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Cotton Science
Volume 33 Issue 6
15 November 2021
  
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    RESEARCH REPORTS
    Cloning and functional analysis of GhBZR1 in Gossypium hirsutum L.
    He Lang,Zhang Huachong,Si Ning,Jian Guiliang
    2021, 33(6):  435-447.  doi:10.11963/cs20200083
    Abstract ( 34 )   HTML ( 89)   PDF (4600KB) ( 23 )  
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    [Objective] BZR1 is a key transcription factor in the brassinosteroid (BR) signaling pathway. After dephosphorylation, it can directly regulate the expression of downstream genes in the BR signaling pathway, and then affect the growth and development, and immune responses of plants. It is of great significance to clarify BZR1 gene function for understanding the molecular mechanism of disease resistance/susceptibility in cotton. [Method] Analyzing the transcriptome sequencing results of the KV-3 inoculated and uninoculated with Verticillium dahliae, a down-regulated gene, GhBZR1 was selected. Rapid amplification of cDNA ends, bioinformatics analysis, real-time quantitative polymerase chain reaction (qRT-PCR), subcellular localization technology, virus induced gene silencing and gene overexpression technology were performed to study the characteristics and function of the gene. [Result] The GhBZR1 gene was cloned from Gossypium hirsutum with a total length of 1 515 bp, and the open reading frame is 942 bp and encodes a protein of 313 amino acids. Bioinformatics analysis shows that GhBZR1 has the highest similarity with the BZR1 protein of G. barbadense, and GhBZR1 has a BES1_N superfamily domain, and is a hydrophilic protein with a variety of phosphorylation sites. There are three cis-acting elements related to pathogen resistance in the 1 000 bp promoter region of this gene, which are TC-rich repeats, ABRE and MBSI. Subcellular localization analysis shows that GhBZR1 is localized in the nucleus. And qRT-PCR analysis shows that GhBZR1 gene is preferentially expressed in cotton stems. With infection of V. dahliae, the transcription level of GhBZR1 is different in resistant cotton varieties and susceptible varieties. And the expression of GhBZR1 can be induced by jasmonic acid and salicylic acid. After GhBZR1 silenced, the susceptible cotton variety 86-1 shows enhanced resistance to Verticillium wilt. Overexpression of GhBZR1 gene weakens the disease resistance of transgenic Arabidopsis and aggravates disease symptoms. [Conclusion] The GhBZR1 gene plays a negative regulatory role in the process of cotton resistance to Verticillium wilt.

    Cloning and functional analysis of epidermis-specific secreted glycoprotein EP1-like gene GhA01EP1 in cotton
    Li Dan,Zhao Cunpeng,Zhao Liying,Liu Xu,Liu Su'en,Wang Kaihui,Wang Zhaoxiao,Geng Junyi,Guo Baosheng
    2021, 33(6):  448-458.  doi:10.11963/cs20200097
    Abstract ( 33 )   HTML ( 37)   PDF (7161KB) ( 4 )  
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    [Objective] The aim of this study is to clone and analyze the epidermis-specific secreted glycoprotein EP1-like gene GhA01EP1 in cotton, which provided a basis for further understanding of the regulatory mechanism of extracellular protein in the resistance to environmental stress. [Method] The sequence of GhA01EP1 from Ji 2658 was obtained by homologous cloning strategy, the expression level of GhA01EP1 was analyzed by quantitative real-time polymerase chain reaction. The physical and chemical properties, structure and subcellular localization of GhA01EP1 protein were analyzed by bioinformatics method. Subcellular localization of protein was carried out by transient expression of tobacco transformation mediated by Agrobacterium. The drought resistance of transgenic Arabidopsis thaliana obtained by Agrobacterium-mediated transformation method was analyzed. [Result] We designated the epidermis-specific secreted glycoprotein EP1-like gene as GhA01EP1, without intron, which has an 1 317 bp open reading frame and encodes 456 amino acids. GhA01EP1 is located on chromosome A01 and contains two domains: B-lectin and Plant PAN/APPLE-like. GhA01EP1 is expressed in root, stem and leaf, and with highest expression level in root. KEGG pathway analysis showed that GhA01EP1 is most likely involved in glycine, serine and threonine metabolism and phenylalanine metabolism. Subcellular localization analysis showed that the GhA01EP1 is a secretory protein. The result of drought resistance analysis showed that transgenic Arabidopsis grew better under drought stress than wild type, with longer root and stronger recovery ability after rehydration. [Conclusion] The GhA01EP1 plays a positive role in drought resistance of cotton and Arabidopsis.

