Functional Identification And Analysis Of The Gossypiumhirsutum L. Mirnvl5 And It Target Gene Ghchr In Response To Salt Stress

MicroRNA (miRNA) is a novel class of endogenous non-coding small RNAs that regulate gene post-transcriptional expression in both plants and animals. Our previous studies identified a novel miRNA through high-throughput sequencing technology from cottone (Gossypium Hirsutum L) which was assigned as miRNVL5 (novel miRNA 5).Its target TC113069 was predicted and validated by 5’RACE. Because the target was identified with rich cysteine (9.6%) and histone (7.4%), here was referred to as CYSTEINE AND HISTIDINE RICH, GhCHR). GhCHR encodes a protein containing 3 PHD zinc finger binding Zn2+ structures and 2 C1 domains. The protein with this type of zinc finger structure is relevant to the regulation of gene expression with an important role played in plant response to biotic and abiotic stresses. However, such a protein is unclear in biological function.In this study, the full-length cDNA sequence of pre-miRNVL5, GhCHR and GhmCHR (GhCHR of point mutantion) was cloned. We analyzed expression of pre-miRNVL5, mature miNVL5 and GhCHR at different stages of cotton development and different tissues, including root, cotyledon, hypocotyl, petal, ovule and fiber. Analysis showed that these genes can be differentially expressed. Generally, there was a contrasting expression pattern between miRNVL5 and GhCHR, indicating that GhCHR can be cleavaged by miRNVL5. Interestingly, expression of miRNVL5 was depressed by 50-400 mM NaCl, whereas its target gene GhCHR was induced by the salt stress. However, both miRNVL5 and GhCHR were not found to be regulated by abscisic acid (ABA), suggesting that miRNVL5 and GhCHR were not involved in ABA transduction pathway.In order to investigate the function of MIRNVL5 and GhmCHR, we constructed transgenic Arabidopsis plants over-expressing MIRNVL5, GhCHR or GhmCHR (MIRNVL5 cleavage resistance version) driven by 35S promoter. Our analysis showed that 35S::GhCHR and 35S::GhmCHR plants were more tolerant to salt stress (100 and 150 mM NaCl) than the wild-type, while 35S::MIRNVL5 plants were more sensitive to the NaCl treatment than the wide type plants, in terms of seed germination and root growth. Expression of the key stress-responsive marker genes such as RD22, RD29A and KIN1 was up-regulated in miRNVL5 over-expressing plants but down-regulated in 35S::GhCHR plants. In addition,35S::MIRNVL5,35S::GhCHR and 35S::GhmCHR plants were insensitive to ABA stress. These results suggested that miRNVL5 and GhCHR regulated the salt stress responses independent on ABA signaling.To study the influence of over-expression of miRNVL5 to the expression of other genes, we constructed four gene expression libraries using 35S::MIRNVL5 Arabidopsis plants including:(1) under normal growing conditions, two libraries of 35S::MIRNVL5 transgenic Arabidopsis (0-miR5) and wild-type Arabidopsis (0-col-0); and (2) under 200 mM NaCl treatment for 1 h, two libraries of 35S::MIRNVL5 transgenic Arabidopsis (200-miR5) and wild-type Arabidopsis (200-col-0). We then used the four libraries to perform the corresponding chip expression analyses. Our results showed that under normal growth conditions,945 differentially expressed genes were detected when we compared 35S::MIRNVL5 transgenic plants with wild-type plants, of which 508 were up-regulated, while 437 were down-regulated. Under 200 mM NaCl condition,1579 differentially expressed genes were detected when we compared 35S::MIRNVL5 transgenic plants with wild-type plants, of which 734 were up-regulated and 845 were down-regulated. Compared with normal growth conditions (0 mM NaCl), treatmenet of WT plants with 200 mM NaCl resulted in 4566 genes differently expressed (of these 2427 were up-regulated, and 2139 were down-regulated), while in transgenic plants there were 4532 differentially expressed genes. Of these,2287 were up-regulated and2245 were down-regulated.We further conducted GO and KEGG analysis to the microarray data. The results of GO analysis showed that over-expression of miRNVL5 caused differentially expression of some genes, of which 38.8% were involved in transcriptional regulation,14% could respond to environmental stress,7.4% were involved in the biosynthesis pathways,5% were involved in material transport pathways, and 1.7% were involved in metabolic pathways; Compared with the WT plants treated with 200 mM NaCl,35S::MIRNVL5 transgenic plants showed that 34.2% of the genes could respond to environmental stress, 26.9% were involved in transcriptional regulation,6.2% were involved in plant growth and genes of the same ratio were involved in material transport,3.8% were involved in metabolic pathways, and 3.1% were involved in the biosynthesis. KEGG results also demonstrated the different gene expression in 35S::MIRNVL5 plants relative to WT. Among these,40% were involved in the biosynthesis of secondary metabolites,17.5% were involved in plant hormone signal transduction,10% were involved in limonene and pinene degradation pathways, etc. For 35S::MIRNVL5 plants under 200 mM NaCl exposure relative to WT,54.5% were involved in the biosynthesis of secondary metabolites and 15.2% were involved in plant-pathogen interactions, etc.