Huaizhong Jiang

Analysis of sea-island cotton and upland cotton in response to Verticillium dahliae infection by RNA sequencing

BackgroundCotton Verticillium wilt is a serious soil-borne vascular disease that causes great economic loss each year. However, due to the lack of resistant varieties of upland cotton, the molecular mechanisms of resistance to this disease, especially to the pathogen Verticillium dahliae, remain unclear.ResultsWe used the RNA-seq method to research the molecular mechanisms of cotton defence responses to different races of Verticillium dahliae by comparing infected sea-island cotton and upland cotton. A total of 77,212 unigenes were obtained, and the unigenes were subjected to BLAST searching and annotated using the GO and KO databases. Six sets of digital gene expression data were mapped to the reference transcriptome. The gene expression profiles of cotton infected with Verticillium dahliae were compared to those of uninfected cotton; 44 differentially expressed genes were identified. Regarding genes involved in the phenylalanine metabolism pathway, the hydroxycinnamoyl transferase gene (HCT) was upregulated in upland cotton whereas PAL, 4CL, CAD, CCoAOMT, and COMT were upregulated in sea-island cotton. Almost no differentially expressed genes in this pathway were identified in sea-island cotton and upland cotton when they were infected with V. dahliae V991 and V. dahliae D07038, respectively.ConclusionsOur comprehensive gene expression data at the transcription level will help elucidate the molecular mechanisms of the cotton defence response to V. dahliae. By identifying the genes involved in the defence response of each type of cotton to V. dahliae, our data not only provide novel molecular information for researchers, but also help accelerate research on genes involved in defences in cotton.

Identification of novel microRNAs in the Verticillium wilt-resistant upland cotton variety KV-1 by high-throughput sequencing

Plant microRNAs (miRNAs) play essential roles in the post-transcriptional regulation of gene expression during development, flowering, plant growth, metabolism, and stress responses. Verticillium wilt is one of the vascular disease in plants, which is caused by the Verticillium dahlia and leads to yellowing, wilting, lodging, damage to the vascular tissue, and death in cotton plants. Upland cotton varieties KV-1 have shown resistance to Verticillium wilt in multiple levels. However, the knowledge regarding the post-transcriptional regulation of the resistance is limited. Here two novel small RNA (sRNA) libraries were constructed from the seedlings of upland cotton variety KV-1, which is highly resistant to Verticillium wilts and inoculated with the V991 and D07038 Verticillium dahliae (V. dahliae) of different virulence strains. Thirty-seven novel miRNAs were identified after sequencing these two libraries by the Illumina Solexa system. According to sequence homology analysis, potential target genes of these miRNAs were predicted. With no more than three sequence mismatches between the novel miRNAs and the potential target mRNAs, we predicted 49 target mRNAs for 24 of the novel miRNAs.These target mRNAs corresponded to genes were found to be involved in plant–pathogen interactions, endocytosis, the mitogen-activated protein kinase (MAPK) signaling pathway, and the biosynthesis of isoquinoline alkaloid, terpenoid backbone, primary bile acid and secondary metabolites. Our results showed that some of these miRNAs and their relative gene are involved in resistance to Verticillium wilts. The identification and characterization of miRNAs from upland cotton could help further studies on the miRNA regulatory mechanisms of resistance to Verticillium wilt.

Gene expression profiling during gland morphogenesis of a mutant and a glandless upland cotton

To identify genes involved in pigment gland morphogenesis in cotton, gene expression was profiled using genechip (Affymetrix) during pigment gland morphogenesis in cotton variety Xiangmian-18, which has glandless seeds but glanded plants, and a glandless line, N5. The results showed that 303 genes were differentially expressed by a factor greater than two during gland morphogenesis; 59% (180) of these genes shared similarity with known genes in GenBank. These genes play roles in defense response, response to oxidative stress, peroxidase activity, and other metabolic pathways. KOBAS (KEGG Orthology-Based Annotation System) indicate that these genes are involved in 68 biochemical pathways. These findings suggest that the related defense response, gossypol biosynthesis pathway and other complex regulation may be associated with pigment gland morphogenesis in cotton. The results may provide a basis for further study and serve as a guide for related research.