    Cloning and functional analysis of GhCRPK1, a gene preferentially expressed during fiber initiation in upland cotton
    Wang Xuehui,Chen Lijin,Zhao Ruolin,Cheng Hailiang,Zhang Youping,Wang Qiaolian,Lü Limin,Song Guoli,Zuo Dongyun
    2021, 33(6):  459-468.  doi:10.11963/cs20210061
    Abstract ( 31 )   HTML ( 43)   PDF (4704KB) ( 15 )  
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    [Objective] The aim of this article is to study the function of GhCRPK1 (Cold-responsive protein kinase 1, CRPK1) during fiber development in upland cotton. [Method] According to the genome-wide association study (GWAS) data and the transcriptome data of fiber development at different stages in cotton, GhCRPK1 may be involved in fiber initiation by gene clone, subcellular localization to study the GhCRPK1. The full length of GhCRPK1 was amplified and transformed into Arabidopsis, and the length and density of trichomes of the GhCRPK1-overexpressed plants and Arabidopsis mutant crpk1 were observed. At last, yeast two-hybrid assay was performed to screen the interaction proteins. [Result] The full-length of GhCRPK1 is 4 594 bp containing a 1 047 bp open reading frame and encoding 348 amino acids. The expression level of GhCRPK1 peaked at the 0-3 days post anthesis. The GhCRPK1 was localized on the plasma membrane. The Arabidopsis plants overexpressing GhCRPK1 had longer trichomes compared with the wild type. While the trichomes were shorter in the crpk1 mutant. Yeast two-hybrid analysis revealed that GhCRPK1 can interact with a heat shock protein Gh_D08G0057. [Conclusion] GhCRPK1, a gene preferentially expressed during fiber initiation and localized on the plasma membrane, regulated the elongation of trichome in Arabidopsis.

    Identification of cotton HDAC gene family and expression analysis of HDAC genes under Verticillium dahliae stress
    Wang Yanqing, Zheng Jie, Xu Yanchao, Cai Xiaoyan, Zhou Zhongli, Hou Yuqing, Wang Kunbo, Wang Yuhong, Chen Haodong, Liu Fang, Li Zhikun
    2021, 33(6):  469-481.  doi:10.11963/cs20210010
    Abstract ( 70 )   HTML ( 452)   PDF (5560KB) ( 26 )  
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    [Objective] Histone deacetylases (HDAC) plays an important role in plant development and disease resistance. Here, we intend to identify the members of HDAC gene family and its function in Verticillium wilt resistance of cotton. [Method] In this study, HDAC genes were identified and their physical and chemical properties, phylogenetic relationships, gene structures were analyzed by bioinformatics analysis. The expression patterns of HDAC genes under Verticillium dahliae stress were tested by qRT-PCR (Quantitative real-time polymerase chain reaction). [Result] A total of 30, 15, and 13 HDAC genes were identified in Gossypium hirsutum, G. arboreum, and G. thurberi, respectively. These genes were divided into RPD3/HDA1, HD2 and SIR2 groups and possessed similar structures and conserved motifs in each group. A large number of cis-acting elements related to hormone response, stress response and plant growth and development were identified in the promoters of HDAC genes. The results also showed that HDAC genes could be induced by ethylene (ET), salicylic acid (SA) and jasmonic acid (JA) and up-regulated under Verticillium dahliae infection, which indicated HDAC genes probably involved in Verticillium wilt resistance by ET/JA and/or SA signding pathways in cotton. [Conclusion] In this study, a total of 58 HDAC genes were identified. Under the treatment of wilt, most of GthHDAC genes were up-regulated in the leaves of G. thurberi, and some GthHDAC genes were induced by ET, SA and JA. These results proved that HDAC can be used in the future research to understand the molecular mechanism of Verticillium wilt tolerance in cotton.

    Metabolomics reveals the variation of flavonoids content in petals of cotton with different colors
    Li Qiulin,Li Yan,Chen Wei,Yao Jinbo,Zhu Shouhong,Yuan Li,Zhang Yongshan
    2021, 33(6):  482-492.  doi:10.11963/cs20210036
    Abstract ( 62 )   HTML ( 14)   PDF (4224KB) ( 12 )  
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    [Objective] This study aims to analyze the composition and difference of flavonoid metabolites in yellow, milky white and white petals of cotton, which provides theoretical basis for the further research and utility of pigmentation in cotton flower. [Method] In this study, firstly, the Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection platform and Analyst 1.6.3 were employed to analyze the flavonoids in three types of cotton petals from Hai 7124(yellow flower), TM-1 (milk white flower), and Shixiya 1 (white flower). Then the multivariate statistical analysis was used to analyze different metabolites. Finally, KEGG and K-means was used to investigate the synthetic pathways of different metabolites. [Result] A total of 184 types of flavonoid metabolites were detected in petals of three colors, among which 76 were flavonol (accounting for 41.30%) and 51 were flavone (accounting for 27.72%). And 171 different metabolites were obtained, which were significantly enriched in the biosynthetic pathway of flavone and flavonols and the biosynthetic pathway of flavonoids. By analyzing the relative content of flavonoids metabolites in yellow, milky white and white petals, 15 different metabolites were found to show an increasing trend and 15 metabolites content were decreasing. Among them, 5 metabolites were annotated in the known KEGG pathway, and the content of chrysoeriol, astragalin and rutin were all increased in the biosynthesis pathway of flavone and flavonoid. In addition, in flavonoid biosynthesis pathway, the content of aromadendrin and epicatechin increased, and the content of isosalipurposide were decreased. [Conclusion] Different flavonoid metabolites in the three cotton materials were found to be mainly involved in the biosynthesis pathway of flavone and flavonol as well as the biosynthesis pathway of flavonoids. As the content of isoaridin was decreased gradually in yellow, milky white and white petals, we speculate that this substance was related to the formation of yellow cotton petals.

