Discovery And Functional Verification Of POD Related Genes In Pear Flower Bud Dormancy

Pear belongs to the temperate deciduous fruit tree of Pyrus L.of Rosaceae and Amygdaloideae,which has the biological characteristics of dormancy in winter.The pears planted in the north of China are mainly late maturing varieties,which generally mature in the middle of September.While the pears planted in the South have the advantage of early maturity,which can be listed before August,and occupy an important position in the market.In recent years,the global climate change is abnormal and the annual temperature shows a gradual upward trend.The insufficient low-temperature accumulation of pear during dormancy in winter leads to the failure of pear flower buds to bloom and bear fruit normally the next year,which affects the fruit yield and quality.And it causes great economic losses to the pear industry.Therefore,the study on the dormancy mechanism of pear flower buds are of great significance in agricultural production.In pear,peach and other fruit trees,it was found that the content of H₂O₂ in flower buds continued to rise with the process of dormancy and decreased sharply with the end of dormancy,which was consistent with the process of natural dormancy.H₂O₂ and other reactive oxygen species in fruit trees might be the key signal molecules regulating the dormancy process.Taking‘Huanghua’pear as the experimental material,when analyzing the H₂O₂ metabolic balance in the process of pear flower bud dormancy.Some POD genes with significant differential expression were screened according to the NCBI pear genome database and the transcriptome database of pyrus pyrifolia at different dormancy stages established in the laboratory.The dormancy model of pear bud induced by low temperature was established.The germination rate in the dormancy process of pear bud was counted and the changes of related substances were measured.And the flower buds in internal dormancy were sprayed with 2%H₂O₂ and 2%DMTU（N,N’-dimethylthiourea,H₂O₂ scavenger）to study the regulation mode of H₂O₂,POD and POD genes during flower bud dormancy.Using the differentially expressed mi RNAs transcriptome database and lnc RNAs transcriptome database established in the early stage of pear flower bud dormancy,a POD gene closely related to H₂O₂and mi RNAs and lnc RNAs interacting with the gene were jointly screened and analyzed.The regulatory relationship between POD genes and corresponding mi RNAs and lnc RNAs were verified by tobacco transient transformation and double luciferase interaction technology.The main results are as follows:1.In this paper,seven significantly differentially expressed POD genes were screened from two databases.Under the dormancy induction treatment at 4℃,the germination rate of pear flower bud in 0～21 days was less than 50%,which was in the internal dormancy stage.21～28 days was the period from dormancy to ecological dormancy.After 28 days,the germination rate was more than 50%,which was in the stage of ecological dormancy.In the process of pear flower bud dormancy,the content of H₂O₂increased first and then decreased.And then it increased rapidly in the process of inner dormancy,decreased rapidly after entering ecological dormancy.The change trend of POD activity was consistent with that of H₂O₂ content.Different POD genes played different roles in different dormancy stages.The results of this study showed that pear flower buds would produce dormancy release signal substance H₂O₂ under the induction of low temperature.Due to the accumulation of H₂O₂,the expression of a large number of POD genes was activated,which affected the activity of POD and regulated the level of H₂O₂ in plants,so as to transmit the signal to flower buds to release dormancy.2.After different treatments on pear flower buds in internal dormant state,the results showed that H₂O₂ promoted the germination of flower buds,and the germination rate increased by 3.1%compared with the control.Compared with the control,the germination rate of DMTU treatment decreased by 0.18%.After H₂O₂ treatment,the content of H₂O₂and the activity of POD in dormant pear flower buds were higher than those in the control.The average content of H₂O₂ and the average activity of POD were 12.92%and 9.06%higher than those in the control.After DMTU treatment,the content of H₂O₂ and the activity of POD of dormant pear flower buds were lower than those of the control.The average content of H₂O₂ and the average activity of POD were 7.57%and 16.95%lower than those of the control.Spraying H₂O₂ could change the expression of POD genes in varying degrees.The results showed that exogenous H₂O₂treatment could break the dormancy of pear flower buds to a certain extent.H₂O₂ could induce the expression of POD genes in varying degrees,and low temperature could improve the POD activity of pear flower buds,thus affecting the dormancy process of pear flower buds.3.In this paper,a PpPOD4-like gene with 984 bp CDS was cloned from‘Huanghua’pear flower bud.And its encoded protein was a stable secretory basic hydrophilic protein.The analysis of promoter cis acting elements showed that PpPOD4-like promoter contained a large number of cis acting elements such as hormone response elements and light response elements.Among them,ABA response elements were associated with stress and signal transduction of corresponding hormones involved in genes.It was speculated that the expression of PpPOD4-like might be induced by ABA,thus affecting the dormancy process of flower buds.The results of subcellular localization showed that PpPOD4-like protein was localized in the cell membrane,and PpPOD4-like protein could regulate the H₂O₂ level of tobacco leaves.The expression of PpPOD4-like showed an upward trend in the process of flower bud dormancy,indicating that PpPOD4-like in pear flower bud regulated the content of H₂O₂ by regulating the activity of POD,so as to participate in the regulation of pear flower bud dormancy.The results showed that in the process of dormancy,low temperature induced the expression of PpPOD4-like,which regulated the content of H₂O₂ through POD,and then participated in the regulation of dormancy release.At the same time,it was speculated that low temperature promotes the production of ABA,and the expression of PpPOD4-like induced by ABA affected the dormancy process of pear flower buds.4.In this paper,mi Run068 and Pp L-T19077 related to PpPOD4-like were screened.The results of q RT-PCR showed that the expression trend of PpPOD4-like and Pp L-T19077 was consistent in the dormancy process of pear buds,while the expression trend of mi Run068 was opposite to that of PpPOD4-like and Pp L-T19077.PpPOD4-like was induced by exogenous H₂O₂,while the expression of mi Run068 was not affected by the change of H₂O₂ content.And the expression of Pp L-T19077 was affected by the change of the content of H₂O₂.The results showed that the release of pear flower bud dormancy might be related to the low expression of mi Run068 and the high expression of PpPOD4-like and Pp L-T19077.At the same time,PpPOD4-like was induced by exogenous H₂O₂.5.The results showed that both PpPOD4-like and Pp L-T19077 were targeted and regulated by miRun068.As a competitive binding target site between e TMs of miRun068 and PpPOD4-like,PpL-T19077 reduced the inhibitory effect of mi Run068 on PpPOD4-like.In this study,we confirmed the inhibitory effect of mi Run068 on PpPOD4-like and PpL-T19077.PpL-T19077 participated in the regulation of pear bud dormancy process by interfering with the inhibitory effect of miRun068 on PpPOD4-like.