Cloning And Functional Analysis Of Tomato Transcription Factor SlNAC73

Plants encounter various stresses in the course of growth and development,which has different effects on the physiological activity and growth of plants.As global temperatures warm up,high temperature stress soars.Under high temperature conditions,reactive oxygen species(ROS)of plants will accumulate,accompaning with increase of membrane damage and photosynthesis inhibititon,which will result in inhibiting the normal physiological metabolism of tomato plants and limiting their growth and development.Therefore,it is significant important to investigate the response and molecular mechanism of tomato to heat stress.This research is important for the cultivation and breeding of heat-sensitive vegetable plants.NAC transcription factor that is unique to plants and one of the largest families of plant transcription factor,which perform a significant function in the plant growth and development and the process of response to adversity.Tomato,an important vegetable crop,contains 102 NAC transcription factors and the function of most them is unknown.In this experiment,we isolated a tomato NAC transcription factor,named SlNAC73 and its function in chlorophyll-biological metabolism and high-temperature stress is discussed.The main results are as follows:(1)The SlNAC73(Solyc07g066330.2)gene was isolated from tomato,which is 1482 bp in length,containing a 1029 bp of open reading frame and encoding 342 amino acids,with a predicted molecular weight of 38.83 kDa and a pI of 9.75.(2)The fusion protein of SlNAC73-GFP was transiently expressed in the protoplast of Arabidopsis.The results suggested that S1NAC73 protein was localized in the nucleus by laser confocal microscopy.It was detected that SlNAC73 had transcriptional activation that was located at the C-terminus of amino acides sequence by the yeast hybrid system.(3)Tissue expression analysis shows that SlNAC73 was expressed in roots,stems,leaves,flowers,fruits and seeds of tomato and among of them the highest expression was noted in seeds.The expression analysis showed that SlNAC73 was induced by extreme temperature and various hormones(ABA,MeJA,GA,ET)and the analysing the promoter element suggested that it contains a variety of response elements,such as HSE,LTR,ABRE and so on.(4)To obtain overexpressed and silenced transgenic tomato plants,eukaryotic expression vector of SlNAC73 gene was constructed and transformed to tomato by leaf disc method.The transgenic lines obtained were identified and screened by PCR and qRT-PCR.(5)The tomato leaves showed premature yellowing when the expression of SlNAC73 gene reaches a certain level under normal growth conditions.What more,the chlorophyll content and the photosynthesis of yellowing leaves was decreased in comparison with the wild type,while there was no significant difference beween WT and the RNAi lines.(6)Both the photosynthetic capacity and the chlorophyll content of tomato plants were reduced under high temperature.However,there was no significant difference between transgenic lines and the wild type.This indicates that SlNAC73 is not involved in plant photosynthesis and chlorophyll metabolism pathway under heat stress.(7)A large accumulation of ROS was found in the overexpression SlNAC73 lines,which have aggravated damage of cell membrane and lower level osmotic adjustment substance,while the RNAi lines showed the opposite situation under high temperature stress.These indicate that SlNAC73 reduced high temperature resistance of tomato.In conclusion,SlNAC73,a typical transcription factor,can reduce the high temperature resistance of tomato and may be affect the photosynthetic capacity by means of involving in the metabolism of chlorophyll.