【Objective】Cotton fiber is the main economic product of cotton. It is the epidermal cells of the ovule outer integument through polar elongation and secondary wall thickening. As one of the longest plant cells, the cotton fiber cells are regarded as an ideal material in the study of plant cell growth and development. Identification of promoters specifically or preferentially expressed in fiber cells is of great significance for basic research on fiber development and molecular breeding for improving fiber traits. 【Method】In this study, we cloned the promoter of GhSLD1 gene, which is predominantly expressed in fiber cells. Through the PlantCARE website for promoter sequence analysis, we identified the important cis-regulatory elements contained in the cloned sequence. According to the distribution of some important cis-regulatory elements, the cloned promoter fragments were deleted at 5′- end. A total of 4 promoter fragments were obtained and the corresponding plant expression vector was constructed. The constructed plant expression vectors were used for genetic transformation of tobacco and cotton. The transgenic plants were identified through molecular identification of transgenic tobacco and cotton. GUS activity in different tissues, organs and fiber cells of transgenic plants at different development stages was also investigated. 【Result】The longest promoter cloned was 2 900 bp in length. In addition to a lot of transcription regulatory elements in the promoter, the sequence also contained multiple abscisic acid response elements, the elements essential for the anaerobic induction, methyl jasmonate response elements, brassinolide response elements, the elements involved in seed-specific regulation, the elements involved in defense and stress responsiveness, and MYB transcription factor binding sites. Four promoter fragments with a length of 2 900 bp (GhSLD-P1), 2 178 bp (GhSLD1-P2), 1 657 bp (GhSLD1-P3) and 1 232 bp (GhSLD-P4) were obtained by the 5′-terminal deletion, respectively. The transgenic tobacco plants were generated after confirmed by molecular identification. GhSLD-P1, GhSLD1-P2 and GhSLD1-P3 did not express in transgenic tobacco, while GhSLD-P4 is widely expressed, and the expression level of GhSLD-P4 was similar to that of CaMV 35S promoter. The different sequence between GhSLD1-P3 and GhSLD-P4 contained four abscisic acid response elements, two brassinolide response elements, and three MYB binding sites. These cis-regulatory elements may be associated with the non-expression of GhSLD1-P1, GhSLD1-P2, and GhSLD1-P3 promoters in transgenic tobacco. The transgenic cotton plants of GhSLD1-P2 were obtained after confirmed by molecular identification. GhSLD1-P2 predominantly expressed in transgenic cotton fibers, and its expression level was higher at the elongation stage (10-15 DPA) of fiber cells while lower in the early developmental stage (5 DPA) of fiber cells and the stage of secondary cell wall deposition (20-30 DPA). 【Conclusion】The GhSLD1-P4 promoter was a widely expressed promoter, and the GhSLD1-P2 promoter was a fiber predominant expression promoter, which was highly expressed during the elongation of fibers. It could be applied to the study on the gene function involved in cotton fiber development and molecular breeding for improving fiber traits.
【Objective】To explore the structure and evolution characteristics of cotton NLP transcription factors in the whole genome, and further understand their expressions patterns, so as to lay a foundation for the further function research and utilization of NLP genes. 【Method】The NLP transcription factor family members in the whole genomes of four cotton species, Gossypium arboreum (G. arboreum, Ga), Gossypium raimondii (G. raimondii, Gr), Gossypium barbadense (G. barbadense, Gb) and Gossypium hirsutum (G. hirsutum, GH), were identified using two strategies, BLASTP and HMM search. Further bioinformatics analysis was carried out on the confirmed cotton NLP family members. The molecular weights, theoretical isoelectric points and other physical and chemical properties were predicted using online software Expasy; the MEGA 7 software was used to build the phylogenetic tree; protein conservative motifs were analyzed through MEME website; online software GSDS 2.0 was used to analyze gene structures; TBtools was used to view the chromosome localizations; McscanX was used to analyze the replication genes of cotton NLP family members; the PlantCARE website was used to predict the cis-acting elements in the promoters of cotton NLP family genes. The heat maps of cotton NLP genes expression levels of different tissues and under abiotic stresses were drawn through TBtools to analyze the tissue expression characteristics and abiotic stresses response characteristics. The expressions of GHNLPs in cotton under nitrogen starvation and nitrogen resupply treatments were analyzed by RT-qPCR. 【Result】A total of 11, 11, 21 and 22 NLP members were screened from the four cotton protein databases of G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. These NLP family genes encoded 693-996 amino acids. The relative molecular masses ranged from 76.92-110.02 kDa and the theoretical isoelectric points were 5.13-7.77. The subcellular localization prediction results showed that almost all the NLP members located in the nucleus. Promoter analysis found a large number of cis-acting elements related to phytohormone and stress response. Phylogenetic analysis showed cotton NLPs were divided into three groups, I, II and III. Gene replication analysis showed that fragment replication was the main force for NLP members expansion in cotton. All the Ka/Ks values were less than 1, indicating that evolution of NLP family in cotton mainly underwent purification selection. The results of expression analysis also confirmed that GHNLPs responded to nitrogen starvation and nitrogen resupply. 【Conclusion】From the whole genome of G. arboreum, G. raimondii, G. barbadense, and G. hirsutum, 11, 11, 21 and 22 NLP transcription factor members were identified respectively. They had high conservatism and some degree of differences. The expression levels of GHNLPs changed significantly during nitrogen starvation and nitrogen resupply processes, which may play a role in the response of cotton to nitrate.
