棉花GhCNGC2基因的克隆与功能鉴定和夏威夷海洋海绵的真菌多样性分析
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摘要
第一部分棉花环化核苷酸调节的离子通道基因GhCNGC2的克隆和功能鉴定
高等植物的生长和发育需要吸收大量的营养物质和离子,它们对植物的信号转导,渗透调节以及代谢都是至关重要的。因此,植物对营养物质吸收的调节成为一种影响植物内部营养平衡的重要机制。近年来,作为一种重要的离子吸收机制,植物细胞对于营养离子的单方向吸收运输引起了越来越多研究者的兴趣,但早期的一些研究却主要集中在那些高选择性的离子通道上。然而最近的研究表明,植物对离子的吸收很大程度上是依靠一些非选择性阳离子离子通道(non-selective cation channel, NSCC)来实现的。通常来说,这类离子通道对阳离子具有很高的透过性,却完全不能传导阴离子;它们对于一价的阳离子具有广泛的渗透性,有时也可以传导一些二价的阳离子。环化核苷酸调节的离子通道(cyclic nucleotide-gated cation channel, CNGC)就是一种非选择性的阳离子通道。
环化核苷酸调节的离子通道蛋白是一种在动、植物中广泛存在的非选择性的阳离子通道。在动物中,CNGC是一种受环化核苷酸(cNMP)调节的视觉和嗅觉的传感器;在植物中,有关CNGC的研究还相对较少,只是在拟南芥(Arabidopsis thaliana)、大麦(Hordeum vulgare)和烟草(Nicotiana tabacum)中有过少量的研究报道,因此对它们功能的了解还是非常有限,通常认为它们与植物的离子传递以及抗病性有关。
棉花是一种纤维用经济作物,它具有广泛的应用价值和研究价值。本文以耐盐棉花品种中棉19(ZM19)为试验材料,构建了盐诱导的棉花幼苗叶片cDNA文库,并通过反向northern差异筛选法从中分离得到了一个与盐胁迫相关的克隆GhCNGC2。通过序列比对发现,该基因可能编码一种环化核苷酸调节的离子通道蛋白,并对该基因的功能和分子机制进行了探索。主要结果如下:
1.利用Clontech Smart~(TM) cDNA Library Construction Kit构建盐胁迫诱导的棉花幼苗叶片cDNA文库,通过反向northern差异筛选法获得一个与盐胁迫相关,长度为670 bp的克隆。设计特异引物,通过5'-RACE和3'-RACE PCR,克隆得到该基因的5'片段和3'片段,根据重叠片段进行拼接,再用特异引物扩增得到2388 bp的全长cDNA,其编码区全长为2145 bp,编码一个715个氨基酸的蛋白,推测其分子量为78 kDa。
2.通过对该基因编码的蛋白同源性比较发现,该蛋白与植物的CNGCs家族具有较高的同源性,其中与拟南芥的AtCNGC2的蛋白同源性达到74.90%。聚类分析也表明,该基因编码的蛋白与拟南芥、水稻、大麦以及烟草中的CNGC序列也有一定的亲缘关系。因此,我们推测,该基因所编码的蛋白产物可能是一种环化核苷酸调节的离子通道,将该基因命名为GhCNGC2。
3.跨膜结构和氨基酸序列的进一步分析表明,GhCNGC2的氨基端有6个保守的跨膜结构域(S1-S6),在第5个和第6个跨膜结构域之间还有一个孔状结构域(Pore region);在羧基端有一个保守的环化核苷酸结合域(CNBD)和一个钙调素结合域(CaMBD),这些都是植物环化核苷酸调节的离子通道的典型序列特征。
4. Northern blot表达分析表明,GhCNGC2在棉花叶片(子叶)中具有较高的表达水平,在茎中的表达水平较低,而在根中检测不到该基因的表达。GhCNGC2还受到多种胁迫的诱导,其中在NaCl处理的棉花幼苗中表达明显增加,而甘露醇和ABA处理也产生显著的诱导,低温、Cu~(2+)和乙烯的诱导相对较弱。对不同萌发时期的棉花幼苗进行处理,发现该基因在不同的萌发时期表达水平基本不变。
