Project description:As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.
Project description:We conduct a genome-wide analysis of the DNA sequences associated with CenH3 using chromatin immunoprecipitation to map the position of centromere regions.
Project description:Low dosages of chloramphenicol (25-50 micrograms/ml) brought about a 2-4-fold stimulation of acid phosphatase activity in 48 h-germinated cotton (Gossypium hirsutum) embryos. However, at high concentrations of chloramphenicol (100-1000 micrograms/ml), there was a progressive decline in enzyme activity. The stimulatory effect of the drug on acid phosphatase activity was relatively specific, since no significant stimulation of activities of proteinase, deoxyribonuclease, ribonuclease, o-diphenolase and peroxidase was observed in germinating cotton embryos. Chloramphenicol, however, did promote the activities of isocitric lyase and alkaline phosphatase. Sephadex G-200 chromatography of the enzyme fraction revealed high (230 000)- and low (106 000)-molecular-weight multiple forms of acid phosphatase in the chloramphenicol-treated embryos, in contrast with a single molecular form (mol.wt. 106 000) in the untreated embryos. Thus the treatment of cotton embryos with chloramphenicol induced both a qualitative and a quantitative change in the acid phosphatase activity. Chloramphenicol-stimulated acid phosphatase activity was strongly inhibited when Pi was included in the germination medium. However, the control embryos showed less pronounced inhibition of enzyme activity in presence of Pi ions.
Project description:BackgroundAn evolutionary model using diploid and allotetraploid cotton species identified 80 % of non-coding transcripts in allotetraploid cotton as being uniquely activated in comparison with its diploid ancestors. The function of the lncRNAs activated in allotetraploid cotton remain largely unknown.ResultsWe employed transcriptome analysis to examine the relationship between the lncRNAs and mRNAs of protein coding genes (PCGs) in cotton leaf tissue under abiotic stresses. LncRNA expression was preferentially associated with that of the flanking PCGs. Selected highly-expressed lncRNA candidates (n = 111) were subjected to a functional screening pilot test in which virus-induced gene silencing was integrated with abiotic stress treatment. From this low-throughput screen, we obtained candidate lncRNAs relating to plant height and tolerance to drought and other abiotic stresses.ConclusionsLow-throughput screen is an effective method to find functional lncRNA for further study. LncRNAs were more active in abiotic stresses than PCG expression, especially temperature stress. LncRNA XLOC107738 may take a cis-regulatory role in response to environmental stimuli. The degree to which lncRNAs are constitutively expressed may impact expression patterns and functions on the individual gene level rather than in genome-wide aggregate.
Project description:Fiber quality is an important economic index and a major breeding goal in cotton, but direct phenotypic selection is often hindered due to environmental influences and linkage with yield traits. A genome-wide association study (GWAS) is a powerful tool to identify genes associated with phenotypic traits. In this study, we identified fiber quality genes in upland cotton (Gossypium hirsutum L.) using GWAS based on a high-density CottonSNP80K array and multiple environment tests. A total of 30 and 23 significant single nucleotide polymorphisms (SNPs) associated with five fiber quality traits were identified across the 408 cotton accessions in six environments and the best linear unbiased predictions, respectively. Among these SNPs, seven loci were the same, and 128 candidate genes were predicted in a 1-Mb region (±500 kb of the peak SNP). Furthermore, two major genome regions (GR1 and GR2) associated with multiple fiber qualities in multiple environments on chromosomes A07 and A13 were identified, and within them, 22 candidate genes were annotated. Of these, 11 genes were expressed [log2(1 + FPKM)>1] in the fiber development stages (5, 10, 20, and 25 dpa) using RNA-Seq. This study provides fundamental insight relevant to identification of genes associated with fiber quality and will accelerate future efforts toward improving fiber quality of upland cotton.
Project description:Cotton, an important natural fiber, is a differentiated epidermal cell. The number of genes that are active in fiber cells is similar to those in leaf, ovule, or root tissues. Through differential screening of a fiber cDNA library, we isolated five cDNA clones that are preferentially expressed in fiber. One of the cDNA clones, pCKE6, corresponded to an abundant mRNA in fiber. Transcripts for E6 were detected throughout the development of the fiber. Immunoprecipitation of in vitro translation products and Western blot analysis of fiber proteins showed two polypeptides in the range of 30-32 kDa as the products of E6 mRNA. Sequence analysis and hybrid-selected RNA translation also suggest that E6 mRNAs encode two polypeptides. Concentrations of E6 mRNA and protein are highest during the late primary cell wall and early secondary cell wall synthesis stages. Sequence comparison of E6 with other known eukaryotic and prokaryotic genes reveals no significant homology (GenBank; December 1991). E6 or a homologous gene(s) is conserved in several members of Malvaceae as well as in one other fiber-producing plant, kapok, but is not found in several other plants examined or in Acetobacter xylinum. A genomic clone corresponding to pCKE6 was isolated, and the promoter element of the E6 gene was shown to direct the expression of a carrot extensin mRNA in a tissue-specific and developmentally regulated fashion in transgenic cotton plants.
