Transcriptional Regulation Of MicroRNA-mRNA In Response To Low Phosphorus Stress Of Pinus Massoniana At Seedings

Phosphorus is an important component of plant biofilm and nucleic acid,and it is also an essential nutrient element in plant growth,development and metabolism.The scarcity of available phosphorus for plants to absorb and utilize in nature limits the increase in agricultural and forestry production.Analyze the response mechanism of plant phosphorus starvation stress in many aspects,so as to discover and utilize the plant phosphorus deficiency stress response gene.Selecting or creating new phosphorus-efficient germplasm is a highly feasible way to solve this problem.Masson pine(Pinus masoniana Lamb.)is one of the coniferous species unique to South China.It produces resin and is used to produce boards and paper,which has very important value in forestry economy.However,the soil of the origin of masson pine is lack of available phosphorus,which limits the growth and production of masson pine.Therefore,it is of great significance for improving the utilization efficiency of phosphorus,screening and cultivating new varieties of masson pine to clarify the response mechanism of masson pine to low phosphorus stress,explore the key regulatory factors of phosphorus deficiency response.Furthermore,it is of great significance for long-term development of the pine industry.In this study,m RNA and miRNA high-throughput sequencing were combined to obtain the differentially expressed m RNA and miRNA of masson pine under phosphorus starvation stress and normal phosphorus supply.Unigenes and miRNAs that might respond to phosphorus starvation stress were screened according to the function of m RNA and miRNA target genes by combining the phenotype and some physiological and biochemical indicators.The response system and molecular mechanism of masson pine tolerant to low phosphorus stress was elucidated from the miRNA and RNA levels.The main contents and results in present study are as follows:(1)Under severe low phosphorus stress(0.5 mg/L),masson pine seedlings had the longest total root length and the largest number of lateral roots.The total root length and the number of lateral roots of masson pine were negatively correlated with the concentration of phosphorus in the medium.However,there was no significant difference in the after 36 d of phosphorus starvation stress in the shoots of P.massoniana.In addition,under low phosphorus stress,the total phosphorus content of masson pine decreased with increasing time.Moderate low phosphorus stress(2 mg/L)was significantly lower than normal phosphorus supply(10 mg/L),severe low phosphorus stress was significantly lower than normal phosphorus supply before 36 days of stress,and the difference was not significant after 48 days.Total phosphorus content under severe low phosphorus stress was higher than that under mild low phosphorus stress,and the difference was significant at 12 days and 60 days of stress.It was speculated that because the number and total length of lateral roots under severe low phosphorus stress were more than those under mild low phosphorus stress,the plants could absorb more phosphorus from the medium,making the content of phosphorus in the plants under severe low phosphorus stress higher than that under mild low phosphorus stress.(2)Transcriptomic analysis showed that 7,049 genes were differentially expressed under phosphorus starvation stress,including 5,162 in roots and 2,955 in shoots.At 48 d of phosphorus deficient stress,the number of differentially expressed genes was the highest.Through the analysis of differentially expressed genes,phosphorus-related genes mainly included transcription factors(WRKY,MYB,b HLH),SPX,transporters,acid phosphatase,hormone signal related genes(auxin and ethylene)and metabolism-related genes.For example,Unigene0011607,which was annotated as MYB transcription factor,was upregulated in the shoots at 24 d under stress,and in the roots at 36 d and 48 d under stress.Ethylene transcription factor Unigene0010383 was down-regulated in 24 d shoots under low phosphorus stress and up-regulated in 36 d roots.PHT transporter,SPX gene and acid phosphatase gene were all up-regulated during phosphorus stress.(3)The miRNA analysis showed 190 miRNAs with differential expression,among which 125 miRNAs were differentially expressed in the underground part and74 miRNAs were differentially expressed in the shoots.The number of miRNAs differentially expressed in the first two periods of root and shoot during the low phosphorus stress was similar,and the number of miRNAs differentially expressed in the underground part was about 7 times that in the aboveground part 48 days after the treatment with low phosphorus stress.By predicting the function of miRNAs target genes,a number of miRNAs that may be related to low phosphorus stress in masson pine were excavated.For example,miR164-y was up-regulated in the intrinsic roots at36 d under stress.Two members of the miR169 family(miR169-x and miR169-y)were inhibited under stress.Three members of the miR399 family(miR399-x,miR399-y and miR399-z)were induced by low phosphorus expression.(4)The precursor sequence of Pma-miR156 and its target gene Pm-RANBP1 B were cloned and verified by genetic transformation.The results showed that the number of lateral roots of plants overexpressing Pma-MIR156 was more than that of wild-type and overexpressing Pm-RANBP1 B,but there was no significant difference between wild-type and overexpressing Pm-RANBP1 B.This indicated that the up-regulated expression of Pma-MIR156 increased lateral root synthesis,and Pm-RANBP1 B may be unrelated to lateral root formation.As one of the target genes of miR156,SPL plays a role in inhibiting the occurrence of lateral roots.It is speculated that miR156 of masson pine regulates the formation of lateral roots in response to low phosphorus stress except Pm-RANBP1 B.