[Objective] The MAPKKK gene family plays an important regulating role in response to multiple abiotic stresses and the development of plant. This study aims to identify MAPKKK genes of Gossypium raimondii and analyze their functions. [Method] In this study, based on G. raimondii genome database and bioinformatics method, G. raimondii MAPKKK family genes were identified and analyzed. Using the MEGA5, GSDS and Mapchart program, the phylogenetic tree, gene structure and chromosomes location analyses were accomplished. Based on the existing microarray data in cotton and comparative profiles of these MAPKKK genes, different expression of them in multiple abiotic stresses and the expression at different cotton fiber developmental stages were analyzed. [Result] A sum of 114 MAPKKK genes were identified systematically in G. raimondii and classified into 3 subfamilies (Raf, ZIK and MEKK) according to the gene stucture and phylogenetic tree analyses. They were distributed on all the 13 chromosomes of G. raimondii, and segmental duplication and tandem duplication events may have occurred. Compared with the recently released 78 genes of G. raimondii MAPKKK family genes, 47 sequences are exactly the same ones. [Conclusion] The results are helpful to understand the evolution and function of MAPKKK gene family. Our results provide a foundation for future functional characterizations of MAPKKK genes in cotton and probably other Gossypium plants.
[Objective] Since 2015, there has been a new cotton boll disease in Xinjiang cotton fields which was obviously different from the common boll rot, leading to stiffness and cracking boll. Olive-black mildew layer was observed on the surface of diseased bolls under wet conditions. It is of great significance to clarify the types of pathogens for the prevention of the disease. [Method] From 20 cotton planting locations of Xinjiang, 38 samples of stiffness and cracking boll disease were collected, and the pathogens were isolated and purified by conventional dilution separation and single-spore separation. And 20 representative strains were selected according to sampling location, cotton varieties and colony characteristics. The selected strains were identified by morphological observation and sequence analysis of rDNA internal transcribed spacer (rDNA-ITS), actin (ACT) and translation elongation factor 1-α (TEF1-α) gene. According to Koch’s law, the spore suspensions of three representative strains C2, C13 and C7 were inoculated to observe and record the incidence of the disease, and the diseased bolls were used for re-separation to observe the similarities and differences between the re-isolated strains and the inoculated strains. [Result] The result shows that these isolates belong to the Cladosporium, and according to its morphological characteristics and molecular biology analysis, 13 of the 20 strains belong to C. cladosporioides, 4 strains belong to C. velox, 3 strains belong to C. limoniforme. The symptoms of three representative strains C2, C13 and C7 were similar to those in the field when inoculated with injuries, and the disease spread was slower when inoculated without injuries. The inoculated strains can be separated from the lesions. [Conclusion] The new cotton boll disease causing stiffness and cracking in Xinjiang cotton fields is caused by three species of Cladosporium, C. cladosporioides, C. velox and C. limoniforme, with C. cladosporioides as the dominant species.
[Objective] Glutathione reductase (GR) gene family is involved in biological processes such as plant growth and abiotic stress response, but its characteristics and functions in cotton have not been known yet. This study aims to explore the role of GR genes in cotton genome evolution and abiotic stress response through the whole genome identification and characterization of GR genes, thus providing a theoretical basis for future studies on the roles of the GR genes in enhancing abiotic stress tolerance in cotton. [Method] The GR genes in Gossypium hirsutum, G. barbadense, G. raimondii and G. arboreum were all identified using bioinformatics software. The physicochemical properties, sequence characteristics, chromosomal location, phylogeny and expression patterns were analyzed. [Result] A total of 18 GR genes were identified. The number of GR genes in G. hirsutum, G. barbadense, G. raimondii and G. arboreum was 6, 6, 3 and 3, respectively. Phylogenetic analysis revealed that GR genes were divided into two sub-groups. The genes in the same subgroup exhibited similar gene structure in relation to exon-intron ratios. The ratios of the non-synonymous mutations (Ka) and homologous mutations (Ks) were all less than 1, indicating that the GR genes underwent strong purification selection during their evolution process. The analysis of the expression patterns of GR genes in upland cotton indicated that all the GR genes responded actively to the stress environment; but under different abiotic stresses, the gene expression patterns were significantly different. [Conclusion] The study explored the evolution and function of the GR gene family in the four cotton genomes, providing a theoretical basis for future studies of cotton GR genes.