5.酵母互补试验证明,GhCNGC2作为一种离子通道,其K+的吸收能力在酵母突变体CY162中受到了内源钙调素的极大抑制,而表达GhCNGC2的缺失形式GhCNGC2ΔCaM和GhCNGC2ΔCNBD则可以使该突变体重新获得K+的吸收能力,从而互补该突变体,这表明CNBD和CaMBD作为羧基端调节区域的两个重要结构域,对GhCNGC2的功能起到了重要的调节作用。
6.转基因植株内源离子含量测定的结果显示,超表达GhCNGC2及其缺失形式的拟南芥植株可以促进K~+的吸收,抑制Na~+的吸收,同时产生一个较高的K~+/Na~+比值,这表明该基因可能与胁迫条件下的种子萌发有密切关系。
7.相比野生型拟南芥,超表达GhCNGC2基因及其缺失形式的转基因种子在高盐、盐碱、干旱、高温等各种胁迫条件下表现出较高的萌发率,说明GhCNGC2可能与种子萌发阶段的胁迫抗性紧密相关。另外,三种转基因种子所表现出来的萌发率高低关系可能是受到了环化核苷酸以及钙调素的双重调节。
第二部分夏威夷海洋海绵的真菌多样性分析
海洋真菌是海洋生态系统中一类重要的生物群落,由于其特殊的生存环境,以前对其研究较少。随着人们对海洋认识的深化,海洋生物的研究将成为二十一世纪生物研究的热点之一。海绵属于多孔动物门,在全球包括15000多种,分属于3个纲:钙质海绵纲(Calcarea)、六放海绵纲(Hexactinellida)和寻常海绵纲(Demopongiae)。作为滤食性动物,一千克的海绵组织一天可以过滤海水达24000升,海绵可以有效的从周围的海水环境中获取食物。各种浮游微生物都可以作为共附生微生物,生长在海绵的中质层中。
共附生微生物的存在对寄主生物的健康和生活环境起到非常重要的作用。虽然在海洋海绵中共附生的原核生物已经被广泛的鉴定,但是人们还尚未使用过分子生物学的方法研究海绵中真菌群落的多样性。真菌是许多海洋和陆地生态系统重要的组成部分,它们也是海洋海绵中非常重要的一种生态群。
本研究对现有的研究海绵中真菌群落的引物进行了可行性分析,选取了8对引物,分别构建rRNA或ITS克隆文库,都没有得到满意的结果;同时选取了11组DGGE引物,这些引物可以很好地扩增一些陆地环境样品中真菌的rRNA或ITS序列。结果发现,有3组能够成功地扩增海洋海绵中的真菌序列。DGGE结果显示,两种不同的夏威夷海洋海绵(Suberites zeteki和Mycale armata)中的真菌群落存在明显的差异,同时在海洋海绵和海水样品中的真菌群落也存在一定的差异。通过对所得DGGE条带的测序,在海绵S. zeteki中鉴定了23种真菌的存在;而在海绵M. armata中鉴定了21种真菌的存在。这些真菌属于11个不同的目,其中包括子囊菌门(Ascomycota)7个目,担子菌门(Basidiomycota)4个目,并且有5个目(Malasseziales, Corticiales, Polyporales, Agaricales和Dothideomycetes et Chaetothyriomcetes incertae sedis)是在海洋海绵中的首次鉴定。在S. zeteki和M. armata中,分别有7个和6个可能的真菌新种被鉴定出来,这些序列与它们在GenBank中的对应序列的序列相似性小于98%。系统进化分析发现,这些来自于海洋海绵的真菌序列与一些其他的来自于海洋生境的序列一起属于“海洋真菌”进化支。
本研究是首次应用分子生物学的方法鉴定海洋海绵中的真菌群落,为以后进一步的研究海绵共附生微生物群落提供了新的思路和方法。
Isolation and Characterization of a Putative Cyclic Nucleotide-Gated Cation Channel Gene GhCNGC2 in Cotton
The 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-, Cu~(2+)- 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.
