Chunsheng Gao

A rice stress-responsive NAC gene enhances tolerance of transgenic wheat to drought and salt stresses

Drought and salinity are the primary factors limiting wheat production worldwide. It has been shown that a rice stress-responsive transcription factor encoded by the rice NAC1 gene (SNAC1) plays an important role in drought stress tolerance. Therefore, we introduced the SNAC1 gene under the control of a maize ubiquitin promoter into an elite Chinese wheat variety Yangmai12. Plants expressing SNAC1 displayed significantly enhanced tolerance to drought and salinity in multiple generations, and contained higher levels of water and chlorophyll in their leaves, as compared to wild type. In addition, the fresh and dry weights of the roots of these plants were also increased, and the plants had increased sensitivities to abscisic acid (ABA), which inhibited root and shoot growth. Furthermore, quantitative real-time polymerase chain reactions revealed that the expressions of genes involved in abiotic stress/ABA signaling, such as wheat 1-phosphatidylinositol-3-phosphate-5-kinase, sucrose phosphate synthase, type 2C protein phosphatases and regulatory components of ABA receptor, were effectively regulated by the alien SNAC1 gene. These results indicated high and functional expression of the rice SNAC1 gene in wheat. And our study provided a promising approach to improve the tolerances of wheat cultivars to drought and salinity through genetic engineering.

Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers

Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.

Wheat-Specific Gene, Ribosomal Protein L21, Used as the Endogenous Reference Gene for Qualitative and Real-Time Quantitative Polymerase Chain Reaction Detection of Transgenes

Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5-4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.

Deletion of the sex-determining gene SXI1α enhances the spread of mitochondrial introns in Cryptococcus neoformans

BackgroundHoming endonuclease genes (HEGs) are widely distributed genetic elements in the mitochondrial genomes of a diversity of eukaryotes. Due to their ability to self-propagate within and between genomes, these elements can spread rapidly in populations. Whether and how such elements are controlled in genomes remains largely unknown.ResultsHere we report that the HEG-containing introns in the mitochondrial COX1 gene in Cryptococcus neoformans are mobile and that their spread in sexual crosses is influenced by mating type (MAT) α-specific homeodomain gene SXI1α. C. neoformans has two mating types, MATa and MATα. In typical crosses between strains of the two mating types, only a small portion (< 7%) of diploid fusants inherited the HEGs from the MATα parent. However, disruption of the SXI1α gene resulted in the majority (> 95%) of the diploid fusants inheriting the HEG-containing introns from the MATα parent, a frequency significantly higher than those of intronless mitochondrial genes.ConclusionsOur results suggest that SXI1α not only determines uniparental mitochondrial inheritance but also inhibits the spread of HEG-containing introns in the mitochondrial genome in C. neoformans.

Embryological and genetic evidence of amphimixis and apomixis in Boehmeria tricuspis

Apomixis is a widespread alternative mode of sexual reproduction resulting in offspring that are genetically identical to the maternal plant. Boehmeria tricuspis (Hance) Makino is a perennial, wind-pollinated, herbaceous plant in the nettle family Urticaceae. The diploid B. tricuspis is monoecious but the triploid B. tricuspis is gynoecious, bearing female inflorescences only. Apomixis in B. tricuspis was first reported 50 years ago, but the mode of apomixis in the species has not been described yet. Here, we provide embryological observations of the embryo sac formation proving that triploid B. tricuspis reproduced apomictically following the Antennaria type of diplospory, and that the diploid individuals were the sexual genotype with the classical Polygonum-type maturation pattern of embryo sac development. A subsequent flow cytometry seed screen (FCSS) showed that the triploids were obligate apomicts with autonomous endosperm development, and the diploids reproduced sexually. In addition, a progeny test by molecular marker assays further demonstrated the above results.

First report of brown root rot caused by Pythium vexans on ramie in Hunan, China

Abstract Ramie is an important natural fibre crop and a perennial herbaceous plant of the nettle family Urticaceae. In 2013, brown root rot disease was observed on ramie plants in Yuangjiang, Hunan Province, China. Symptoms included stunted plants, reduced number of ramets per plant, and brown to purple-black discolouration of the root. The pathogen was identified as Pythium vexans according to morphological features and sequence identity (99–100%) to the published data for internal transcribed spacer (ITS), 18S and 28S rDNA sequences. The pathogenicity was confirmed on healthy fibrous roots of seedlings and on detached storage roots under greenhouse conditions. To our knowledge, this is the first report of brown root rot disease caused by Pythium vexans on ramie.

