Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

DNA-Barcoding of Some Medicinal Plant Species in Saudi Arabia Using <i>rbcL</i> and <i>matK</i> Genes

In the Kingdom of Saudi Arabia (KSA), thousands of plants are considered to have therapeutic value. The ambiguous use of identification mainly morphological characteristics of many plants has resulted in the adulteration and displacement of plant products which undermine their therapeutic value and weak documentation of plant resources. The aims of this study were therefore to evaluate genetic variability and explore the phylogeographic architecture for Saudi medicinal plant samples using rbcL and matK genes as barcodes for genomic identification. The matK and rbcL sequences collected for these samples were used as key markers for examining the relationship between Saudi medicinal plant species based on genetic diversity. During our study we were successful in identifying and documenting 4 different species (Foeniculum vulgare, Nitraria retusa, Dodonaea viscosa, and Rumex nervosus) located in Saudi Arabia using DNA barcoding technique. A total number of 8 sequences were obtained with a total sequence length of 6176 bp, where it ranged from 617 bp to 878 bp with an average length of 772 bp. The total number of rbcL sequences length is 2801 bp, where it ranges from 617 bp to 807 bp with an average length of 700.2 bp. Out of the 4 plant samples used, only three samples were identified correctly on the species level with an identity percentage higher than 95% using rbcL gene. Additionally, 4 matK sequences have been retrieved belong to 4 species. The total number of matK sequences length is 3375 bp, where it ranges from 819 bp to 878 bp with an average length of 843.8 bp. Out of the 4 plant samples used, only two samples were identified correctly on the species level with an identity percentage higher than 98% using matK gene. Both rbcL and matK have been able to identify most of our collected plant samples by genus, and some by species. Using only one DNA-barcoding technique was not reliable for plant identification, where matK and rbcL must be used as a dual DNA-barcoding procedure.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Molecular cloning and characterization of an inducible RNA-dependent RNA polymerase gene, <Emphasis Type="Italic">GhRdRP</Emphasis>, from cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

The RNA-dependent RNA polymerase (RdRP) cDNA, designated as Gossypium hirsutum RdRP (GhRdRP) was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 3,672 bp in size and encoded an open reading frame (ORF) of 1,110 amino acids which contained the RdRP conserved functional domain and the signature motif DbDGD. Amino acid sequence alignment indicated that GhRdRP shared the highest identity (66.37%) with AtRdRP1 and had homology with other plant, fungal, yeast and nematode RdRPs. The corresponding genomic DNA containing five exons and four introns, was isolated and analyzed. Also a 5′-flanking region was cloned, and a group of putative cis-acting elements were identified. Southern blot analysis revealed a single copy of the GhRdRP gene in cotton genome. The expression analysis by semi-quantitative RT-PCR showed that GhRdRP was induced by salicylic acid (SA), 5-chloroSA (5-CSA) and fungal infection of Rhizoctonia solani Kuhn. The cloning and characterization of the GhRdRP gene will be useful for further studies of biological roles of GhRdRP in plants.     

Characterization of <Emphasis Type="Italic">Glossy1</Emphasis>-homologous genes in rice involved in leaf wax accumulation and drought resistance

The outermost surfaces of plants are covered with an epicuticular wax layer that provides a primary waterproof barrier and protection against different environmental stresses. Glossy 1 (GL1) is one of the reported genes controlling wax synthesis. This study analyzed GL1-homologous genes in Oryza sativa and characterized the key members of this family involved in wax synthesis and stress resistance. Sequence analysis revealed 11 homologous genes of GL1 in rice, designated OsGL1-1 to  OsGL1-11. OsGL1-1, -2 and -3 are closely related to GL1. OsGL1-4, -5, -6, and -7 are closely related to Arabidopsis CER1 that is involved in cuticular wax biosynthesis. OsGL1-8, -9, -10 and -11 are closely related to SUR2 encoding a putative sterol desaturase also involved in epicuticular wax biosynthesis. These genes showed variable expression levels in different tissues and organs of rice, and most of them were induced by abiotic stresses. Compared to the wild type, the OsGL1-2-over-expression rice exhibited more wax crystallization and a thicker epicuticular layer; while the mutant of this gene showed less wax crystallization and a thinner cuticular layer. Chlorophyll leaching experiment suggested that the cuticular permeability was decreased and increased in the over-expression lines and the mutant, respectively. Quantification analysis of wax composition by GC–MS revealed a significant reduction of total cuticular wax in the mutant and increase of total cuticular wax in the over-expression plants. Compared to the over-expression and wild type plants, the osgl1-2 mutant was more sensitive to drought stress at reproductive stage, suggesting an important role of this gene in drought resistance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

一种强启动子的分离与功能

A bidirectional promoter of cotton leaf curl virus (CLCuV) was obtained from the total of DNA CLCuV infected tomato leaves by polymerase chain reaction, and the amplified DNA fragment was cloned into the vector. DNA sequences analysis and homology comparison with the promotor of four kinds of isolates recently found indicated that the cloned promoter fragment composed of 436 bp was 99.32% homolog was up to in nucleotides with that of the isolates. Transient expression vectors were constructed by fusing the promoter fragment with gus reporter gene and nopaline terminator in different orientation. These constructs were delivered into the tobacco (Nicotiana tabacum L.) and cotton ( Gossypium hirsutum L.) leaf cells for transient expression by particle bombardment. The results indicated that complementary sense promoter was a strong promoter with high activity in leaf mesophyll and vascular tissues, but virion sense promoter was weaker. The experiments suggested that isolated bidirectional promoter, as a novel strong promoter, could be used for dicots and especially cotton genetic transformation.     

一种强启动子的分离与功能

以棉花曲叶病毒（ＣＬＣｕＶ）侵染的番茄叶片组织总ＤＮＡ为模板,通过ＰＣＲ反应扩增ＣＬＣｕＶ双向启动子片段并插入克隆载体。序列分析和同源性比较表明,克隆的启动子长４３６ｂｐ,与目前发现的４类ＣＬＣｕＶ分离物的启动子序列的同源性最高为９９．３２％。将启动子片段分别以不同方向与ｇｕｓ报告基因和ｎｏｓ终止子融合,构建了瞬时表达载体。通过基因枪法将质粒载体导入烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.