Functional Analysis Of MYB113 Gene In Tomato Under Chilling Stress

Tomato is a widely planted vegetable crop in northern China.As a kind of warm vegetable,tomato is easily damaged by low temperature stress in autumn and winter,which affects the yield and quality and causes great economic loss.Low temperature stress is divided into chilling stress(0 to 10 ?)and freezing injury(below 0 ?).When tomato suffers chilling stress,it will accumulate a large amount of reactive oxygen species,causing membrane lipid peroxidation and aggravating plant damage.In addition,chilling stress will also have a serious impact on the photosynthetic mechanism and photosynthesis of tomato,resulting in growth retardation,fruit setting rate decline,and increase in the number of abnormal fruit.In serious cases,it will lead to plant death.Anthocyanin is a natural water-soluble pigment,which can alleviate the harm of chilling stress to tomatoes to some extent due to its antioxidant capacity.Anthocyanin synthesis is regulated by a variety of environmental factors and multiple genes,and the transcription factors regulating anthocyanin synthesis mainly include MYB,bHLH,WD40 and other families.MYB transcription factor family is one of the largest transcription factor families in plants,which is involved in the regulation of various physiological and biochemical processes in different plants.In addition,MYB transcription factor family is also involved in the response of plants to stresses such as chilling stress.There are 127 MYB transcription factors in tomato genome,and there are few reports about their functions.In this experiment,a MYB transcription factor(SlMYB113)was isolated from tomatoes.The transgenic tomato with overexpression of SlMYB113 and wild-type plants were used as experimental materials to investigate the effect of this gene on tomato low-temperature resistance and anthocyanin synthesis under chilling stress.The results of this experiment have important significance for elucidating the function of MYB transcription factor in tomato resistance to chilling stress.The main research results are as follows:(1)The full length CDS of SlMYB113 gene(950 bp,with a 780 bp open reading frame)which encoded 259 amino acids was cloned from the tomato cDNA.Primers were designedaccording to the gene sequence of SlMYB113 on SGN website.Protein alignment and a blast search revealed that SlMYB113 belong to MYB transcription factor of R2R3 type.(2)SlMYB113-GFP vector and SlMYB113-FLAG vector were constructed and transformed into Agrobacterium strains GV3101 and LBA4404.Then the GV3101 strain containing SlMYB113-GFP plasmid and the LBA4404 strain containing SlMYB113-FLAG plasmid were transformed into tobacco and tomato,respectively.Confocal scanning microscopy and western blot assay showed that the SlMYB113 protein was localized in the nucleus.(3)The expression patterns of SlMYB113 under different stresses were analyzed by qRT-PCR.It was found that the expression of SlMYB113 was induced by chilling stress,drought and signal molecule gibberellin.Organ expression analysis showed that SlMYB113 had the highest expression in leaves.(4)A plant specific expression vector pBI121 without any tag containing SlMYB113 was constructed and transformed into Agrobacterium LBA4404 strains which was transformed into tomato plants immediately with Agrobacterium-mediated leaf disc method.The SlMYB113 over-expression transgenic tomato plants were obtained.Transgenic lines were screened and identified,and the lines with high expression(OE-8,OE-11 and OE-22)were selected for next experiments.(5)Phenotype analysis showed that the leaves anthocyanin accumulation of the SlMYB113 transgenic lines were more than that in the wild-type(WT).Moreover,the changed expression of some anthocyanin synthesis-related genes in transgenic tomatoes suggested that SlMYB113 may be involved in the anthocyanin synthesis process.(6)The value of MDA and REC(two indicators of membrane damage)increased in both the WT and SlMYB113 transgenic plants after chilling stress,but this increase was less pronounced in the SlMYB113 transgenic plants than in the WT,indicating that the cell membrane of transgenic tomato was less damaged and the resistance of transgenic tomato to chilling stress was higher.In addition,SlMYB113 transgenic lines showed a less ROS accumulation than WT under chilling stress.However,the activity of ROS scavenging enzymes in transgenic tomatoes was lower than that of wild type,which suggested that ROS in SlMYB113 transgenic plants may be mainly scavenged by anthocyanins.(7)Yeast one-hybrid assay and transcript activation experiment proved that SlWHY1 could bound to the promoter of SlMYB113 and regulated the expression of SlMYB113.This suggests that SlMYB113 was a downstream target gene of SlWHY1.Conclusion: We cloned a R2R3 type MYB transcription factor SlMYB113 from tomato.The full length of SlMYB113 was 950 bp,containing an open reading frame of 780 bp which encoded 259 amino acids.The product of this gene was located in the nucleus.The expression of SlMYB113 was induced by various stresses and hormones such as low temperature,drought and gibberellin,and the expression level was the highest in the leaves.Overexpression of SlMYB113 increased content of anthocyanin,thus improving the low temperature resistance of transgenic tomato.SlMYB113 was a downstream target gene of SlWHY1.