Study Of LncRNA Lnc3in Betula Platyphylla Involved In Drought Stress

Birch(Betula platyphylla)is an important industrial timber plant species,which is widely distributed in Eurasia.Long non-coding RNA is a class of transcripts with a length of more than 200 bp and no protein coding ability.lncRNA have been reported to be involved in many biological processes,including resistance to abiotic stress in plants.However,there are few studies on the regulation of abiotic stress by lncRNA in woody plants,especially birch.In this study,the total RNA of birch treated with PEG-6000 was extracted,and the library was constructed with the Illumina Hi Seq platform.A total of 93.39 Gb Clean Data was obtained.The accuracy of RNA-seq data was verified by q RT-PCR with 18 randomly selected genes,a total of 5950 lncRNA were identified,and 15843 target genes were predicted to be regulated by trans and cis regulation.KEGG analysis showed that target genes were involved in different biological processes,such as "plant hormone signal transduction","phenylalanine metabolism",and "photosynthesis".GO classification showed that these target genes were mainly involved in " response to stimulus "," biological regulation "," metabolic process " and other biological processes.A total of 53 drought-responsive lncRNA were identified.Among them,31 lncRNA were significantly induced by drought,and 22 lncRNA were significantly inhibited by drought.The6 lncRNA with high differential expression were selected for further study,and were cloned into the plant expression vector p ROKII and RNAi vector p FGC5941,then were respectively constructed and transferred into Agrobacterium EHA105.Transgenic birch was generated by transient transformation method,and q RT-PCR was used to detect the expression of 97 target genes in transgenic birch and control plants.The results showed that the expression of 73 target genes was consistent with the RNA-seq data,and the target genes predicted by trans regulation were more reliable than those predicted by cis regulationn,and physiological and biochemical parameters related to drought tolerance were determined.Among the 6 lncRNA studied,4lncRNA were drought tolerant to transgenic plants,and the other 2 lncRNA were sensitive to drought stress.These results indicated that many drought-responsive lncRNA had drought resistance or sensitivity.One of the drought-positive lncRNA,lnc3,was selected and stably transformed by Agrobacterium-mediated leaf discus method.A total of 10 overexpressed birch lines and 6inhibited birch lines were obtained for DNA detection.The expression level was detected by q RT-PCR,indicating that it has been successfully overexpressed or inhibited by RNAi.OE6 with the relatively middle expression level and OE10 with the highest expression level in the overexpression lines were selected,and SE3 and SE6 with the lowest expression level in the suppressed expression lines were selected for subsequent experiments.Na Cl and PEG-6000 were used for salt and drought stress respectively,the phenotype was observed,and the physiological and biochemical indicators related to adversity stress were determined.At the same time,q RT-PCR analysis of POD,SOD and other related genes was carried out.The results showed that lnc3 overexpressed birch lines increased the activity of antioxidant enzymes by increasing the expression of related genes such as POD and SOD.Increased proline and chlorophyll content,decreased MDA content and relative electrical conductivity improved salt tolerance and drought resistance of birch,while the birch line with lnc3 inhibited expression was the opposite,but decreased drought tolerance and salt tolerance,suggesting that lnc3 could improve the salt and drought stress of birch.RNA was extracted from the overexpressed lnc3 from birch and wild-type after stress,and RNA-seq research was performed.A total of 666 differentially expressed genes were obtained,including 243 up-regulated genes and 423 down-regulated genes.The GO function annotations and enrichment of differentially expressed genes showed that lnc3 was mainly involved in plant hormone signal transduction,phenylpropanoid biosynthesis,plant pathogen interaction,starch and sugar metabolism,carbon metabolism,amino acid and ribonucleic acid biosynthesis and other processes.