| Loquat(Eriobotrya japonica Lindl.)is a subtropical evergreen fruit tree in the genus Eribotrya of Rosaceae.There are about 32 species(varieties),but only one cultivated species.Its characteristics such as narrow genetic base,poor resistance,low seed edibility and low photosynthetic efficiency cannot be improved for a long time.In addition,loquat trees bloom in early winter,mature in early summer,fruit and fruit are easily hurt by low temperature,seriously affecting the yield and quality.Therefore,it is of great significance for industrial development to cultivate new varieties with excellent comprehensive characters such as cold resistance,seedless,high light efficiency and so on.Triploid loquat is characterized by strong resistance,seedless and increased contents.Therefore,it is one of the important breeding objectives to select a new variety of cold-resistant triploid loquat.In the early stage,the research group created a batch of triploid F1 generation from wild Guizhou loquat(2X,GZ23)and Longquan 1(4X,B431).Under low temperature stress,it was found that the triploid F1 generation had the characteristics of frost resistance.Transcriptome analysis showed that EjTIFY6 B and EjMYC2 genes were closely related to triploid frost resistance.Previous studies have shown that in the jasmonic acid pathway,TIFY protein with complete JAS domain specifically identifies with Co I1 gene,leading to ubiquitination degradation of TIFY protein and release of MYC2 class transcription factors to regulate downstream JA response genes.Moreover,TIFY protein family plays an important role in plant response to biological and abiotic stresses,and MYC transcription factors also directly regulate plant growth,development and stress tolerance.However,the functions and regulatory mechanisms of EjTIFY6 B and EjMYC2 genes in loquat are still unclear.Therefore,EjTIFY6 B,EjMYC2 and EjCOI1 genes were cloned in this study,and their transcriptional activation activities and interactions were verified by yeast biheterozygous analysis.The expression patterns of EjTIFY6 B and EjMYC2 were further analyzed,and the function of the gene was verified by transplanting Arabidopsis thaliana,and then the function of the gene in response to low temperature stress in triploid loquat was analyzed.The main research results are as follows:(1)Changes in endogenous JA content and oxidase activity of triploid loquat under cold stressIn this study,each ploidy loquat was treated at-5℃,-3℃,0℃ and CK(25℃).After 3 days of treatment,the endogenous JA content of loquat was measured.The results showed that the content of endogenous JA in each ploid loquat increased significantly with the decrease of temperature,and the content of endogenous JA in triploid loquat was significantly higher than that in diploid and tetraploid loquat at room temperature.The leaves of triploid loquat were treated at-3℃ and MeJA was applied to measure the changes of oxidase activity within 2 days.The results showed that the activity of oxidase increased significantly with the decrease of temperature.After the application of MeJA,the activities of SOD,POD and CAT oxidase increased significantly.Except for SOD oxidase,the activities of POD and CAT oxidase increased gradually with the longer the low temperature treatment time,and reached the peak at24 h,while the activity of SOD oxidase reached the peak at 6h,and then decreased gradually.(2)Gene cloning,expression and bioinformatics analysisThe CDS lengths of EjTIFY6 B,EjMYC2 and EjCOI1 were 1188 bp,2058bp and1764 bp,respectively,encoding 395,685 and 587 amino acids and a stop codon from triploid loquat,respectively.By sequence comparison of the amino acids encoded by EjTIFY6 B and EjMYC2,it was found that the intermediate sequence and C terminal of EjTIFY6 B gene had conserved and complete TIFY/ZIM and Jas domains,respectively,and the EjMYC2 gene sequence had conserved JID domain,Tad domain and b HLH domain.The phylogenetic tree analysis showed that the EjTIFY6 B and EjMYC2 genes were closely related to TIFY6 B and MYC2 genes in apples and pears.Loquat,apple and pear all belong to Rosaceae,which is consistent with traditional taxonomy.Real-time fluorescence quantitative analysis showed that compared with CK,the expression level of EjTIFY6 B gene under-3 ℃ low temperature treatment had a significant tendency to decrease,while the expression level of EjMYC2 gene had a significant trend to increase.After MeJA application,the expression of EjTIF6 B gene reached the peak at 12 h and then decreased,and the possible downstream low-temperature response genes ICE,CBF1 and CBF2 showed a corresponding trend of change.Therefore,the author preliminary speculated that in the Jasmonic acid signaling pathway in response to low temperature in Loquat,EjTIFY6 B may act as a repressor of transcription factor to respond to Jasmonic acid signal,and EjMYC2 as a transcription factor can induce the expression of downstream COR cold-resistant genes through the ICE-CBF pathway.Furthermore,we constructed the plant fluorescence expression 35S::EjTIFY6B-GFP,which was displayed in the nucleus by subcellular localization,indicating that it functions in the nucleus and has the characteristics of complete nuclear localization of jas domain.(3)yeast double hybrid analysisIn order to verify the interaction between EjTIFY6 B,EjMYC2 and EjCOI1 genes,A yeast double hybrid vector p GBKT7-EjMYC2,p GADT7-EjMYC2 and p GADT7-EjCOI1 was constructed.The results showed that EjTIFY6 B had no transcriptional autoactivation activity,while EjMYC2 had transcriptional autoactivation activity.Co-transformation experiments showed that EjTIFY6 B interacted with EjMYC2 and EjCOI1,respectively.(4)Functional analysis of transformed Arabidopsis thalianaThe transgenic plants of EjTIFY6 B and EjMYC2 were successfully obtained by inflorescence infection.The leaves of transgenic Arabidopsis thaliana were larger than that of wild-type Arabidopsis.Root sensitivity experiments showed that,compared with wild-type Arabidopsis thaliana in MS medium containing MeJA,the roots of transgenic Arabidopsis EjTIFY6 B grew significantly and were insensitive to MeJA.However,the roots of transgenic Arabidopsis thaliana with EjMYC2 were significantly shortened and showed strong sensitivity to MeJA.Under low temperature treatment,the expression pattern of transgenic plants EjTIFY6 B and EjMYC2 was as follows: EjTIFY6 B was rapidly induced within 1h,and the expression level gradually decreased,while the expression level of EjMYC2 increased with the longer the low temperature treatment time.After low temperature treatment,EjMYC2 transgenic Arabidopsis thaliana showed no obvious wilting,and its relative conductivity was lower than that of wild type.After returning to normal temperature,the survival rate was higher than that of the wild type,and the growth rate was also significantly stronger than that of the wild type.The leaves of transgenic Arabidopsis EjTIFY6 B showed a lot of wilting,and the relative electrical conductivity was significantly higher than that of wild type.After returning to normal temperature,the survival rate was lower than that of the wild type,and the growth rate was also significantly weaker than that of the wild type.In conclusion,EjTIFY6 B and EjMYC2 jointly respond to low temperature stress response of loquat,with the former negatively regulating and EjMYC2 positively regulating.However,the changes of their expression patterns in different ploidy loquats,especially the expression patterns and molecular regulation mechanisms related to freezing resistance of triploid loquats,remain to be further studied. |
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