Identification Of Drought Responsive Non-coding Rnas And Constructions Of Their Regulatory Networks In Cotton

Non codingRNA(ncRNA),including microRNA(miRNA),long noncodingRNA(lncRNA)and circularRNA(circRNA),is a kind ofRNA with weak coding or no protein coding ability,which can participate in the process of plant growth and development and stress response.Cotton is an important strategic material.Drought is the main abiotic factor affecting cotton production.Studying the molecular mechanism of cotton under drought stress,mining important drought tolerant genes and regulatory factors and using them to cultivate drought tolerant cotton varieties is an important way to solve this important demand for drought resistance.However,the study on ncRNA of cotton under drought stress is relatively few.In this study,the basic characteristics of three types of ncRNA(miRNA,lncRNA and circRNA)in drought sensitive cultivar ZY7 and drought tolerant cultivar ZY168 before and after drought treatment were identified,and to explore the molecular mechanism of ncRNA regulating cotton plasticity development under drought stress,construct a ceRNA regulatory network under drought stress,and identify the important regulatory nodes ncRNA and their expression patterns.The main results of the study are as follows:1.A total of 574 miRNA were identified by miRNA sequencing,of which 96 miRNA had significant changes in expression after drought treatment,and these differentially expressed miRNA(DE-miRNA)had strong tissue specificity and variety specificity.The expression patterns of some DE-miRNA were also verified by q RT-PCR.According to the expression of DE-miRNA,they were divided into basic drought response class,tissue-specific class,variety-specific class and other class.The annotation of transcription factor types and target gene functions of these four DE-miRNA showed that some DE-miRNA could participate in the response of cotton to drought stress through transcriptional regulation;Different types of DE-miRNA could play a regulatory role in a variety of biological processes and participate in the response to cotton drought.2.4810 lncRNA were identified by lncRNA sequencing.The proportion of lncRNA types in intergenic regions were the highest.Compared with mRNA,the length distribution of lncRNA was more concentrated and tended to be composed of 1-3 exons.A total of 1393 differentially expressed lncRNA(DE-lncRNA)were identified after drought treatment.Some DE-lncRNA expression patterns were also verified by q RT-PCR.DE-lncRNA could also be divided into four categories.The functional annotation of target genes of different types of DE-lncRNA indicated that different types of lncRNA might perform different functions under drought conditions.3.A total of 79 circRNA were identified by circRNA sequencing.Most of the circRNA(70.87%)were loop by exons.A total of 28 differentially expressed circRNA(DE-circRNA)were identified after drought treatment.We verified the cyclization of some DE-circRNA by PCR amplification,and their expression pattern were also verified by q RT-PCR.The enrichment results of DE-circRNA parent genes showed that DE-circRNA could respond to cotton drought stress by regulating the response to light stimulation and the expression of key factors in the starch biosynthesis pathway.4.Combining the differentially expressedRNA found previously,A total of 66 lncRNA,55 miRNA and 157 mRNA were predicted to be used to construct tissue-specific lncRNA-miRNA-mRNA networks.Among them,6 key lncRNA(TCONS＿150021,TCONS＿72099,TCONS＿82802,TCONS＿72858,TCONS＿59536,TCONS＿111741)could bind with at least 5 miRNA and play an important role in the network.In the cultivar-specific regulatory network constructed by lncRNA-miRNA-mRNA,the key lncRNA in the drought-tolerant cultivar ZY168 dominated.Eight circRNA,17 miRNA and 402 mRNA could construct circRNA-miRNA-mRNA networks and participate in the response of cotton to drought stress.Among them,circ＿62 can be used as a key circRNA to combine with multiple miRNA to indirectly regulate important target genes such as CKX5,TGA7,EIN2,etc.to participate in drought response process.The q RT-PCR validation of some circRNA-miRNA-mRNA regulatory networks indicated that they might be involved in the response of cotton to adversity through the regulation of circ＿62-mi R7484b-TGA7/CKX5.