Cloning And Functional Analysis Of Cotton GhCNGC2 Gene And Molecular Detection Of Fungal Communities In Hawaiian Marine Sponges

Isolation and Characterization of a Putative Cyclic Nucleotide-Gated Cation Channel Gene GhCNGC2 in CottonThe growth and development of higher plants require the uptake of sufficient nutrients and ions, which are helpful for signaling, osmotica and metabolites, and the regulation of nutrient ions uptake is one process affecting the internal nutrient balance of plants. The unidirectional influx of nutrient ions into the plant cell as an important ion uptake mechanism has been of great interest to most researchers in the field of plant ion transport. While early research mainly focused on highly selective ion channels, it has recently become clear that a diverse array of cations is transported via non-selective cation channels (NSCCs). NSCCs are ion channels that are selectively permeable for cations over anions, but that do not discriminate strongly between monovalent cations. Cyclic nucleotide-gated cation channels (CNGCs), which are a type of NSCC, have been identified by some researchers recently.Cyclic nucleotide-gated cation channel (CNGC) is one kind of non-selective cation channel in animals and plants. In animal cells, CNGCs have been verified to function as a kind of sensors transducing olfactory and visual stimuli which is regulated by cellular cNMP, whereas in plants, only several CNGCs have been isolated from Arabidopsis, barley and tobacco, but their functions are still obscure, they are thought to be related with ion transportation and disease resistance.Cotton is one of the most important and valuable fiber and oil crops. In this research, a cDNA library was constructed by using mRNA isolated from salt-induced cotton seedlings and screened by differential hybridization. We present the isolation and characterization of a cDNA clone, GhCNGC2, encoding a novel cyclic nucleotide-gated cation channel from cotton seedlings. The main results are as follows:1. A salt-induced cDNA library of cotton seedlings was constructed with Clontech Smart^TM cDNA Library Construction Kit. We obtained a salt-induced clone by differential hybridization, and the length of the fragment was 670 bp. The 5'-end and 3'-end of the gene was further isolated by RACE-PCR then used RT-PCR to amplify the full length cDNA of the gene. The isolated cDNA is 2388 bp in length and harbors an opening reading frame of 2145 bp encoding a 78 kDa protein of 715 amino acids.2. Sequence and homology comparison revealed that the isolated cDNA shared high sequence similarity with CNGCs in plants, and the sequence identity to AtCNGC2 is up to 74.90%. The phylogenetic analysis also indicated that the kinship of this isolated cDNA was close to CNGCs in Arabidopsis, rice, barley and tobacco. Therefore, it is suggested that the isolated cDNA might belong to the cyclic nucleotide-gated cation channel in cotton, and it is designated GhCNGC2.3. The Transmembrane domains prediction and amino acids analysis further indicated that there were 6 conserved transmembrane domains in the N-terminus of GhCNGC2, and a pore region is located between the 5th and 6th transmembrane domains; while, one cyclic nucleotides binding domain (CNBD) and one calmodulin-binding domain (CaMBD) exist in the C-terminus of this protein. All of them are typical sequence characteristics of plant CNGCs.4. Northern blot analysis revealed that GhCNGC2 transcripts were easily detected in the leaves, less detected in the stems, and could not be detected in roots. The GhCNGC2 mRNA levels were also assayed in different stress-treated seedlings, and it was strongly and rapidly induced in salt stress conditions, obviously induced in mannitol- and ABA-treated seedlings, and less induced in low temperature-, Cu²⁺- and ethylene-treated seedlings. Moreover, no evident difference could be observed in mRNA accumulation between different germination stages.5. Yeast complementation test demonstrated that the K+ uptake ability of GhCNGC2 in yeast was blocked by endogenous calmodulin (CaM), and expression of the truncated forms of GhCNGC2 could completely rescue the K⁺ uptake ability of yeast mutant CY162, the results suggested that the regulation domains (CNBD and CaMBD) in C-terminus played an important role in the function of GhCNGC2.6. Ion content assay showed that overexpression of the full length and truncated GhCNGC2 could promote the uptake of K⁺ and inhibit the uptake of Na⁺, which led to a high K⁺/Na⁺ ratio. These results suggested that GhCNGC2 might be involved in seed germination under stress conditions.7. The transgenic seeds exhibited higher germination percentages than wild type seeds under different stress conditions, suggesting that GhCNGC2 is involved in stress tolerance at the germination stage. Moreover, the germination percentages of three kinds of transgenic seeds revealed that GhCNGC2 might accommodate the dual regulatory functions of cyclic nucleotide and calmodulin. Sponges (phylum Porifera) contain an estimated 15,000 species in three taxonomic classes: Calcarea (Calcareous sponges), Hexactinellida (Glass sponges), and Demospongiae (Demosponges). As sedentary filter-feeding organisms, sponges are remarkably efficient at obtaining food from the surrounding water and can pump up to 24,000 liters of seawater through a 1-kg sponge per day. Any planktonic microbe can become a resident in sponges, provided that it can survive the digestion and immune response in sponges as well as capable of growing in the microenvironment of the sponge mesohyl.Symbiotic microbes play a variety of fundamental roles in the health and habitat range of their hosts. While prokaryotes in marine sponges have been broadly characterized, the diversity of sponge-inhabiting fungi has never been explored using molecular approaches. Fungi are an important component of many marine and terrestrial ecosystems, and they may be an ecologically significant group in sponge-microbial interaction.This study tested the feasibility of existing fungal primers for molecular analysis of sponge-associated fungal communities. None of 8 selected primer pairs yielded satisfactory results in fungal rRNA gene or ITS library constructions. However, 3 of 11 DGGE primer sets, which were designed to preferentially amplify fungal rRNA gene or ITS regions from terrestrial environmental samples, were successful to amplify fungal targets in marine sponges. DGGE analysis indicated that fungal communities differ among different sponge species (Suberites zeteki and Mycale armata) and also varies between sponges and seawater. Sequence analysis of DGGE bands identified 23 fungal"species"from sponge S. zeteki and 21 fungal"species"from sponge M. armata. These"species"were representatives of 11 taxonomic orders and belonged to the phyla of Ascomycota (7 orders) and Basidiomycota (4 orders). Five of these taxonomic orders (Malasseziales, Corticiales, Polyporales, Agaricales, and Dothideomycetes et Chaetothyriomcetes incertae sedis) are identified for the first time in marine sponges. Seven and six fungal"species"from S. zeteki and M. armata, respectively, were potentially new species because of their low sequence identity (≤98%) with their references in GenBank. Phylogenetic analysis indicated sponge-derived sequences were clustered into"marine fungus clades"with those from other marine habitats.This is the first report of molecular analysis of fungal communities in marine sponges, adding new depth and dimension to our understanding of sponge-associated microbial communities.