Molecular Mechanism Of GABA Synthesis Key Gene PuGAD Regulated By Transcription Factor PuMYB10/91 Mediating The Pericarp Browning Of Refrigerate ‘Nanguo’ Pears

‘Nanguo’pear is important cultivar in characteristic pear production areas.The harvested‘Nanguo’pears began to enter the senescence stage after being placed at room temperature for about 15 days,and the symptom of browning of the core and pulp appeared and lost its edible value.Therefore,cold storage is usually used in production to extend the market supply period and storage period of‘Nanguo’pears.However,many years of production and practice have found that although cold storage can effectively delay the storage period of‘Nanguo’pears,long-term cold storage will cause the peel to brown.Therefore,study the mechanism of peel browning and explore the regulation technology is important.Previous studies in our laboratory found that energy deficit caused by abnormal energy metabolism is closely related to peel browning.This paper firstly analyzes the occurrence law of peel browning after different storage periods,and further analyzes the relationship between changes in the number,structure and function of mitochondria and browning,focusing on the key substances related to browning with mitochondria as the synthesis site Gamma-aminobutyric acid（GABA）,focused on the transcriptional regulation mechanism of transcription factors on their key genes in their synthesis,and further explored the corresponding regulation technology based on the above.Findings:（1）Occurrence of peel browning of‘Nanguo’pears refrigerated for 60 d,120 d and 180 d was significantly different during the shelf life at room temperature.Among them,the fruits refrigerated for 60 d did not show browning symptoms during the entire shelf life.However,different degrees of peel browning occurred in pears that were refrigerated for 120 days and180 days.The longer the period of refrigeration,the earlier and more severe the symptoms of peeling browning.The mitochondrial structure of‘Nanguo’pear peels that were browned during long-term storage at room temperature was significantly damaged,the mitochondria were severely swollen,and the mitochondrial membrane permeability was significantly increased.During cold storage,the content of ROS in fruits increased with the prolongation of cold storage time,while the contents of GABA and Spm did not change significantly,while the contents of Put and Spd decreased.During storage at room temperature after refrigeration,the content of ROS in fruits after refrigeration for 60 d was relatively stable,while the content of ROS in fruits after refrigeration for 120 d and 180 days increased rapidly,and the longer the refrigeration time,the greater the increase;the GABA content of fruits in different refrigeration periods In the early stage of shelf life,there was a gradual upward trend,but the GABA content of the fruits after 120 d and 180 d of refrigeration showed a sudden drop in the later stage of shelf life;the contents of Arg and Spm increased gradually with the prolongation of shelf life,and Put and Spd showed a decreasing trend,and the longer the refrigeration time,the greater the changes of the above indicators.The results of correlation analysis showed that the browning rate and browning index of fruits were significantly negatively correlated with GABA content after 120 d and 180 d of cold storage.The activity and relative expression level of GAD,a key gene of GABA synthesis in long-term refrigerated fruits,were higher than those in short-term storage fruits at the time of delivery,but their levels were significantly lower than those in short-term storage fruits at the later stage of shelf life.This indicated that the GABA synthesis ability of the fruit after long-term refrigeration was insufficient in the later shelf life.（2）In the fruits treated with 5 mmol/L GABA before refrigeration,the GABA content was significantly higher than that of the control during the refrigeration process and during the normal temperature shelf life after refrigeration.The ROS and MDA contents of the treated fruits were significantly reduced,and the mitochondrial structure was well maintained.After refrigeration,during the shelf life,the occurrence of peel browning was significantly alleviated.This proves that GABA is involved in regulating the peel browning process of refrigerated‘Nanguo’pear fruits.（3）Based on the results of transcriptome sequencing,the promoter sequence of PuGAD,a key enzyme gene for GABA synthesis in‘Nanguo’pear,was cloned.Two transcription factors,PuMYB10 and PuMYB91,were screened by yeast one-hybridization to bind to the PuGAD promoter;EMSA assay showed that PuMYB10 and PuMYB91 transcription factors could specifically bind to the cis-acting elements of CAACTG and TAACCA on the PuGAD promoter,respectively;dual fluorescein Enzyme assay and GUS activity analysis assay showed that both PuMYB10 and PuMYB91 could activate the expression of PuGAD,and PuMYB10 had higher transcriptional activation activity,and there was no interaction between the two transcription factors.RT-q PCR showed that PuMYB10 and PuMYB91 expression levels decreased under long-term low temperature stress;in different varieties of‘Nanguo’pear and‘Huagai’pear,the expression of PuGAD gene was also observed to be gradually up-regulated along with the up-regulation of PuMYB10 and PuMYB91 gene expression.Subcellular localization results showed that both PuMYB10 and PuMYB91 were located in the nucleus;phylogenetic tree analysis found that PuMYB10 was closely related to Co MYB10 of quince,and PuMYB91 of‘Nanguo’pear was closely related to Pd MYB1 of almond.Domain and protein structure analysis showed that both PuMYB10 and PuMYB91 belong to the R2R3-MYB family;The prediction results of protein phosphorylation sites and protein transmembrane structure suggest that both PuMYB10 and PuMYB91 proteins are non-secreted proteins,and both are phosphorylated mainly by serine to regulate the biological function of‘Nanguo’pear fruit.The above results confirmed that PuMYB10 and PuMYB91 positively regulate the transcription of PuGAD by targeting the PuGAD promoter,thereby participating in GABA’s resistance to the peel browning of‘Nanguo’pear caused by long-term low temperature stress.（4）The browning phenomenon of‘Nanguo’pear treated with 4%CaCl₂ was obviously alleviated after 180 days of storage.The contents of Ca²⁺and calmodulin in the pericarp of the treated fruits were significantly increased,the GABA content,GAD enzyme activity and gene expression,GABA branch-related enzyme activity and gene expression,and the gene expression levels of two transcription factors that regulate PuGAD transcription,PuMYB10and PuMYB91,were up-regulated.Structural integrity is maintained well.It can be seen that Ca²⁺treatment can promote the biosynthesis of GABA,thereby alleviating the peel browning of refrigerated‘Nanguo’pears.