| Bell pepper(Capsicum annuum L.)is one of the indispensable and important vegetables in people’s lives.It is rich in nutrients and has a crisp taste,which is popular to consumers.After harvest,the physiological metabolism of green pepper fruit is vigorous,and its quality rapidly decreases at room temperature,with severe decay occurring.Low temperature storage and transportation is the main preservation measure.However,bell pepper is native to tropical and subtropical regions such as South America and Mesoamerica,and is sensitive to low temperatures.Under unsuitable low temperatures,fruits can suffer from cold damage symptoms,manifested by pitting and watering stains on the surface,browning of seeds,calyxes and stalks,which seriously affect their quality.At the same time,the ability of fruits subjected to resist pathogenic microorganisms has significantly decreased,making them prone to decay.Therefore,it is extremely important to study the mechanism of chilling injury of bell pepper under low temperature stress and explore regulatory techniques.In this paper,firstly,by observing and monitoring the changes in the apparent and microscopic morphology of bell pepper under different temperatures,physiological and biochemical indicators related to chilling injury,fatty acid composition and content,and changes in the expression of desaturase genes,key desaturase genes were selected,and their promoters were used as target sequences to screen and identify upstream key transcription factors.With a series of molecular biological techniques,the transcriptional regulation mechanism was studied,and on this basis,the transcriptional regulation of this transcription factor on key genes in cold signal transduction pathway was further studied.The level of DNA methylation of the promoter of transcription factor genes under low temperature stress was explored,revealing the molecular mechanism of cold injury in bell pepper,providing theoretical basis and new insights for improving post-harvest processing technology of bell pepper.The main research results are as follows:(1)At 10°C,there was no chilling injury in pepper,while at 4°C,there were significant chilling injury symptoms in pepper fruit,which were characterized by large areas of water stains on the surface,and browning of the calyx,fruit handle,and seeds.Under the transmission electron microscope,it can be seen that the parenchyma cells of the fruit pulp after chilling injury have undergone plasmolysis,the membrane system of the system has been severely damaged,the chloroplasts have swelled,and the lamellar structure has disintegrated.The LOX activity,relative electrical conductivity,MDA and proline contents of fruits at 4°C significantly increased,while the GSH and As A contents significantly decreased.This indicates that low temperature may cause an imbalance of active oxygen species and a decrease in antioxidant capacity in pepper,leading to oxidative damage to cell membranes,and exacerbating the symptoms of chilling injury in green pepper.GC-MS found the presence of four fatty acid components in green pepper,namely,C16:0,C18:0,C18:2,and C18:3.The quantitative results and unsaturation evaluation indicators showed that 4°C significantly reduced the content of C18:2 and C18:3 unsaturated fatty acids and fatty acid unsaturation in pepper.The results of gene expression analysis showed that low temperature can simultaneously cause changes in the expression of multiple desaturase genes,among which the changes in the expression of Ca SAD2 and Ca FAD7 are significantly negatively correlated with the increase in membrane permeability(**P<0.01),indicating that the decrease in the expression of Ca SAD2 and Ca FAD7 at low temperature causes a decrease in the content and unsaturation of unsaturated fatty acids,affecting the fluidity and permeability of cell membranes,this leads to the leak of cellular content,which destroys the cellular structure,and ultimately leads to the occurrence of chilling damage to bell pepper.(2)The promoter sequences of Ca SAD2 and Ca FAD7 were cloned and the key upstream transcription factor Ca MYB340 was selected from the transcriptome data using yeast single hybridization(Y1H).Electrophoretic mobility shift assay(EMSA),bioinformatics analysis,subcellular localization,yeast two hybrid(Y2H),GUS activity and double luciferase experiments showed that Ca MYB340 is a cold induced R2R3 type MYB transcription factor located in the nucleus,which can specifically recognize the core motif of Type ⅡG MYB in Ca SAD2 and Ca FAD7 promoter sequences and bind to it,Moreover,it can negatively regulate the transcription and expression of downstream target genes by combining with itself to form homodimers or with Cab HLH96/Ca MYB1R1 to form heterodimers.Finally,this negative regulatory model was further validated by transient overexpressing and silencing of Ca MYB340 in bell pepper.(3)RT-q PCR was used to analyze the gene expression of Ca CBFs in ICE-CBF cold signal transduction pathway under low temperature,and Ca CBF3 was identified as a key gene involved in the occurrence of chilling injury in bell pepper.Y1 H and EMSA experiments have shown that Ca MYB340 can specifically recognize the Type ⅡG MYB core motif in the Ca CBF3 promoter sequence and bind to it.Meanwhile,GUS and dual luciferase experiments showed that Ca MYB340 and its interacting protein can significantly inhibit the transcription and expression of Ca CBF3.The above results indicate that the transcription factor Ca MYB340 and its interacting protein can not only inhibit the expression of the key desaturase genes Ca SAD2 and Ca FAD7 involved in unsaturated fatty acid metabolism,but also participate in the occurrence of chilling injury through negative regulation of key genes in the ICE-CBF cold signal transduction pathway.(4)GUS and RT-q PCR experiments showed that the Ca MYB340 promoter is cold sensitive,and its expression level can be significantly upregulated within 1 hour of exposure to low temperature,reaching a peak at the second day.Mcr BC experiments have shown that low temperature can significantly increase the DNA methylation level of the Ca MYB340promoter-1491 bp —-1774 bp fragment.Sulfite transformed DNA and PCR experiments(BSP)showed that the sequence of Ca MYB340 promoter from-1491 bp to-1774 bp contained 46 methylation sites,including 4 CG,10 CHG,and 32 CHH.The lowtemperature induced DNA methylation modification occurred at the 13 th CHH site,indicating that low temperature activates Ca MYB340 promoter activity and gene transcriptional expression by causing changes in its methylation level,thereby inhibiting its downstream target desaturase genes and ICE-CBF cold signal transduction pathway,ultimately triggering the occurrence of chilling injury in bell pepper fruit. |
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