Identification and characterization of hemp WRKY transcription factors in response to abiotic stresses

Plant WRKY genes encode a complex and ancient class of zinc-finger transcription factors that are involved in multiple biological processes, especially in regulating defense against abiotic stresses. Despite a growing number of studies on the genomic organization of the WRKY gene family in various species, little information is available about this family in hemp (Cannabis sativa L.). In this study, based on the hemp genome sequence, 40 hemp WRKY (CsWRKY) genes were classified into three main groups and five subgroups according to their orthologs in Arabidopsis. Among these, 23, 15, and 14 CsWRKY genes were responsive to drought, NaCl, and Cd stress, respectively. Interestingly, the expressions of all of the 23 drought stress-responsive genes were up-regulated. Moreover, 18 CsWRKY genes were induced by abscisic acid (ABA) treatment. A total of six up-regulated genes related to all three stresses were identified. Among these, five were up-regulated, and one was down-regulated by ABA. These results indicate a diverse function of the CsWRKY genes, which provides a basis for future clarification of their function in hemp tolerance to abiotic stresses.

Isolation and identification of a Bacillus subtilis HZ-72 exhibiting biocontrol activity against flax seedling blight

Seedling blight caused by Rhizoctonia solani is a serious soil-borne disease on flax. In this study, we isolated a bacterial strain HZ-72 from the rhizosphere soil of flax with obvious inhibitory effect on R. solani and other six plant fungal pathogens. Strain HZ-72 was identified as Bacillus subtilis based on morphological, physiological, biochemical characteristics and 16S rDNA sequence analysis. In greenhouse experiments, the control efficiency of strain HZ-72 reached 83.34%. Additionally, in vitro assays indicated that cell wall-degrading enzymes such as protease and cellulase, volatile compounds, proteins and lipopeptides produced by strain HZ-72 all contributed to its antagonistic activity against R. solani. To our knowledge, this is the first report on the use of a rhizosphere B. subtilis strain as a biocontrol agent for the biocontrol of flax seedling blight caused by R. solani.

Genome-Wide Expression Profiles of Hemp (Cannabis sativa L.) in Response to Drought Stress

Drought is the main environmental factor impairing hemp growth and yield. In order to decipher the molecular responses of hemp to drought stress, transcriptome changes of drought-stressed hemp (DS1 and DS2), compared to well-watered control hemp (CK1 and CK2), were studied with RNA-Seq technology. RNA-Seq generated 9.83, 11.30, 11.66, and 11.31 M clean reads in the CK1, CK2, DS1, and DS2 libraries, respectively. A total of 1292 differentially expressed genes (DEGs), including 409 (31.66%) upregulated and 883 (68.34%) downregulated genes, were identified. The expression patterns of 12 selected genes were validated by qRT-PCR, and the results were accordant with Illumina analysis. Gene Ontology (GO) and KEGG analysis illuminated particular important biological processes and pathways, which enriched many candidate genes such as NAC, B3, peroxidase, expansin, and inositol oxygenase that may play important roles in hemp tolerance to drought. Eleven KEGG pathways were significantly influenced, the most influenced being the plant hormone signal transduction pathway with 15 differentially expressed genes. A similar expression pattern of genes involved in the abscisic acid (ABA) pathway under drought, and ABA induction, suggested that ABA is important in the drought stress response of hemp. These findings provide useful insights into the drought stress regulatory mechanism in hemp.

Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.)

Phytocystatins play multiple roles in plant growth, development and resistance to pests and other environmental stresses. A ramie (Boehmeria nivea L.) phytocystatin gene, designated as BnCPI, was isolated from a ramie cDNA library and its full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence (691 bp) consisted of a 303 bp open reading frame (ORF) encoding a protein of 100 amino acids with deduced molecular mass of 11.06 kDa and a theoretical isoelectric point (pI) of 6.0. The alignment of genome DNA (accession No. MF153097) and cDNA sequences of BnCPI showed that an intron (~104 bp) exists in the coding region. The BnCPI protein contains most of the highly conserved blocks including Gly5-Gly6 at the N-terminal, the reactive site motif QxVxG (Q49V50V51S52G53), the L79-W80 block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L22G23R24 F25A26V27 D28D29H30 N31) block that is common among plant cystatins. BLAST analysis indicated that BnCPI is similar to cystatins from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%). The BnCPI was subcloned into expression vector pSmart-I and then overexpressed in Escherichia coli BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of 11.4 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA (Nα-benzoyl-L-arginine-2-naphthyamide), as well as the mycelium growth of some important plant pathogenic fungi. The data further contribute to our understanding of the molecular functions of BnCPI.