Study On The Function Mode Of Key Genes And Regulatory Mech Anism Of Non-coding RNAs Involved In Eu-rubber Biosynthesis

Eucommia ulmoides Oliver.,which belongs to Eucommia,Eucommiaceae,is an important hardy rubber-producing species native to China.Its leaf,flower,fruit,bark,and root contain cotton-like rubber.The rubber produced by E.ulmoides,also called Eu-rubber,has great potential for development due to its “rubber and plastic” character.However,the relatively low rubber content results in high cost in the commercial process of Eu-rubber.Our previous studies found the main pathways for Eu-rubber biosynthesis,identified Eu-rubber related genes and found that E.ulmoides has specifically differentiated a gene group FPSII（including FPS1,FPS3,and FPS5）,which can synthesized the long-chain trans-polyisoprene.FPS5 was considered as the key gene for Eu-rubber accumulation in fruits.However,the action mode of key genes involved in Eu-rubber biosynthesis and regulation role of non-coding RNAs during Eu-rubber biosynthesis are still unclear.In this study,we revealed the regulation mechanism of the FPS5 promoter through cis-element prediction,promoter activity detection,and trans-acting elements screening;identified the interaction proteins of FPSII through yeast two-hybrid,BIFC,and subcellular localization methods;constructed the non-coding RNA regulation network of FPS5 by the identification of lncRNAs and miRNAs involved in Eu-rubber biosynthesis.Our result is of great theoretical and practical value in the breeding of E.ulmoides with high rubber yield.The main findings are as follows:（1）The regulation mechanism of trans-acting factor on FPS5 was revealed.Bioinformatics analysis of the promoter regions revealed that the promoter of FPS5 specifically contained ehtylene responsive element and salicylate responsive element compared with FPS1,suggesting that the expression of FPS5 may be more susceptible to enthylene and salicylic acid.Through yeast one-hybrid,two the trans-acting element interacting with the FPS5 promoter was screened out-ethylene responsive transcription factor（ERF）,and DNase I.By gene expression analysis,we found that ERF negatively regulates the expression of FPS5.We also found that the expression patterns of genes involved in ethylene transtuction pathway（such as ETR1,CTR,and ERF）were negatively correlated with the accumulation of Eu-rubber.Whereas,at least one gene’ expression in each step in ethylene biosynthesis pathway（such as SAMS1,ACS1,and ACO1）was significanly positively correlated with the accumulation of Eu-rubber.These results indicated that the ethylene synthesized in fruit regulates the expression of FPS5 via ERF involved in ethylene transduction pathway,and ultimately affecting the biosynthesis of Eu-rubber.（2）The interacting proteins of key enzymes involved in Eu-rubber biosynthesis were screened out.Through yeast two-hybrid,we screened out 34 and 21 positive clones for baits FPS5 and FPS1,respectively.Among them,31 out of 34 clones screened out by FPS5 were small rubber particle proteins（1 SRPP1 and 31 SRPP7）,and the other 3 clones were bHLH,SWAP,and PDF1 B.16 out of 21 clones screened out by FPS1 were SRPP（1 SRPP1 and 15 SRPP7）,and the other 5 clones were ERD7,RNA biding proteins,and so on.Both SRPP1 and SRPP7 interacts with FPS1 and FPS5,respectively,and the expressions of SRPP1 and SRPP7 were significantly correlated with Eu-rubber accumulation.The interactions of SRPP1+FPS1 and SRPP7+FPS1 were verified by BIFC.These results indicated that SRPP and FPS might function as a complex in Eu-rubber biosyhthesis.（3）The co-expression network of key gene FPS5,which is regulated by lncRNA,was constructed.The middle of the pericarp has a rich Eu-rubber content,whereas the margin of the pericarp is almost free of Eu-rubber.The Eu-rubber accumulates quickly in May and almost stagnetes in June.Transcriptomic studies indicated that the expression of FPS5 in the middle part of pericarp in May was significantly higher than that in the margin of the pericarp and in the middle and margin of the pericarp in June with log2 foldchange ≥ 3.By differential gene expression analyses,we identified 11 differentially expressed lncRNAs（DE-lncRNAs）and 18 DE-TUCPs（transcripts with uncertain coding potential）and their 103 target DE-mRNAs.The Pearson correlation coefficint of DE-lncRNAs/TUCPs with FPS5 ranged from 0.95 to 1.0.Through constructing the co-expression network of 103 DE-mRNAs and FPS5,we identified 13 hub genes.The expressions of hub genes and FPS5 were extreamly significantly correlated with Pearson correlation coefficient ranging 0.9-1.0.The function of 8 out of 13 hub genes are correlated with cell cycle,cell division,cell wall,microtube,and nucleotide metabolism.Since the density of laticifers in the middle of the pericarp（covering the seed）is higher than that in the margin of the pericarp,and the Eu-rubber accumulation was alonged with the development of laticifers,we proposed that these 8 hub genes might be involved in the development of laticifers to provide sufficient “room” for Eu-rubber granules,which indicated that lncRNAs/TUCPs regulated the expression of FPS5 and laticifers development-related genes at the same time,ultimately affecting the synthesis of Eu-rubber.（4）The ceRNA network of FPS5 during Eu-rubber biosynthesis was constructed.Through high-throughput sRNA transcriptome sequencing of the same samples colletected in（3）and bioinformatics analysis,we 12 miRNAs could be targeted by FPS5.Among them,3 miRNAs（miR858a,novel₂26,and novel₂27）could also be target by 4 lncRNAs,indicating that these 4 lncRNAs might regulate the expression of FPS5 by these 3 miRNAs.We also found 7 miRNAs（such as mi R164 a,miR169h,and novel₇4）could also be targeted by 6 mRNAs,indicating that these mRNAs might regulate the expression of FPS5 by competing with FPS5 for binding to the same miRNAs.Function analysis of 6 mRNAs revealed that HMGS1,HMGS2,and HMGR2 were involved in Eu-rubber biosynthesis;KIN14E might be involved in the development of pericarp;MYB61 could indirectly regulate the expression of FPS5 through miR858a;HMT-1 encodes an enzyme catalyzing the synthesis of ethylene precursors,which further demonstrated that the ethylene biosynthesis pathway could interact with the Eu-rubber biosynthesis pathway through ERF and miRNA（novel₇4）,and these two pathways were positively correlated.