Project description:MBI9915 is an introgression cotton line with excellent fiber quality. It was obtained by advanced backcrossing and continuous inbreeding from an interspecific cross between the upland cotton (Gossypium hirsutum) cultivar CCRI36 as the recurrent parent and the sea island cotton (G. barbadense) cultivar Hai1, as the donor parent. To study the genetic effects of the introgressed chromosome segments in G. hirsutum, an F2 secondary segregating population of 1537 individuals was created by crossing MBI9915 and CCRI36, and an F2:3 population was created by randomly selecting 347 individuals from the F2 generation. Quantitative trait locus (QTL) mapping and interaction for fiber length and strength were identified using IciMapping software. The genotype analysis showed that the recovery rate for MBI9915 was 97.9%, with a total 6 heterozygous segments and 13 homozygous segments. A total of 18 QTLs for fiber quality and 6 QTLs for yield related traits were detected using the two segregating generations. These QTLs were distributed across 7 chromosomes and collectively explained 0.81%-9.51% of the observed phenotypic variations. Six QTLs were consistently detected in two generations and 6 QTLs were identified in previous studies. A total of 13 pairs of interaction for fiber length and 13 pairs of interaction for fiber strength were identified in two generations. Among them, 3 pairs of interaction for fiber length and 3 pairs of interaction for fiber strength could be identified in all generations; 4 pairs of interactions affected fiber length and fiber strength simultaneously. The results clearly showed that 5 chromosome segments (Seg-5-1, Seg-7-1, Seg-8-1, Seg-20-2 and Seg-20-3) have important effects on fiber yield and quality. This study provides the useful information for gene cloning and marker-assisted breeding for excellent fiber related quality.
Project description:BackgroundWe previously reported the development of a set of Gossypium hirsutum-G. australe alien chromosome addition lines. Naturally, however, G. hirsutum-G. australe chromosome exchanges were very limited, impeding the stable transference of useful genes from G. australe (G2G2 genome) into the most cultivated cotton, G. hirsutum (AADD).ResultsIn the present report, the pollen from a pentaploid (2n = AADDG2) of G. hirsutum-G. australe was irradiated with seven different doses ranging from 10 to 40 Grays and used to pollinate emasculated flowers of G. hirsutum over three consecutive years. Irradiation greatly increased the genetic recombination rates of the G. hirsutum and G. australe chromosomes and a total of 107 chromosome introgression individuals in 192 GISH-negative (with no GISH signal on chromosome) survived individuals, 11 chromosome translocation individuals (containing 12 chromosome translocation events) and 67 chromosome addition individuals were obtained in 70 GISH-positive (with GISH signal(s) on chromosome(s)) survived individuals, which are invaluable for mining desirable genes from G. australe. Multicolor genomic in situ hybridization results showed that there were three types of translocation, whole arm translocation, large alien segment translocation and small alien segment translocation, and that all translocations occurred between the G2-genome and the A-subgenome chromosomes in G. hirsutum. We also found that higher doses induced much higher rates of chromosome variation but also greatly lowered the seed viability and seedling survivability.ConclusionsIrradiation has been successfully employed to induce chromosome introgressions and chromosome translocations and promote chromosome exchanges between cultivated and wild species. In addition, by balancing the rates of chromosome introgression and translocation to those of seed set, seed germination, and seedling rates in the M1 generation, we conclude that the dosage of 20 Grays is the most suitable. The established methodology may guide the utilization of the tertiary gene pool of Gossypium species such as G. australe in cotton breeding in the future.
Project description:BackgroundORP (Oxysterol-binding protein-related proteins) genes play a role in lipid metabolism, vesicular transferring and signaling, and non-vesicular sterol transport. However, no systematic identification and analysis of ORP genes have been reported in cotton.ResultIn this study, we identified 14, 14, 7, and 7 ORP genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Phylogenetic analysis showed that all ORP genes could be classified into four groups. Gene structure and conserved motif analysis suggest that the function of this gene family was conserved. The Ka/Ks analysis showed that this gene family was exposed to purifying selection during evolution. Transcriptome data showed that four ORP genes, especially GhORP_A02, were induced by abiotic stress treatment. The cis-acting elements in the ORP promoters were responsive to phytohormones and various abiotic stresses. The silenced plants of GhORP_A02 were more sensitive to drought stress when compared to control.ConclusionThe major finding of this study shed light on the potential role of ORP genes in abiotic stress and provided a fundamental resource for further analysis in cotton.