高等植物的生长和发育需要吸收大量的营养物质和离子,它们对植物的信号转导,渗透调节以及代谢都是至关重要的。因此,植物对营养物质吸收的调节成为一种影响植物内部营养平衡的重要机制。近年来,作为一种重要的离子吸收机制,植物细胞对于营养离子的单方向吸收运输引起了越来越多研究者的兴趣,但早期的一些研究却主要集中在那些高选择性的离子通道上。然而最近的研究表明,植物对离子的吸收很大程度上是依靠一些非选择性阳离子离子通道(non-selective cation channel, NSCC)来实现的。通常来说,这类离子通道对阳离子具有很高的透过性,却完全不能传导阴离子;它们对于一价的阳离子具有广泛的渗透性,有时也可以传导一些二价的阳离子。环化核苷酸调节的离子通道(cyclic nucleotide-gated cation channel, CNGC)就是一种非选择性的阳离子通道。
环化核苷酸调节的离子通道蛋白是一种在动、植物中广泛存在的非选择性的阳离子通道。在动物中,CNGC是一种受环化核苷酸(cNMP)调节的视觉和嗅觉的传感器;在植物中,有关CNGC的研究还相对较少,只是在拟南芥(Arabidopsis thaliana)、大麦(Hordeum vulgare)和烟草(Nicotiana tabacum)中有过少量的研究报道,因此对它们功能的了解还是非常有限,通常认为它们与植物的离子传递以及抗病性有关。
棉花是一种纤维用经济作物,它具有广泛的应用价值和研究价值。本文以耐盐棉花品种中棉19(ZM19)为试验材料,构建了盐诱导的棉花幼苗叶片cDNA文库,并通过反向northern差异筛选法从中分离得到了一个与盐胁迫相关的克隆GhCNGC2。通过序列比对发现,该基因可能编码一种环化核苷酸调节的离子通道蛋白,并对该基因的功能和分子机制进行了探索。主要结果如下:
1.利用Clontech Smart~(TM) cDNA Library Construction Kit构建盐胁迫诱导的棉花幼苗叶片cDNA文库,通过反向northern差异筛选法获得一个与盐胁迫相关,长度为670 bp的克隆。设计特异引物,通过5'-RACE和3'-RACE PCR,克隆得到该基因的5'片段和3'片段,根据重叠片段进行拼接,再用特异引物扩增得到2388 bp的全长cDNA,其编码区全长为2145 bp,编码一个715个氨基酸的蛋白,推测其分子量为78 kDa。
2.通过对该基因编码的蛋白同源性比较发现,该蛋白与植物的CNGCs家族具有较高的同源性,其中与拟南芥的AtCNGC2的蛋白同源性达到74.90%。聚类分析也表明,该基因编码的蛋白与拟南芥、水稻、大麦以及烟草中的CNGC序列也有一定的亲缘关系。因此,我们推测,该基因所编码的蛋白产物可能是一种环化核苷酸调节的离子通道,将该基因命名为GhCNGC2。
3.跨膜结构和氨基酸序列的进一步分析表明,GhCNGC2的氨基端有6个保守的跨膜结构域(S1-S6),在第5个和第6个跨膜结构域之间还有一个孔状结构域(Pore region);在羧基端有一个保守的环化核苷酸结合域(CNBD)和一个钙调素结合域(CaMBD),这些都是植物环化核苷酸调节的离子通道的典型序列特征。
4. Northern blot表达分析表明,GhCNGC2在棉花叶片(子叶)中具有较高的表达水平,在茎中的表达水平较低,而在根中检测不到该基因的表达。GhCNGC2还受到多种胁迫的诱导,其中在NaCl处理的棉花幼苗中表达明显增加,而甘露醇和ABA处理也产生显著的诱导,低温、Cu~(2+)和乙烯的诱导相对较弱。对不同萌发时期的棉花幼苗进行处理,发现该基因在不同的萌发时期表达水平基本不变。
5.酵母互补试验证明,GhCNGC2作为一种离子通道,其K+的吸收能力在酵母突变体CY162中受到了内源钙调素的极大抑制,而表达GhCNGC2的缺失形式GhCNGC2ΔCaM和GhCNGC2ΔCNBD则可以使该突变体重新获得K+的吸收能力,从而互补该突变体,这表明CNBD和CaMBD作为羧基端调节区域的两个重要结构域,对GhCNGC2的功能起到了重要的调节作用。
