Molecular Mechanism Study On MiRNA Regulation Of Tamarix Chinensis Salt Tolerance

High salt stress caused by saline-alkali land has a direct impact on crop yield.China’s saline-alkali land covers an area of more than 200,000 square kilometers.It is of great significance to improve overall agricultural production capacity and solve the food crisis by understaning the mechanism of plant salt tolerance and making full use of saline-alkali land with anti-stress breeding.Compared with herbaceous plants,salt-tolerant tree species have excellent characteristics of high salt-tolerance.Non-coding small molecular micro RNA(micro RNA,mi RNA)plays an important regulatory role in plants’ response to stress.By study on post-transcriptional regulatory mechanism,mi RNA molecular mechanism of forest for regulating salt-tolerance would be elucidated,which has important guiding significance for the evaluation of salt-tolerant germplasm resources and the genetic improvement of plant stress tolerance.As one of the most salt-tolerant species,Tamarix chinensis Lour.is an excellent material for the study of tree salt-tolerance.In this study,s RNA-seq was used to scan the whole genome of T.chinenses roots.The regulatory network of mi RNA and target genes was established.Two stress-induced micro RNAs(tch-mi R156 and tch-mi R164)with target genes were predicted and verified.The heterologous overexpression of the resistance target gene confirmed that tch-mi R156 and tch-mi R164 were positive regulators of salt tolerance.The main results are as follows.I.Small RNA sequencing was performed on the root tissues of T.chinensis under salt stress.288 mi RNAs were predicted and their expression profiles in the root tissues were established.191 differentially expressed micro RNAs and 706 target genes were predicted.A regulatory network of micro RNAs and salt-tolerant-target genes was predicted by combining salt stress related genes annotated in transcriptome data.II.Three mi RNAs(ath-mi R164 a,ath-mi R156 a and ath-mi R157a)were selected as candidates according to the mi RNA expression profile and the quantitative PCR verification with stem-loop primers.The expression profiles of target gene families under salt stress were constructed by RT-q PCR.NAC(No Apical Meristem,ATAF1/2 and CUC2)family and SBP(SQUAMOSA promoter binding protein)family were selected as candidate target genes.III.Based on the de novo prediction process,the primary transcripts corresponding to ath-mi R164 a and ath-mi R156 a were predicted.The two genes’ full length were verified by RACE.Five targets of mi RNA156(SBP1-5)and three targets of mi RNA164(NAC1-3)were verified by constructing transient overexpression vector of mi RNA and co-transforming protoplast with dual luciferase reporter vector.IV.The full length of 5 SBP target genes and 3 NAC target genes were cloned by RACE.The expressions model of target genes were analyzed.NAC1,which was specifically expressed in roots and was quickly inhibited by salt stress,and SBP5,which had consistent expression patterns in roots,stems and leaves and was significantly negatively correlated with mi RNA156 expression,were selected as the target genes for functional verification.Through multi-point mutation of mi RNA response elements of NAC1 and SBP5,mi R156 resistance targets r SBP5 and mi R164 resistance targets r NAC1 were obtained.The two resistance targets were verified to be typical nuclear localization.V.Genetic transformation of resistance targets r SBP5 and r NAC1 showed that,under 0.3%salt stress,the germination rates of NAC1-over-expressrd and SBP5-over-expressrd Arabidopsis thaliana were significantly lower than that of the wild type.And the growth of seedling roots and cotyledons was significantly slowed down for stress inhibition.In addition,compared with the over expression of SBP5,NAC1 overexpression inhibited the germination rate and seedling growth more significantly.In summary,the results of this study showed that tch-mi R156 could inhibit salt-sensitive induction by silencing target gene SBP5,and tch-mi R164 could inhibit salt-sensitive induction by silencing target gene NAC1.Based on our results and literature,we speculated the model for salt-tolerance regulation in T.chinenses.Under salt stress,tch-mi R156/164 genes were induced quickly to accumulate a large number of mi R156/164.The post-transcriptional silencing of SBP\NAC mediated by tch-mi R156\tch-mi R164 correspondingly decreased transcription activity of downstream genes and expression level.At last,T.chinensis high salt tolerance was induced by relieving salt sensitive state.In conclusion,tch-mi R156 and tch-mi R164 regulated T.chinensis to adapt to salt stress by transcriptional silencing NAC1 and SBP5.