[Objective] DUF642 (Domain of unknown function 642 genes), an unknown functional gene family, plays an important role in plant stress response. The aim of this study is to analyze its function in cotton stress response. [Method] The members of DUF642 gene family in cotton were systematically identified base on the information from Gossypium hirsutum genome. The gene structure, physiochemical properties, subcellular localization, phylogenetic evolution and promoters of DUF642 were analyzed by bioinformatics. The expression patterns of DUF642 genes in different tissues and under various stresses, including cold, hot, drought, salt and Verticillium dahliae infection, were analyzed by transcriptome data and real-time quantitative polymerase chain reaction (RT-qPCR). [Result] The results showed that 23 DUF642 genes in G. hirsutum distributed on 14 chromosomes and one scaffold. Most of GhDUF642 proteins were predicated to be in the plasma membrane and contain 1―2 conserved DUF642 domains. GhDUF642 genes were divided into four subgroups and highly conservative in evolution. GhDUF642 genes have a wide range of tissue expression types, most of which are highly expressed in roots and leaves. Combining promoter analysis and expression analysis, it was speculated that GhDUF642-09 participated in cotton resistance to salt stress, GhDUF642-08 and GhDUF642-19 were involved into the interaction between cotton and V. dahliae, and GhDUF642-02, GhDUF642-14 and GhDUF642-17 were related to cotton growth and stress tolerance. [Conclusion] These results could provide reference for further study on the function of DUF642 genes and the molecular mechanism for stress resistance in cotton.
[Objective] In this study, we cloned the R2R3 MYB (v-myb avian myeloblastosis viral oncogene homolog, MYB) transcription factor gene Gh_A12G2460, which is highly expressed in the second cell wall (SCW) development stage during cotton fiber formation, and preliminarily investigated its functional role in fiber development at SCW stage. [Method] The Gh_A12G2460 gene was isolated from Gossypium hirsutum acc. TM-1 by one-step cloning strategy. Sequence alignment showed it is similar to AtMYB52 in Arabidopsis, so it was renamed as GhMYB52. The gene structure, protein structure, expression profile, subcellular location and protein-protein interaction were analyzed by phylogenetic analysis, multiple alignments of amino acid sequences, qRT-PCR, Agrobacterium tumefaciens-mediated transient transformation system of tobacco, and yeast two hybridization assay. [Result] The results showed that GhMYB52 contains a 672 bp open reading frame, encoding 223 amino acid residues, with a predicted nuclear localization signal region. qRT-PCR results indicated that GhMYB52 is predominantly expressed in the fiber at 15―25 DPA (days post anthesis), suggesting it may be an SCW-associated gene. GhMYB52 localized in the nucleus examined by tobacco transient expression, consistent with the characteristics of transcriptional factors. Yeast transformation test demonstrated it has transcriptional activation activity. Further protein-protein interaction analysis revealed that GhMYB52 strongly interacts with GhFSN1, a NAC transcription factor. [Conclusion] These results demonstrated that GhMYB52 is a nuclear-localized transcription activator, which might participate in the SCW process of cotton fiber development by forming a corresponding regulatory network with NAC transcription factors. This study lays a foundation for further verification of the biological function of GhMYB52 in cotton fiber development at the molecular level.
[Objective] Maintaining intracellular ion homeostasis is one of the important salt-tolerant mechanisms of crops. This study aims to analyze differences in response characteristics of cotton ionome and salt-tolerant gene expression under different saline-alkali stresses, which provides a basis for understanding the mechanism of salt tolerance and improving salt tolerance of cotton. [Method] Using Lumianyan 24 as the experimental material, three kinds of salt and alkali stress types (salt stress, alkali stress, and mixed salt-alkali stress) and two concentration gradients (low and high concentrations) were set under pot cultivation conditions. Meanwhile, non-saline-alkali stress treatment was set as control. The dry matter weight of cotton plants and root morphological parameters including root length, root surface area, and root volume were measured in this study. The concentrations of 13 elements such as P, Na, K, Ca and Mg in different organs of cotton plants were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The relative expressions of salt tolerance related genes GhDFR1, GhSOS1, GhNHX1 and GhAKT1 were determined by the quantitative real-time polymerase chain reaction method. [Result] 1) Salt and alkali stresses significantly inhibited cotton growth. The growth inhibition rate of cotton under mixed salt-alkali stress treatments (48.7%-57.9%) was significantly higher than that under salt stress (27.6%-49.9%) and alkali stress (21.2%-35.5%) treatments. Under salt stress and mixed salt-alkali stress treatments, both shoot and root growth of cotton were significantly inhibited, dry matter weight, root length, root surface area and root volume were significantly reduced, while root growth was less inhibited under alkali stress treatments. 2) Under three types of saline-alkali stresses, Na content and Mo content in different organs of cotton plant increased significantly, but N content in leaves and roots decreased. 3) Under salt stress treatments, the uptake of Ca, Mg, Fe, Mn and Zn in cotton was inhibited, and the ion balance was maintained by promoting the transport of these ions and P, K. 4) In addition to Ca, Mg, Fe, Mn and Zn, P uptake was also inhibited under alkali stress treatments, but K uptake and P, K, Ca, Mg, Fe, Mn and Zn transport were promoted. 5) Under mixed salt-alkali stress treatments, especially under high salinity and pH conditions, most of nutrients uptake was inhibited, and the transport capacity of Ca, Mg, Zn, Mn and Fe was reduced. 6) The relative expression of GhSOS1 and GhAKT1 genes increased significantly under salt stress treatments, but increased first and then decreased under alkali stress and mixed salt-alkali stress treatments. The relative expression levels of GhSOS1 and GhAKT1 genes under three types of saline-alkali stresses were alkali>salt>mixed saline-alkali stress. With the increase of soil salinity and pH value, the relative expression of GhNHX1 gene increased first and then decreased. The expression levels of GhNHX1 gene were salt>alkali>mixed salt-alkali stress. [Conclusion] Due to high salinity and pH value, mixed salt-alkali stress significantly inhibits cotton growth and ions uptake, which restricts the transport of P, K, Ca, Mg, Zn, Mn and Fe. The decrease of K and Na regulation ability leads to ions imbalance.