    CHEMICAL CONTROL
    Mepiquat chloride improves drought tolerance by adjusting water balance and photosynthetic performance in cotton seedling
    Wang Yuxian, Dong Yingying, Li Fangjun, Du Mingwei, Tian Xiaoli, Li Zhaohu
    2021, 33(6):  493-503.  doi:10.11963/cs20210054
    Abstract ( 59 )   HTML ( 186)   PDF (3210KB) ( 24 )  
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    [Objective] The aim of this research is to study the regulation mechanism of plant growth regulator mepiquat chloride (MC/DPC) in improving the drought tolerance of cotton seedlings. [Method] In this study, CCRI 41(Gossypium hirsutum L.) was used as the test material, and four treatments including CK-Ctrl (sufficient water supply + spraying water), CK-DPC (sufficient water supply + spraying 30 mg·L-1 DPC), DS-Ctrl (drought stress + spraying water), and DS-DPC (drought stress + spraying 30 mg·L-1 DPC) were performed. At the two-leaf stage, DPC or water was sprayed on leaves, and then irrigation was stopped at five days as drought treatment, and using sufficient water supply as the control. [Result] The experimental results showed that under sufficient water conditions, DPC has no significant effect on the relative water content, total chlorophyll content, net photosynthetic rate, and water use efficiency of leaves. Under drought stress, DPC can significantly increase the accumulation of proline and relative water content. Meanwhile, DPC can slightly enhance the stomatal conductance and transpiration rate of leaves, and significantly increase the net photosynthetic rate, to improve the water use efficiency finally. In addition, DPC can also promote the accumulation of soluble protein of leaves in response to drought stress. [Conclusion] Spraying DPC can significantly increase the relative water content, net photosynthetic rate and water use efficiency of cotton seedlings, and can enhance the ability of cotton to resist drought stress by adjusting water balance and photosynthetic performance.

    RESEARCH NOTES
    Optimization of key parameters for next-generation sequencing bioinformatics analysis of polyploid homologous segments
    Wu Jiannan,Chen Ken,Wang Huan,Pang Boshi,Zhou Yuxun,Xiao Junhua,Li Kai
    2021, 33(6):  504-512.  doi:10.11963/cs20200085
    Abstract ( 34 )   HTML ( 8)   PDF (3316KB) ( 2 )  
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    [Objective] Single nucleotide polymorphism (SNP) detection in polyploidy plant is complicated due to the presence of homologous segments. Here, amplicon dataset of homologous segments from the tetraploid upland cotton is used as an example to observe the influence of the homologous segments and optimize the bioinformatics pipeline. [Method] Firstly, three segments with potential variations were amplified and sequenced from 136 upland cotton (Gossypium hirsutum L.) samples. Then, mapping and SNP detection were conducted with different parameters. Finally, Different schemes were compared and optimized with their advantages. [Result] In routine method, variants in segment 1 and segment 2 were identified as correct genotypes (homozygous), while almost all variants in segment 3 seemed to be undistinguished. Blast analysis showed that the similarity between segment 3 (located on chromosome A12) and its homologous sequence (located on chromosome D12) is 96.28%. While only its homologous sequence was used as reference genome for mapping, the genotyping results didn't change. However, correct genotypes were called if both segment 3 and its homologous segment were used as reference sequence. By this way, the proportion of different sub-genome reads of target segment 3 and its homologous segment was 48% and 52%, respectively. Therefore, the genotyping error of segment 3 is due to the presence of the homologous segment. Actually, the genotype of the potential SNPs in segment 3 should be homozygous TT. In addition, by comparing the GATK results, two new SNPs were found in segment 3 and three false positive SNPs were excluded caused by homoeologous sequence variant. [Conclusion] This study confirms that the existence of homologous sequences seriously affects SNP genotyping and bioinformatics analysis in polyploid plant. Optimization of key parameters, especially using polyploid homologous sequences as reference genomes at the same time, can improve the accuracy of SNP detection in polyploid plant.