Identification And Biological Function Analysis Of Long Non-coding RNA Involed In Phytoplasma Infection In Mulberry

Mulberry yellow dwarf disease is a devastating mulberry disease,which seriously affects the realization of the economic and ecological value of mulberry trees.However,the molecular mechanism of the disease is still unclear.In this study,the chain-specific transcriptome sequencing technology was used to analyze the expression profiles of mRNAs and lncRNAs in healthy and phytoplasma-infected(infected)leaves,and the mRNAs and lncRNAs that involved in the response to phytoplasma infection in mulberry were identified.Moreover,the metabolic pathways of the differentially expressed mRNAs and lncRNAs were analyzed using bioinformatics technology.Based on the achievement of the transcriptomic analysis the biological functions of one of the differentially expressed LRR receptor-like serine/threonine-kinase gene(putative LRR receptor-like serine/threonine-Protein kinase)(named as Mul-LRR)and its antisense lncRNA(named as Mul-LRR-AS)and their interactions were studied in depth.The main results of this study are as follows:(1)Construction and sequencing analysis of the chain-specific transcriptome sequencing libraries of mulberry leaves The chain-specific transcriptome sequencing libraries of healthy and phytoplasma-infected mulberry leaves was successfully constructed and sequenced by Illumina Solexa sequencing technology,respectively.A total of 99 933 mRNAs and 16 805 lncRNAs was identified.Among the mRNAs identified,there were 9600 mRNAs differentially expressed in healthy and infected leaves,and there were 3049 genes up-regulated and 6551 down-regulated in the infected leaves.Differentially expressed mRNAs were predicted to be involved in various physiological processes such as growth and development,metabolism,signal transduction,stress response and etc.There were 943 lncRNAs were detected differentially expressed in the healthy and infected leaves,and 220 lncRNAs were up-regulated and 723 were down-regulated in the infected leaves.Among the differentially expressed lncRNA and mRNAs,there were 318 lncRNA and mRNAs predicted to have antisense interactionsand and 49 of them were associated with plant disease resistance.(2)Functional analysis of Mul-LRR gene and its antisense lncRNA(Mul-LRR-AS)The Mul-LRR gene was cloned by PCR and its plant expression vector was constructed successfully.The transgenic Mul-LRR gene Arabidopsis was obtained,and it was found that overexpression of Mul-LRR gene in Arabidopsis can enhance the resistance of transgenicplants to Pseudomonas syringae pv.Tomato DC3000,(Pst.DC3000).Meanwhile,the Mul-LRR-AS gene was also cloned by PCR,and its plant expression vector was constructed.The transgenic Mul-LRR-AS plants were obtained and used to interbreed with the transgenic Mul-LRR-AS plants to obtain hybrid plants.Real-time PCR analysis showed that the LRR-AS was successfully expressed in the hybrid plants,and the expression of Mul-LRR gene was inhibited by the expression of Mul-LRR-AS.The resistance of the hybrid plants to Pst.DC3000 was significantly reduced compared with that of the transgenic Mul-LRR gene plants.These indicated that Mul-LRR-AS can negatively regulate the resistance of plants to Pst.DC3000 by inhibiting the expression of Mul-LRR gene.(3)Expression activity analysis of the promoters of Mul-LRR and Mul-LRR-AS The promoter sequences of Mul-LRR and Mul-LRR-AS genes were cloned by PCR and were named pMul-LRR and pMul-LRR-AS,respectively.Both the two promoter nucleotide sequences contain multiple promoter core elements such as CAAT-Box and TATA,but they contain different hormone and environmental responsive elements,which indicated that they may have different inducible expression activities.Combined tobacco transient infection expression system with GUS histochemical staining,it was demonstrated that it was the activity of pMul-LRR not of pMul-LRR-AS was induced by pathogenic bacteria,jasmonic acid and salicylic acid.The information obtained in this study will laid a foundation for further study of the biological functions of Mul-LRR and its antisense lncRNA.It will be is of great significance to broaden and enrich the understanding of the function and mechanism of lncRNA in plants,and to elucidate the molecular mechanism that operates under phytoplasma infection comprehensively.Moreover,it will be fundamental to advance the research of the functional genomics of mulberry and provide effective candidate genes for resistant breeding of mulberry trees.