Lili Geng

Mining Tissue-specific Contigs from Peanut (Arachis hypogaea L.) for Promoter Cloning by Deep Transcriptome Sequencing

Peanut (Arachis hypogaea L.), one of the most important oil legumes in the world, is heavily damaged by white grubs. Tissue-specific promoters are needed to incorporate insect resistance genes into peanut by genetic transformation to control the subterranean pests. Transcriptome sequencing is the most effective way to analyze differential gene expression in this non-model species and contribute to promoter cloning. The transcriptomes of the roots, seeds and leaves of peanut were sequenced using Illumina technology. A simple digital expression profile was established based on number of transcripts per million clean tags (TPM) from different tissues. Subsequently, 584 root-specific candidate transcript assembly contigs (TACs) and 316 seed-specific candidate TACs were identified. Among these candidate TACs, 55.3% were root-specific and 64.6% were seed-specific by semi-quantitative RT-PCR analysis. Moreover, the consistency of semi-quantitative RT-PCR with the simple digital expression profile was correlated with the length and TPM value of TACs. The results of gene ontology showed that some root-specific TACs are involved in stress resistance and respond to auxin stimulus, whereas, seed-specific candidate TACs are involved in embryo development, lipid storage and long-chain fatty acid biosynthesis. One root-specific promoter was cloned and characterized. We developed a high-yield screening system in peanut by establishing a simple digital expression profile based on Illumina sequencing. The feasible and rapid method presented by this study can be used for other non-model crops to explore tissue-specific or spatially specific promoters.

Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis

Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects.

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela

Key messagePeanuts transformed with the syntheticcry8Ea1gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times.AbstractWhite grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.

The influence of Bt-transgenic maize pollen on the bacterial diversity in the midgut of Apis mellifera ligustica

Honeybees are subjected to direct contact with transgenic maize pollen due to their feeding activities on pollen. The potential side effects of transgenic cry1Ah-maize pollen on the midgut bacteria of the larvae and worker bees of Apis mellifera ligustica were investigated through denaturing gradient gel electrophoresis under both laboratory and field conditions. Newly emerged bees were fed transgenic cry1Ah-maize pollen, normal maize pollen, linear cry1Ah gene DNA, supercoiled plasmid DNA, and sugar syrup under the laboratory conditions. The results showed that there were no significant differences in the midgut bacterial community composition among the five treatments. No significant effects were observed in the midgut communities between larvae and adult honeybees fed transgenic cry1Ah-maize pollen and non-transgenic maize pollen in the field trials.

High-efficiency regeneration of peanut ( Arachis hypogaea L.) plants from leaf discs

A high-efficiency regeneration system for peanut plants was established. The regeneration frequency of leaf discs reached 40.9% on Murashige and Skoog medium supplemented with 0.5 mg l -1 naphthylacetic acid and 0.5 mg l -1 thidiazuron. The regenerated shoots elongated, developed roots and produced seeds. This procedure was highly efficient and is feasible for the genetic transformation of peanuts. Key words : Peanut, regeneration, high efficiency.

The Influence of Bt-Transgenic Maize Pollen on the Bacterial Diversity in the Midgut of Chinese Honeybees, Apis cerana cerana

Abstract Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana , was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear cry1Ah gene (800 ng mL −1 ) and supercoiled plasmid DNA (800 ng mL −1 ) under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannons index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic cry1Ah maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.

