Molecular Mechanism Of Sly-miR319b Regulating TCPs In Response To Low Potassium Stress In Tomato

Tomato,as a kind of important vegetable crop,is susceptible to the environment(low temperature,low illumination,soil p H,and low potassium content in the soil)and its own development period(mid-to-late development period).Potassium deficiency symptoms during the growth and development affect the quality and yield in tomato.The research team has found that the expression level of mi R319 b in low-potassium-tolerant tomato line JZ34 and potassium-sensitive line JZ18 is extremely different by sequencing the small RNA in the early stage.As a non-coding small molecule RNA in vivo,first mi RNA cut or inhibit the target m RNA by specifically recognizing it,and then negatively regulates gene expression at the transcription level.Previous studies have shown that mi R319 can target TCP transcription factors to regulate the growth and development in plant.However,there is no report on the function of mi R319 in response to low potassium stress.On the one hand,this study uses RLM-5'RACE to identify Slmi R319 b target gene break sites,using yeast one-hybrid and EMSA methods to establish the molecular pathway of Slmi R319 b regulating target gene Sl TCP10 in response to low potassium stress;On the other hand,Slmi R319 b over-expressing tomato transgenic material was constructed and the root morphology,potassium ion content,ROS content and jasmonic acid content of the transgenic lines under low potassium conditions were determined.The results will give the theoretical guidance to deepen analysis the mi RNA signal transduction pathways in response to low potassium stress,stress-resistant cultivation and quality improvement in tomato.The main findings are as follows:1.Clarify the distribution characteristics and evolution relationship of mi R319 family members in plants.The statistics on the mi RBase database showed that mi R319 members can be found in 41 plant species,including highly evolved angiosperms and less evolved ferns,but not found in algae and bryophytes.2.The results veritied that the targets gene of mi R319 b is TCP10 transcription factor by q RT-PCR and RLM-5'RACE.Analysis of the JA2 promoter revealed that it contains a TGGTCC binding site t which can bind to TCP.Yeast one-hybrid and EMSA tests were used to verify that TCP10 can bind to the JA2 promoter.3.An overexpression vector of p CAMBIA3301-35S-mi R319 b were construct,and obtained an overexpressing 35S-mi R319 b transgenic line with low potassium tolerance tomato JZ34 by using Agrobacterium-mediated leaf disc method.The T0 generation was tested positive for transgenic plants,and 17 overexpressing lines were obtained.4.Through further screening in T0 line,there were 8 positive plants with high expression level of mi R319 b and low expression level of TCP10.The seeds were collected by individual plant,the T1 plants were treated with low potassium stress.It was found that the root structure in the transgenic lines under control conditions was slower than those of in non-transgenic lines.After low potassium treatment,the root growth was significantly inhibited.The potassium ion content,ROS content,and JA content were measured on this basis.The results showed that compared with WT,the transgenic line had less potassium ion content,increased ROS content,increased JA content,and was more susceptible to low potassium stress.The above results indicate that Slmi R319 b mediated by Sl TCP10 can regulate the downstream pathway through the jasmonic acid pathway to respond to low potassium stress.