[Objective] To promote the study on color improvement in cotton fiber, it is a key to use transgenic approach. [Method] In this study, the maize Lc gene, under the control of the fiber specific promoter of GhACT1, was introduced into cotton by Agrobacterium-mediated method. The integration of maize Lc gene was confirmed by the polymerase chain reaction (PCR) and southern blot, meanwhile follow-up observation of color change during the transgenic plants growing. The expression of transcription level was analyzed by reverse transcription (RT)-PCR amplification. [Result] Our data showed there were 25 lines of positive plants that had no obvious color change. The expression test results of Lc gene showed that there was no expression signal in vegetative parts, including leaf, petiole and stem. However, there were high expression signals in fiber of ovules at 1―12 days post anthesis in transcription level. The result of culturing in vitro showed that red color appeared on the ovules’ surface and the initial fiber turned red under light for 1 week. [Conclusion] The Lc gene driven by the promoter of GhACT1 was introduced into cotton successfully and expressed in ovule and fiber specially, leading to red fiber appeared under light. These data indicate that it is potential to engineer colored fibers through transgenic approach in cotton.
[Objective] The aim of this study is to investigate the effect of cotton genotypes, growth environment, and their interaction on major components in cottonseeds. [Method] Eleven upland cotton cultivars were planted in 10 different locations in the middle and lower reaches of Yangtze River region as research materials. Cottonseeds were collected to determine the contents of six main components including protein content (PC), oil content (OC), gossypol, phytic acid (PA), α-tocopherol and γ-tocopherol, then the genotypes, environment, and their interaction effects were analyzed accordingly. [Result] The results showed that the genotypes, environment, and their interaction had extremely significant effect on the protein and oil content in cottonseeds. The environment had extremely significant effect on the gossypol and phytic acid content in cottonseed. The genotypes had extremely significant effect on the gossypol content and had significant effect on the phytic acid content in cottonseed, but their interaction was insignificant. The genotype effects of α-tocopherol and γ-tocopherol were not significant, but the environmental effects were significant. The differences of tocopherols between different regions reached the extremely significant level. In addition, protein content and oil content were significantly negatively correlated and the sum of the protein and oil contents remained stably at 75%. [Conclusion] In cotton seed quality breeding, not only the differences in varieties, but also the environment and the interaction between varieties and environment should be considered. The expression of the main components of cottonseed is the result of a combination of genotype and ecological environment.
[Objective] The colonization in soil, promoting cotton growth effects, inducing resistance of cotton and controlling effects to cotton Verticillium wilt of Bacillus vanillea SMT-24, B. velezensis BHZ-29, B. subtilis SHT-15 and B. atrophaeus SHZ-24 were studied in this paper, which provides a scientific basis for better control of cotton Verticillium wilt. [Method] The antagonistic resistance of these bacteria to Verticillium dahliae was judged by observing the inhibition zone; the number of colonization in the rhizosphere soil was tested to represent the colonization ability of antagonistic bacteria; the growth promoting effect of strains on cotton was determined by analyzing cotton plant height, root length, root hair number and number of leaves; the effect of antagonistic strains on cotton induced resistance was explored by determining catalase (CAT), superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activities in cotton leaves, and investigating the disease index of cotton after inoculation with antagonistic bacteria solutions. [Result] SMT-24, BHZ-29, SHT-15 and SHZ-24 strains significantly inhibited the growth of V. dahliae and formed a clear zone of inhibition. After 2 to 22 days of the third inoculation, these strains of bacteria could survive in the root soil, increase the plant height, leaf number, and root hair number of cotton plants, as well as the activities of cotton CAT, SOD, PAL and POD enzymes, but reduced the disease index. [Conclusion] SMT-24, BHZ-29, SHT-15 and SHZ-24 strains can promote cotton growth, increase the activities of some related defense enzymes, and effectively control cotton Verticillium wilt.
