Differential Expression Of MiRNA In Different Salt-tolerant Cotton Under Salt Stress

Cotton (Gossypium hirsutum L.) is one of the most salt-tolerant crops.The different kinds of variety,growth stage,organ and soil salinity result in the dissimilarities of salinity tolerance. The ion toxicity to membrane composition and function of new organs or newly developing stages is the major damage of cotton under salt stress which inhibits the growth,affects development course,decreases total fruit knots,yields and qualities. Soil salinization, by inhibiting growth and crop yield, is a severe and increasing constraint on agricultural productivity. Therefore, it is essential to analyze the expression and the function of salt-induced genes.Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that have also been linked to stress responses. At present, the salt-tolerance research on cotton mainly focused on the morphological, physiological and biochemical aspects, while the relationship between cotton salt-tolerance and miRNA has not been reported. This study used salt-tolerant cotton cultivar Shannong 91-11 and salt-sensitive cultivar Lumian 6 as the research materials to detect the differential expressions of miRNAs between two cultivars and two treatments by miRNA microarray. In order to verify the reliability of miRNA microarray results, we adopted Northern blot to detect the expressions of the salt-induced miRNAs under salt stress, the main research results are as follows:1. Extraction and purification of the total RNA, isolated miRNAs from salt-tolerant cultivar Shannong 91-11 and salt-sensitive cultivar Lumian 6, labeled them with CU-cy3 and CU-cy5 respectively, then hybridized with miRNA microarray which contained 426 miRNA probes to study the differential expressions of miRNAs. Hybridization results showed that microarray was relatively stable, chip repeat point was reproducible, signal was homogeneous, the data had no polluted effect, leak rate was no more than 3‰, correction factor of inter-chip was stable, microarray quality was good.2. The results analyzed by SAM showed that: there were 17 miRNAs whose expressions differed significantly between the control materials and salt-treated materials of Shannong 91-11, compared with control materials, only one predicted miRNA up-regulated its expression significantly, 16 miRNAs down-regulated their expressions significantly, of them, the function of osa-miR156k, ath-miR159a, osa-miR159a, osa-miR159c, osa-miR159d, osa-miR159f, osa-miR160e, osa-miR166k, osa-miR166m, osa-miR169f have been known.3. There were 18 miRNAs whose expressions differed significantly between the control materials and salt-treated materials of Lumian 6, compared with control materials, miR529,miR160h and a predicted miRNA up-regulated their expressions significantly, 15 miRNAs down-regulated their expressions significantly, of them, the function of ath-miR159a, osa-miR159a, ath-miR159b, ath-miR159c, osa-miR159c, osa-miR159d, osa-miR159e, osa-miR159f, ath-miR167a, ptc-miR169x, osa-miR399i have been known.4.There were 14 miRNAs whose expressions differed significantly between the control materials of Shannong 91-11 and Lumian 6, compared with Lumian 6, ath-miR160a, osa-miR535, ath-miR827 up-regulated their expressions significantly, 11 miRNAs down-regulated their expressions significantly, of them, the function of ath-miR159a has been known. 5. There were 27 miRNAs whose expressions differed significantly between the salt-treated materials of Shannong 91-11 and Lumian 6, compared with Lumian 6, 11 miRNAs up-regulated their expressions significantly, of them, 10 miRNAs whose functions have been known belong to the miRNA family of miR156, miR164, miR167, miR397 and miR399 respectively; 16 miRNAs down-regulated their expressions significantly, of them, 2 miRNAs whose functions have been known belong to the miRNA family of miR156, miR172.6. miR156, miR159, miR167, miR169 and miR399 which expressed differentially significantly in salt-tolerant and salt-sensitive cultivar were selected to verify. Northern blot proved that the expressions of miR156, miR169, miR167, miR399, miR159 expression were fully consistent with the results of microarray, and the these miRNAs can be regulated by environment of salt stress.