【Objective】This study on the function and mechanism of GhCPR5 in response to Verticillium dahliae (V. dahliae) and Botrytis cinerea (B. cinerea) in cotton analysed nucleotide sequence, protein structure, expression pattern, and biological function to provide a theoretical basis and genetic resources for cotton disease resistance and breeding mechanism research. 【Method】GhCPR5 was identified from the unpublished transcriptome data of cotton responses to V. dahliae infection. The full-length coding sequence of GhCPR5 was amplified from upland cotton TM-1. The conserved domain, protein structure, and phylogenetic relationship of GhCPR5 and homologous genes were analysed using bioinformatics techniques. Real-time fluorescence quantitative PCR (qPCR) was used to analyse the GhCPR5 expression patterns in cotton roots, stems, leaves, ovules, fibres, and petals, and the induced expression of GhCPR5 by V. dahliae infection. The silencing fragments of GhCPR5 were amplified and inserted into the VIGS vector to generate the gene silencing construct TRV:CPR5. GhCPR5-silencing plants were created via Agrobacterium-mediated transformation methods. RT-PCR and qPCR were used to analyse the silencing efficiency of GhCPR5 in TRV:CPR5 plants. TRV:00 and TRV:CPR5 plants were inoculated with V. dahliae and B. cinerea, respectively, to analyse the difference in resistance in TRV:00 and TRV:CPR5 plants in response to pathogens. To analyse defence signal pathways involving GhCPR5, the expression levels of defence-related genes in TRV:00 and TRV:CPR5 plants were detected by qPCR. 【Result】The GhCPR5 cloned from G. hirsutum TM-1 is 1 683 bp long and encodes a 560 amino acid protein. The relative molecular weight and isoelectric point of GhCPR5 are 62.883 kDa and 9.01, respectively. Multiple-sequence alignment and phylogenetic analysis showed that GhCPR5 is highly homologous with the CPR5 of Durio zibethinus and Theobroma cacao. Furthermore, the C-terminals of GhCPR5 and CPR5 of other species are highly conserved and contain 4-5 transmembrane domains. The GhCPR5 expression level was induced by V. dahliae infection and is highest in leaves and lowest in stems. Under normal conditions, no significant developmental differences were observed between the GhCPR5-silencing plants, TRV:CPR5, and the TRV:00 control plants. After inoculation with V. dahliae, the rate of disease and the disease index of TRV:CPR5 plants were significantly higher than those of the control plants. Analysis of detached leaves inoculated with V. dahliae and B. cinerea and lactophenol-trypan blue staining showed that the lesions on the leaves of TRV:CPR5 plants were much bigger than those of TRV:00 plants, indicating that silencing GhCPR5 made cotton less resistant to V. dahliae and B. cinerea. In addition, the JAZ1 expression levels in TRV:CPR5 plants were significantly higher than in TRV:00 plants, whereas the PR3, PR4, and PR5 expression levels were markedly lower in TRV:CPR5 plants. 【Conclusion】GhCPR5 positively regulates cotton disease resistance; the downregulated expression of GhCPR5 significantly reduced cotton resistance to V. dahliae and B. cinerea.
【Objective】Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin synthesis pathway, which plays an important role in enhancing plant mechanical strength and resisting pathogen invasion. The aim of this study is to identify CAD and CAD-Like (CADL) gene family members in Gossypium barbadense and to analyze their expression characteristics and their role in Verticillium wilt resistance, which provides reference for the mechanism elucidation and disease resistance breeding of cotton against Verticillium wilt. 【Method】The CAD and CADL gene family members in G. barbadense genome were identified by bioinformatics method, and their chromosomal location, phylogenetic relationship, gene structure and promoter cis-element prediction were systematically analyzed. The expression characteristics of GbCAD and GbCADL were analyzed by obtaining publicly released transcriptome data and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Functional analysis of GbCAD and GbCADL genes was performed by viral-induced gene silencing (VIGS) technique. 【Result】A total of 25 GbCAD and 34 GbCADL genes were identified from G. barbadense and distributed on 10 and 17 different chromosomes, respectively. GbCAD and GbCADL genes are divided into 3 and 4 subgroups, respectively. The genes in the same group contain similar exon-intron structures and conserved domains. GbCAD and GbCADL genes have different transcriptional expression characteristics, and the promoters of GbCAD and GbCADL genes contain various hormone response elements and stress response elements. Transcriptome data and qRT-PCR showed that the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL5A/D, GbCADL6A/D, and GbCADL7A/D were induced by Verticillium dahliae, especially the GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D indicated significant increased expressions under V. dahliae treatment. The genes of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D were respectively silenced in cotton by virus-induced gene silencing (VIGS) technology, to analyze the changes of VIGS plant lines against V. dahliae treatment. The results showed that, compared with the control plants, the VIGS plant lines indicated significant decreased resistance to V. dahliae. The results of diaminobenzidine (DAB) histochemical stain displayed that, both control and VIGS plants showed similar normal phenotype without V. dahliae addition; after 6 h treatment of V. dahliae, the VIGS plant lines silencing GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D expressions demonstrated a deeper brown coloring, indicating a higher reactive oxygen species (ROS) accumulation in the VIGS plant lines. The results of stem sectioning showed that, the stem vascular tissues of VIGS plant lines TRV:GbCAD10A/D, TRV:GbCADL4A/D, TRV:GbCADL6A/D, and TRV:GbCADL7A/D showed obvious dark brown enrichment after V. dahliae treatment, indicating the significant decreased resistance to V. dahliae. 【Conclusion】 Suppressing the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D could significantly reduce the cotton resistance to V. dahliae.
