Low Temperature Response And Functional Study Of MiR157c In Arabidopsis Thaliana

Low temperature is one of the main environmental factors affecting plant growth and geographical distribution.Brassinolide(BR)plays an important role in the response of plants to low temperature,but the mechanism is still unclear.In this paper,bioinformatics methods were used to analyze the transcriptome data of Arabidopsis substrate response which obtained in the early stage of the laboratory.It was found that MIR841 A and MIR157 C may play an important role in the response of BR mutants to low temperature.Therefore,this study further used physiological,biochemical and molecular biological methods to analyze the expression response and biological function of miR157 c under low temperature stress.The paper obtained the following results:1.Using Arabidopsis transcriptome data which resposed by wild-type and BR mutants in low temperature,it was found that MIR841 A and MIR157 C have the greatest betweenness in the transcriptional regulatory network which constructing by low temperature response genes,speculating that the two genes may be have an important role in the plants low temperature response which related to BR.2.To verify the results of transcriptome and bioinformatics analysis,the expression of miR157 c in wild-type Col-0 and BR synthesis or signaling mutants was analyzed by qPCR.It was found that in wild-type Col-0,the expression of miR157 c reduced low temperature treatment,while the expression of miR157 c was not affected by exogenous eBL.The expression of miR157 c reduced in the BR signal negative regulation factor BIN2 function-gained mutant bin2-1 and bzr1-D/bin2-1 by low temperature,and the BR synthetic mutants dwf4 and bri1-116/bzr1-D were not affected by low temperature.At room temperature,compared with wild type Col-0,the expression of miR157 c in BR receptor mutants bri1-116,dwf4 and bin2-1 was significantly decreased;after cold acclimation,the expression of miR157 c was significantly decreased in bin2-1 and was no difference in bri1-116 and dwf4,while bzr1-D restored the expression of miR157 c in bri1-116 and bin2-1 under both treatment conditions.The above results indicated that BR mutation affected the response of miR157 c to low temperature.3.SPL6(SQUAMOSA promoter binding like),SPL9,SPL10,SPL11,SPL15 may be potential target genes for miR157 c by informatics prediction.qPCR quantitative analysis showed that the expression of the above SPLs was opposite to that of miR157 c at low temperature.These results indicate that SPL6,SPL9,SPL10,SPL11 and SPL15 may be target genes of miR157 c.4.The miR157 c transgenic plant(oxmiR157c)with Arabidopsis wild type Col-0 as background was obtained.The miR157 c transgenic plant of BR synthesis mutant dwf4,BR receptor mutant bri1-116,BR signal negative regulation factor BIN2 function-gained mutant bin2-1 were obtained by artificial hybridization(dwf4/oxmiR157 c,bri1-116/oxmiR157 c,bin2-1/oxmiR157c).By observing the growth phenotype,compared with wild-type Col-0,the primary root length of miR157 c transgenic plants became shorter at normal temperature,the number of rosette leaves increased,the flowering was delayed,the number of tillers increased,the plant height became shorter,the number of lateral branches and lateral branches diverage increased,the number of pods increased and the length of the pod is shortened.5.By measuring the electron leakage rate and low temperature survival rate,BR synthesis mutant dwf4 and the receptor mutant bri1-116 showed stronger constitutive antifreeze ability than the wild type,while the BR signal negative regulation factor BIN2 function-gained mutant bin2-1 showed a sensitive phenotype to freezing damage;under cold acclimation conditions,dwf4,bri1-116 and bin2-1 showed a sensitive phenotype to freezing damage.Compared with wild type plants,the freeze resistance of transgenic plants did not change significantly,CBFs and CORs gene expression have no significant difference with or without cold acclimation.The results of physiological indicators showed that the fresh weight and dry weight of the transgenic plants were significantly higher than those of the wild type,but the relative water content was not significant.At normal temperature,there was no significant difference in chlorophyll and proline content in the transgenic plants,but the two indicators are reduced after cold acclimation.The above results indicated that BR is involved in the low temperature response process of Arabidopsis thaliana,but miR157 c does not affect the freeze resistance of Arabidopsis thaliana,and the expression of cold response genes such as CBF1,CBF2,CBF3,COR15 A,COR47 and COR78 is not dependent on miR157 c.6.By analyzing the expression of several potential target genes of miR157 c in transgenic plants,we found that the expression trend was not completely opposite to that of miR157 c,indicating that the expression of SPLs may be affected by other factors besides miR157 c.In summary,the results of this paper displayed that the expression of miR157 c in Arabidopsis thaliana is regulated by low temperature and BR signal.miR157 c does not affect the freezing resistance of plants,but participates in the growth and development of plants.This study is helpful to understand the interaction mechanism between BR and miRNA in response to stress,and it plays an important role in further exploring the detailed molecular mechanism of plant regulation between low temperature and growth.