No adverse effects of transgenic maize on population dynamics of endophytic Bacillus subtilis strain B916-gfp

Endophytic bacterial communities play a key role in promoting plant growth and combating plant diseases. However, little is known about their population dynamics in plant tissues and bulk soil, especially in transgenic crops. This study investigated the colonization of transgenic maize harboring the Bacillus thuringiensis (Bt) cry1Ah gene by Bacillus subtilis strain B916‐gfp present in plant tissues and soil. Bt and nontransgenic maize were inoculated with B916‐gfp by seed soaking, or root irrigation under both laboratory greenhouse and field conditions. During the growing season, B916‐gfp colonized transgenic as well as nontransgenic plants by both inoculation methods. No differences were observed in B916‐gfp population size between transgenic and nontransgenic plants, except at one or two time points in the roots and stems that did not persist over the examination period. Furthermore, planting transgenic maize did not affect the number of B916‐gfp in bulk soil in either laboratory or field trials. These results indicate that transgenic modification of maize with the cry1Ah gene has no influence on colonization by the endophytic bacteria B916‐gfp present in the plant and in bulk soil.

Template Preparation Affects 16S rRNA High-Throughput Sequencing Analysis of Phyllosphere Microbial Communities

Phyllosphere microbial communities are highly diverse and have important ecological implications; in that context, bacterial identification based on 16S rRNA genes is an important research issue. In studies of phyllosphere microbial communities, microporous filtration and centrifugation are used to collect microorganism samples, but it is unclear which one has a better collection efficiency. In this study, we compared these two microorganism collection methods and investigated the effects of the DNA extraction process on the estimation of microbial community composition and organization. The following four treatments were examined: (A) filtration, resuspension, and direct PCR; (B) filtration, DNA isolation, and PCR; (C) centrifugation, resuspension, and direct PCR; (D) centrifugation, DNA isolation, and PCR. Our results showed that the percentage of chloroplast sequence contaminants was affected by the DNA extraction process. The bacterial compositions clearly differed between treatments A and C, suggesting that the collection method has an influence on the determination of community structure. Compared with treatments B and D, treatments A and C resulted in higher Shannon index values, indicating that the DNA extraction process might reduce the observed phyllosphere microbial alpha diversity. However, with respect to community structure, treatments B and D yielded very similar results, suggesting that the DNA extraction process erases the effect of the collection method. Our findings provide key information to ensure accurate estimates of diversity and community composition in studies of phyllosphere microorganisms.

Subterranean infestation by Holotrichia parallela larvae is associated with changes in the peanut (Arachis hypogaea L.) rhizosphere microbiome

Rhizosphere microorganisms contribute to the health and development of crops and these beneficial microbes are recruited to the root-zone when plants experience biotic/abiotic stress. The subterranean pests Holotrichia parallela cause severe crop loss in peanut (Arachis hypogaea L.) fields. Hypothesizing that infestation by H. parallela larva may influence the composition of rhizosphere microbial communities, deep sequencing of V3 and V4 hypervariable regions of 16S rRNA gene was used to characterize the rhizosphere bacteria of infested and uninfested peanuts. A total of 2,673,656 reads were generated and an average of 2558 OTUs were obtained for each sample. Comparisons of rhizosphere bacterial community structure of peanuts with those infested by H. parallela larva revealed that the relative abundance of Proteobacteria and Bacteroidetes increased, while that of Actinobacteria decreased in the rhizosphere with infestation. A significant shift in bacterial communities was observed within 24 h after infestation by principal coordinate analysis. For the 332 genera identified in 24 h treatment, infestation of white grubs led to the significant changes of abundance of 67 genera. An increase in the Pseudomonas genus of infested-samples for 24 h was verified by real-time qPCR. Our results indicate H. parallela larvae infestation can quickly leads to the change of peanut rhizosphere microbiome and enrichment of specific bacterial species. But the effects were not persistent. This study provides the insight into the function of rhizosphere microbiome in the interaction between subterranean pests and crops.

Complete genome sequence of Bacillus thuringiensis Bt185, a potential soil insect biocontrol agent

Bacillus thuringiensis Bt185 and its insecticidal spectrum-expanded engineering strains are considered as potential biocontrol agents to soil insect Holotrichia parallela, Holotrichia oblita or Anomala corpulenta. Here we reported the complete genome of strain Bt185, it harbors eight plasmids, and plasmid pBT1850294 carries three cry8 genes.