Molecular phylogeny of Ranunculaceae based on rbc L sequences

A phylogenetic tree was constructed by sequencing rbcL genes of 33 species representing 19 genera of Ranunculaceae, and three related species, Mahonia bealei, Mahonia fortunei and Nandina domestica. The results showed that the rbcL sequences of these Ranunculaceae range from 1,346 bp to 1,393 bp. The results based on the phylogenetic tree indicated that Caltha and Trollius should not be put in the same tribe, and a close relationship between Adonis and Trollius is supported by our research, while Aquilegia should be in Thalictroideae. In combination with the morphological and chemical evidence, the generic classification of Ranunculaceae should be revised into five subfamilies: Hydrastidoideae, Coptidoideae, Helleboroideae, Thalictroideae and Ranunculoideae. We demonstrate that the rbcL gene is of great value for investigating generic to subfamilial relationships in Ranunculaceae.

Profiling Gene Expression During Gland Morphogenesis of a Glanded and a Glandless Upland Cotton

The pigment gland is an important character of the Gossypium plant. With the aim of identifying genes involved in pigment gland morphogenesis in cotton, gene expression during pigment gland morphogenesis in Chuan 2802, which is glanded both in seed and plant, and a glandless line N5 was profiled using Affymetrix Cotton microarray. The results showed that there were 564 differentially expressed genes greater than twofold during gland morphogenesis. About 60.2% of these genes shares similarity with known genes on GenBank and about 39.8% with no functional description in the database. These described genes may play roles in defense response, response to oxidative stress, peroxidase activity, and the other metabolic pathways. The KEGG Orthology-Based Annotation System indicated that these above twofold expressed genes involved seven biochemical pathways on KEGG. These findings suggest that a complicated regulation is associated with pigment gland morphogenesis and the associated defense response including gossypol biosynthesis in cotton.

Molecular cloning and expression analysis of a new WD40 repeat protein gene in upland cotton

A new member of the WD repeat protein family, named GhWD40, was cloned from a near-isogenic line for glands in cotton. It has 2629 bp cDNA and a complete opening reading frame (ORF) of 1239 bp, containing the initial code (ATG) and terminal code (TAG); there is a 1061 bp non-coding sequence at the 5′-end, and a 329 bp non-coding sequence at the 3′-end, including the poly(A) sequence (accession number: JN714279). The predicted protein of the complete ORF comprised 412 amino acids with a calculated molecular mass of 47.1 kDa and an isoelectric point of 8.88. Protein domain scanning showed that the novel protein has five wd40 motifs and belongs to the WD40 family. From a search for GhWD40 cDNA and amino acid sequences in the database, it has 77% sequence identity and was 90% sequence positive with the WD-40 repeat protein from Trifolium pratense (accession number BAE71307.1), and 80% sequence identity and 89% sequence positivity with the ribosome biogenesis protein bop1 from Ricinus communis (accession number XP 002529002.1). We propose that GhWD40 may play the same role as bop1. In addition, expression of GhWD40 in near-isogenic lines 11 and 3 (with and without glands, respectively) was studied by quantitative RT-polymerase chain reaction, and the level in near-isogenic line 11 was higher than that in near-isogenic line 3, suggesting that GhWD40 may be related to gland formation.

Molecular characterization and alternative splicing of a MYB transcription factor gene in tumourous stem mustard and its response to abiotic stresses

Abstract Tumourous stem mustard, a variant of Brassica juncea (Cruciferae), is an economically important crop. In a preliminary study, the expression of a fragment of a MYB (v-myb avian myeloblastosis viral oncogene homolog) gene was screened out during stem swelling. The four complete cDNA sequences of novel MYB genes were cloned by rapid amplification of cDNA ends based on the MYB sequence fragment. These sequences, which represent distinct alternatively spliced transcripts of a BjMYB1 (Brassica juncea var. tumida Tsen et Lee MYB gene 1), possessed a similar alternative splicing pattern to several reported MYB genes of other species. RT-PCR analysis revealed different expression patterns for these four spliced transcripts in various tissues. However, stem expression of BjMYB1 gene was detected in the non-swelling growth phase, and different expression patterns were detected at other stages during stem swelling. The expression levels of the transcripts differed after treatment with various phytohormones (e.g., ABA) and NaCl. In addition, prokaryotic expression and Western blotting showed that BjMYB1-2 encodes a 5.3 kDa protein while BjMYB1-3 encodes a 21.6 kDa protein. The overexpression of BjMYB1-3 and BjMYB1-4 in Arabidopsis thaliana showed earlier germination, promoting growth and having tolerance to abiotic stress such as osmotic stress or high salt. The research of this article set the foundation for further study of the function of MYB genes in tumourous stem mustard and other Brassica plants.