6.转基因植株内源离子含量测定的结果显示,超表达GhCNGC2及其缺失形式的拟南芥植株可以促进K~+的吸收,抑制Na~+的吸收,同时产生一个较高的K~+/Na~+比值,这表明该基因可能与胁迫条件下的种子萌发有密切关系。
7.相比野生型拟南芥,超表达GhCNGC2基因及其缺失形式的转基因种子在高盐、盐碱、干旱、高温等各种胁迫条件下表现出较高的萌发率,说明GhCNGC2可能与种子萌发阶段的胁迫抗性紧密相关。另外,三种转基因种子所表现出来的萌发率高低关系可能是受到了环化核苷酸以及钙调素的双重调节。
第二部分夏威夷海洋海绵的真菌多样性分析
海洋真菌是海洋生态系统中一类重要的生物群落,由于其特殊的生存环境,以前对其研究较少。随着人们对海洋认识的深化,海洋生物的研究将成为二十一世纪生物研究的热点之一。海绵属于多孔动物门,在全球包括15000多种,分属于3个纲:钙质海绵纲(Calcarea)、六放海绵纲(Hexactinellida)和寻常海绵纲(Demopongiae)。作为滤食性动物,一千克的海绵组织一天可以过滤海水达24000升,海绵可以有效的从周围的海水环境中获取食物。各种浮游微生物都可以作为共附生微生物,生长在海绵的中质层中。
共附生微生物的存在对寄主生物的健康和生活环境起到非常重要的作用。虽然在海洋海绵中共附生的原核生物已经被广泛的鉴定,但是人们还尚未使用过分子生物学的方法研究海绵中真菌群落的多样性。真菌是许多海洋和陆地生态系统重要的组成部分,它们也是海洋海绵中非常重要的一种生态群。
本研究对现有的研究海绵中真菌群落的引物进行了可行性分析,选取了8对引物,分别构建rRNA或ITS克隆文库,都没有得到满意的结果;同时选取了11组DGGE引物,这些引物可以很好地扩增一些陆地环境样品中真菌的rRNA或ITS序列。结果发现,有3组能够成功地扩增海洋海绵中的真菌序列。DGGE结果显示,两种不同的夏威夷海洋海绵(Suberites zeteki和Mycale armata)中的真菌群落存在明显的差异,同时在海洋海绵和海水样品中的真菌群落也存在一定的差异。通过对所得DGGE条带的测序,在海绵S. zeteki中鉴定了23种真菌的存在;而在海绵M. armata中鉴定了21种真菌的存在。这些真菌属于11个不同的目,其中包括子囊菌门(Ascomycota)7个目,担子菌门(Basidiomycota)4个目,并且有5个目(Malasseziales, Corticiales, Polyporales, Agaricales和Dothideomycetes et Chaetothyriomcetes incertae sedis)是在海洋海绵中的首次鉴定。在S. zeteki和M. armata中,分别有7个和6个可能的真菌新种被鉴定出来,这些序列与它们在GenBank中的对应序列的序列相似性小于98%。系统进化分析发现,这些来自于海洋海绵的真菌序列与一些其他的来自于海洋生境的序列一起属于“海洋真菌”进化支。
本研究是首次应用分子生物学的方法鉴定海洋海绵中的真菌群落,为以后进一步的研究海绵共附生微生物群落提供了新的思路和方法。
Isolation and Characterization of a Putative Cyclic Nucleotide-Gated Cation Channel Gene GhCNGC2 in Cotton
The 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-, Cu~(2+)- 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.
引文
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