in different "source-sink" status.In addition,an antisense RNA(asCySTS)and a miRNA(miR2610)were obtained by target sequence prediction and screening which may play a regulatory role in CsSTS expression regulation.The effects of these non-coding RNAs on the expression of three CsSTS transcripts were further studied,The results provide some cues for regulating cucumber source-sink relationship by cultivation practice.The results obtained are as follows:1.The translation experiment revealed that there was no significant difference of translation efficiency among three CsSTS isoforms,at least under this in vitro condition.Among three iosoforms in cucumber seedling stage leaves,we found the half-life of CsSTS3 was significantly longer than those of CsSTS1 and CsSTS2.But in fruiting-node leaves,the half-life of CsSTS1 was significantly longer than those of CsSTS2 and CsSTS3.2.Whether it contains fruit on fruiting-node leaves,it had a significant effect on the expression of the three transcripts.When the cucumber plant grows to 20th nodes and it's not retain fruit,expression of three mRNAs remained at a quite low level.But when the 12th leaves of 20-leaf stage plants with a fruit,CsSTSs expression peak shifts to the lower leaves and the expression of three CsSTS transcrpits are increased in near the 12th leaves.In the leaf at the fruit-carrying node(12th leaves),the expression of CsSTS2 and CsSTS3 was severely inhibited and only CsSTSl was highly expressed.This "fruit-carrying inhibition" after flowering 5 days start to appear,but picked the fruit after 3 days had to disappear.The "fruit carrying inhibition" still exists when carrying two fruits.It indicates that the leaves of cucumber fruit-carrying node can ensure the output of assimilation by selecting the stable CsSTS transcript.Weakening either source or sink had a significant negative impact on the CsSTS1 expression,and this adverse effect could be more pronounced when both source and sink were impaired.3.The expression of miR2610 in fruiting-node leaves is significantly higher than that of non-fruiting-node leaves,and the relative levels of asCsSTS in the two types of leaves showed the opposite tendency of miR2610.asCsSTS mainly exists in cytoplasm,indicating a post-transcript regulation role this cis-nature antisense transcripts.By overexpressing or inhibiting miR2610 in cucumber protoplasts and the fusion expression test of the enhanced green fluorescent protein,we found that miR2610 can negatively regulate asCsSTS,and asCsSTS further negatively regulates the expression of CsSTS2 and CsSTS3.Interestingly,and asCsSTS has no regulatory effect on CsSTSl.We conclude that miR2610 and asCsSTS may play roles in cucumber source-library regulation by affecting the expression of three CsSTSs.The potential application of this regulation in the cultivation and breeding of cucumber were discussed. |
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