Identification of the genes and pathways associated with pigment gland morphogenesis in cotton by transcriptome profiling of near-isogenic lines

Cottonseed protein is underutilized due to the presence of pigment gland containing a toxic compound called gossypol. Cotton produces gossypol and related compounds in various tissues to protect itself against microbial, insect, and rodent attacks. Understanding the mechanism of cotton pigment gland formation and regulation of gossypol biosynthesis will greatly facilitate the research efforts in developing a cotton variety with a gossypol free seed and normally glanded foliage. In this study we make use of near-isogenic lines of cotton pigment gland to screen the genes related to gland morphogenesis applying both GeneChip and suppression subtractive hybridization (SSH) methods.We identified 880 differentially expressed genes associated with gland morphogenesis in cotton by comparing transcriptome profiles of cotton from glandless and glanded near-isogenic lines using a GeneChip. Gene ontology (GO) analysis showed that 880 genes were distributed mainly among the following GO categories: cellular process (14.45%), physiological process (14.23%), catalytic activity (9.21%), metabolism (8.99%), and cell parts (5.24%). Molecular pathway analysis revealed that these differentially expressed genes were involved in 58 KEGG pathways. Differentially expressed genes were also identified and isolated using suppression subtractive hybridization (SSH) with the same near-isogenic lines. A total of 147 ESTs were identified whose expression was either up- or down-regulated. Sequencing and BLAST analysis indicated that some of these genes were novel, while others were related to energy metabolism, transcription factors, and biotic responses. 13 genes were found to be differentially expressed both in SSH and GeneChip analysis. The expression pattern of these genes was verified by real-time PCR. The gene expression profiles produced in this study provide useful information on the molecular mechanism and regulation of gland formation and the related process in cotton. Of particular interest for future study are the genes identified by both SSH and GeneChip analysis. The outcomes are helping for our understanding of the development of specialised structures such as trichomes in plant species, from an applied and basic science perspective and promoting the application in molecular breeding.

Molecular cloning, structural analysis and expression of a zinc binding protein in cotton

The full-length zinc-binding protein (ZnBP) gene was cloned from a normalized cDNA library constructed from a cotton mutant (Xiangmian-18) during the gland-forming stage. The clone was sequenced and analysed. BLASTP analysis showed that the deduced amino acid sequence of ZnBP in Xiangmian-18 is similar to that in Arabidopsis thaliana (GenBank accession no. EFH46337.1) with an overall similarity of 77%. The cDNA insert comprises 654 base pairs (bp) and 217 amino acid residues. Its molecular weight is 24.6 kDa, and the theoretical pI is 9.33. The cotton ZnBP gene was cloned from the gDNA from Xiangmian-18 leaves. After sequencing the two fragments, a 1731 bp cotton ZnBP gene with three introns was identified. Using pET-28a(+) as a prokaryotic expression vector, the gene was expressed in Escherichia coli BL21(DE3). The conditions for achieving optimal ZnBP expression were 37°C, IPTG 1 mmol/L, 8 h and a shaker speed of 150 rpm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed the correct expression of the protein. pCAMBIA2300-35S-OCS was used as a eukaryotic expression vector. The recombinant plasmid pCAMBIA2300-ZnBP was used to transform competent Agrobacterium GV3101 by the freeze-thaw method. Then, A. thaliana plants were transformed by the floral dipping method. Transformed plants were grown to maturity in a growth chamber. After screening on kanamycin-resistant half-strength Murashige and Skoog plates and polymerase chain reaction (PCR) analysis, two transgenic plant strains were obtained. Northern blot analysis showed that ZnBP expression was higher in homozygous plants than in wild-type plants. The differences between the phenotypes of homozygous and wild-type plants indicate that the ZnBP gene affects the growth and development of A. thaliana. The results of prokaryotic expression of ZnBP and overexpression of the ZnBP gene in A. thaliana improve our understanding of the function of this gene. Future studies should investigate the molecular mechanisms involved in gland morphogenesis in cotton. Key words : Gossypium hirsutum, pigment gland, zinc binding protein, prokaryotic expression, overexpression.