Cloning Of SlMYB4 And Its Functional Analysis In Tomato Response To Chilling Stress

Temperature is one of the important environmental factors affecting crop growth,development and yield.Some crops originating in the tropics or subtropics,such as tomato,cucumber and corn are particularly sensitive to low temperature stress.Low temperature hazards often occur in vegetable cultivation and production in protected area in northern China,resulting in the hindrance of crop growth,development,the decline of yield and quality.To cope with low temperature stress,plants have evolved many adaptive physiological and biochemical mechanisms.In recent years,with the development of research,great progress has been made in the mechanism of plant response to low temperature stress.The ICE-CBF-COR signaling pathway is believed to play an important role in plant responses to low temperature stress,in which a variety of transcription factors are involved and play a key regulatory role.MYB transcription factor,one of the largest transcription factor families in plants,plays a regulatory role in all stages of plant growth and development,and also participates in the response of plants to environmental stress.We found that SlMYB41 gene was significantly induced by low temperature from the cold transcriptome in our laboratory,and its regulatory mechanism under low temperature stress has not been reported yet.Therefore,we investigated the function and mechanism of SlMYB41 transcription factor in tomato response to low temperature in this study,so as to provide genetic resources for the cultivation of new tomato varieties resistant to low temperature.In this study,a tomato MYB transcription factor(SlMYB41)was cloned and transformed into tomato to obtain overexpressed and RNAi lines.The function of SlMYB41 under chilling stress was disscussed.The main results are as follows:(1)SlMYB41(Solyc07g054840)was isolated from tomato leaves.The full length of the gene is 1244 bp,which contains a CDS sequence of 1002 bp and 23 bp of 3?-UTR.It encodes a protein of 333 amino acids,and is located on the 7th chromosome of the tomato genome.The molecular weight of SlMYB41 protein is 38.07 kDa and its isoelectric point is 5.71.It has two conserved MYB domains,which are located from the 14 th to the 61 st amino acid and from the 67 th to 111 st amino acid at the N-terminal.(2)The fusion protein of SlMYB41-GFP was transiently expressed in tobacco leaves,and it was found that the SlMYB41 protein was localized in the nucleus by laser confocal microscopy.(3)qRT-PCR analysis showed that SlMYB41 was expressed in tomato roots,stems,leaves,flowers and fruits,among which the expression was the highest in flowers,followed by roots,less in fruits and leaves,and the lowest in stem.Moreover,SlMYB41 was induced not only by low temperature,but also induced by high temperature and NaCl.(4)The overexpression and RNAi silencing vector of SlMYB41 were constructed and transformed into tomato by Agrobacterium-mediated leaf disc method.The transgenic tomato lines with overexpression and RNAi silencing were obtained.The transgenic tomato plants were identified by PCR and qRT-PCR.The over-expressed and RNAi tomato transgenic lines were successfully obtained.(5)Under chilling stress,the overexpressing lines were more sensitive to low temperature than the wild type,while the RNAi-silencing lines were more resistant to low temperature.Under chilling stress,compared with the wild type,the cell membrane damage of over-expressing plants was more serious,the content of hydrogen peroxide was significantly higher,and the activities of APX and CAT were lower,while the changes of these indicators of the RNAi plants were opposite to those of the overexpressing plants.These results indicate that SlMYB41 may be a negative regulator of tomato under chilling stress.(6)The results of qRT-PCR showed that there was no significant difference in the expression of related genes in the CBF signaling pathway between transgenic lines and wild-type after low-temperature treatment,suggesting that the regulation of low-temperature resistance of tomato by SlMYB41 may not depend on the CBF signaling pathway.However,we performed transcriptomic analysis of tomato overexpressed lines and wild-type lines at low temperature and found that SlGRF1,SlPPO,SlACO-like,SlACO and SlEIL3 may be potential downstream target genes of SlMYB41.