Identification And Functional Prediction Of Long Non-coding RNAs In The Genesis Of Populus Euphratica Oliv.heteromorphic Leaves

Populus euphratica.Oliver is a unique precious forest resource in desert areas and the only natural tree species in the Taklimakan Desert.P.euphratica has strong vitality,and has the characteristics of salt resistance,alkali resistance,cold resistance,drought resistance and wind and sand resistance.Many P.euphratica forests are distributed in the middle,upstream,and downstream areas of the Tarim River in southern Xinjiang.These P.euphratica forests provide wind protection and sand fixation in areas with perennial sand and dust weather,regulate the regional climate,effectively block the northward movement of the desert,and protect the stability of the residents around the greenbelt,played an important positive role.P.euphratica has obvious heteromorphism,and the shape of its leaves will change with the age of the tree.The leaves of the young P.euphratica are all lanceolate,like willow leaves.With the increase of the age of the tree,the upper crown gradually appears diamond-shaped,kidney-shaped,the oval and broad oval leaves are shaped like poplar leaves.The typical heterotypic leaf nature of P.euphratica is the performance of P.euphratica in long-term adaptation to the desert environment at different stages of growth and development,which plays an important role in resisting the bad environment.At present,most researches on the heterophyllous leaf of P.euphratica mainly focus on the morphological structure level,however,the molecular mechanism of studying the occurrence of P.euphratica heteromorphic leaves on the molecular level is still few.Long non-coding RNA?lnc RNA?is a type of transcript that is widespread in eukaryotes and exceeds 200 nt and does not encode protein.Studies have shown that lnc RNA is closely related to chromatin modification,X chromosome silencing,regulated transcription,and regulated cascade reactions,and is also involved in regulating vital life activities such as growth and development of organisms and cell growth,differentiation,proliferation,and apoptosis.lnc RNA also plays an important role in the regulation of plant nutritional stages and reproductive development.In this study,the P.euphratica leaves,lanceolate leaves,oval leaves,and broad oval leaves were used as materials.By performing strand-specific transcriptome sequencing and small RNA sequencing,lnc RNA of P.euphratica was identified using bioinformatics methods,and further constructed.Identify the molecular regulatory network of lnc RNA-mi RNA-m RNA in the occurrence of P.euphratica heteromorphic leaves,further comparatively analyze and predict the lnc RNA and target gene functions of the above four leaf shapes of P.euphratica,and combine q PCR and other molecular techniques to detect the key RNAs in the regulatory network in P.euphratica.The expressions of the four-leaf shapes provide a preliminary analysis of the role of lnc RNAs in the occurrence of P.euphratica.The results obtained in this study are as follows:1.A total of 1977 novel lncRNAs were found in P.euphratica leaf tissue.Through second-generation sequencing and third-generation full-length transcriptome sequencing,high-quality transcriptome data of P.euphratica heterophylla were obtained.Take transcript length??200 bp?,exon?number ? 2?as basic screening conditions,and then pass CPC?score< 0?,CNCI?score < 0?,PLEK?score < 0?,pfam protein structure domain analysis potential coding ability screening?E-value < 0.001?and other bioinformatics analysis tools,and then obtain lnc RNA where the above screening results intersect.Finally,1977 lnc RNAs were obtained from P.euphratica leaf tissues.2.552 lnc RNAs related to the occurrence of heterotypic leaves of P.euphratica were identified.In this study,using the transcriptome data of four types of leaf tissues of P.euphratica,which are linear,lanceolate,oval and broad oval leaves,obtained by this research group,the expression difference analysis was carried out to investigate whether lnc RNA of P.euphratica affected the heterotypic leaves of P.euphratica morphogenesis.When the difference screening conditions were p < 0.05 and Foldchange > 2,552 differential lnc RNAs related to the occurrence of heterotypic leaves of P.euphratica were finally obtained.Among them,linear leaves were used as controls.In this study,there were 157 differential lnc RNAs in linear leaves and lanceolate leaves,of which 57 lnc RNAs were down-regulated and 100 lnc RNAs were up-regulated;in linear and oval leaves a total of 207 differential lnc RNAs,75 of which were down-regulated and 132 lnc RNAs were up-regulated;there were 215 differential lnc RNAs in linear leaves and broad oval leaves,of which 105 lnc RNAs were down-regulated and 110 lnc RNAs were up-regulated.Taking lanceolate leaves as a control,a total of 55 differential lnc RNAs were found in lanceolate leaves and oval,22 of which were down-regulated and 33 of lnc RNA were up-regulated;in lanceolate and broad oval leaves There were 142 differential lnc RNAs,72 of which were down-regulated and 70 of them were up-regulated.Taking the oval leaf as a control,a total of 156 differential lnc RNAs were found in the oval and broad oval leaves,of which 86 lnc RNAs were down-regulated and 70 lnc RNAs were up-regulated.The expression of some selected RNAs was verified by real-time fluorescence quantitative PCR method,which was basically consistent with the sequencing results.3.The lnc RNA-mi RNA-m RNA expression regulatory networks were constructed in the P.euphratica leaves heteromorphic leaves.Based on the competitive endogenous RNA hypothesis and co-expression analysis,an lnc RNA-mi RNA-m RNA expression regulatory network was constructed.It was found that 50 lnc RNAs,25 mi RNAs and 284 m RNAs were involved in the expression control networks.The real-time fluorescence quantitative PCR method was also used to verify some of the selected regulatory relationships,which was basically consistent with the predicted results.4.The biological function of lnc RNAs in heteromorphic leaves of P.euphratica was predicted.Functionally annotate the target genes of lnc RNA related to P.euphratica heteromorphic leaves.It was found that related lnc RNAs in P.euphratica abnormity involves a variety of biological functions,for example,lnc RNAs can respond to a variety of stimuli,such as light stimulation,chemical stimulation,temperature stimulation,etc.;participate in a variety of biological processes,such as developmental processes,metabolic processes,reproductive processes,growth and development process.For instance,TCONS₀0004524,TCONS₀0017878,TCONS₀0056727,TCONS₀0065259 and TCONS₀0057022 were up-regulated expression in the oval leaves to affect their downstream target gene yoda?XM₀11030993.1?up-regulated expression,and lead to local cell proliferation,which may promote the broadening of P.euphratica leaves;XR₈44921.1 in P.euphratica heterotypic leaves.It played a regulatory role in the occurrence of oval leaves,increasing the number of cells,which can promote the broadening of P.euphratica leaves.This study shows that lnc RNAs could participate in regulating the occurrence of P.euphratica by regulating mi RNAs and corresponding coding genes.In summary,through identification and functional analysis of lnc RNA in P.euphratica heteromorphic leaves in this study,it was found that there are a large number of new lnc RNA in P.euphratica heteromorphic leaves,and these lnc RNAs were involved in the biological processes of P.euphratica growth and development,material metabolism,signal transduction,etc.There may be a complex lnc RNA expression regulation mechanism.This study provides abundant long-chain non-coding RNA data of P.euphratica heteromorphic leaves,and also provides useful resources and reference value for the subsequent research on the molecular mechanism of P.euphratica heterophylla and plant morphology.