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棉花学报
2021年 第33卷 第6期
刊出日期:2021-11-15
  
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    研究与进展
    陆地棉GhBZR1基因的克隆及功能研究
    贺浪,张华崇,司宁,简桂良
    棉花学报. 2021, 33(6):  435-447.  doi:10.11963/cs20200083
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    【目的】BZR1是油菜素内酯(Brassinosteroid,BR)信号通路中关键的转录因子,去磷酸化后可直接调控BR信号通路下游基因的表达,影响植物的生长发育和免疫反应。明确其功能对了解棉花的抗/感病分子机制具有重要意义。【方法】分析未接种和接种黄萎病菌(大丽轮枝菌,Verticilium dahliae)的中植棉KV-3的转录组测序结果,筛选到1个下调表达基因GhBZR1,并通过cDNA末端快速扩增(Rapid amplification of cDNA ends,RACE)、生物信息学分析、实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,qRT-PCR)、亚细胞定位、病毒诱导的基因沉默(Virus-induced gene silencing,VIGS)和超表达,对该基因进行了克隆与功能验证。【结果】在陆地棉中克隆出GhBZR1基因全长1 515 bp,开放阅读框为942 bp,编码313个氨基酸。生物信息学分析表明,GhBZR1蛋白与海岛棉中的BZR1蛋白同源性最高,具有BES1_N超家族结构域,属于亲水性蛋白,具有多个磷酸化位点,在其启动子区域存在3个与病原菌抗性相关的顺式作用元件,分别是TC-rich repeats、ABRE和MBSI元件。亚细胞定位显示,GhBZR1蛋白定位在细胞核。qRT-PCR检测结果表明,GhBZR1基因在茎中优势表达;大丽轮枝菌诱导后,该基因在不同抗病、感病品种中的转录水平不同,且能被茉莉酸和水杨酸诱导表达;抑制GhBZR1基因表达能增强感病陆地棉品种86-1对黄萎病菌的抗性;超表达GhBZR1基因使转基因拟南芥抗黄萎病菌能力减弱、病症加重。【结论】GhBZR1基因在棉花抗黄萎病的过程中起负调控作用。


    [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.

    棉花类表皮特异性分泌糖蛋白基因GhA01EP1的克隆和功能分析
    李丹,赵存鹏,赵丽英,刘旭,刘素恩,王凯辉,王兆晓,耿军义,郭宝生
    棉花学报. 2021, 33(6):  448-458.  doi:10.11963/cs20200097
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    【目的】本研究对类棉花表皮特异性分泌糖蛋白基因GhA01EP1的克隆及功能分析,为进一步了解胞外蛋白抵抗环境胁迫的调控机制提供了基础。【方法】利用同源克隆法获得了冀2658中GhA01EP1的序列,通过实时定量聚合酶链式反应检测GhA01EP1基因的组织表达及干旱胁迫前后表达变化。对GhA01EP1蛋白的理化性质、结构、亚细胞定位等进行了生物信息学分析。用烟草瞬时转化试验对GhA01EP1蛋白进行亚细胞定位。利用农杆菌蘸花法获得转基因拟南芥,并对转基因植株进行抗旱鉴定。【结果】将克隆到的与抗旱相关的类表皮特异性分泌糖蛋白基因命名为GhA01EP1,位于A01号染色体。该基因无内含子,开放阅读框长1 371 bp,编码456个氨基酸,包含B-lectin、Plant PAN/APPLE-like 2个结构域。GhA01EP1基因在根、茎和叶片中均有表达,在根中表达量最高。KEGG通路分析发现,GhA01EP1参与甘氨酸、丝氨酸和苏氨酸代谢以及苯丙氨酸代谢的可信度较高。烟草瞬时转化试验结果显示,GhA01EP1蛋白为分泌蛋白。抗旱鉴定发现,与野生型相比,转基因拟南芥在干旱胁迫下长势好、根系长、胁迫复水后恢复能力强。【结论】GhA01EP1基因在棉花和拟南芥抗旱中起积极作用。


    [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.

    陆地棉纤维起始期优势表达基因GhCRPK1的克隆及功能研究
    王雪慧,陈丽锦,赵若林,程海亮,张友平,王巧连,吕丽敏,宋国立,左东云
    棉花学报. 2021, 33(6):  459-468.  doi:10.11963/cs20210061
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    【目的】研究陆地棉类受体激酶GhCRPK1(Cold-responsive protein kinase 1,CRPK1)在棉花纤维发育中的功能。【方法】以陆地棉全基因组关联分析(Genome-wide association study,GWAS)数据及陆地棉纤维发育不同时期的表达数据,筛选到GhCRPK1在棉花纤维起始期优势表达,通过基因克隆、亚细胞定位等方法研究了GhCRPK1的功能,对过表达GhCRPK1的拟南芥及突变体crpk1的表皮毛长度和密度进行观察统计,利用酵母双杂交筛选与GhCRPK1相互作用的蛋白。【结果】GhCRPK1基因全长为4 594 bp,开放阅读框长度为1 047 bp,编码348个氨基酸,在开花当天到开花后3 d持续优势表达,编码产物定位在细胞膜上。GhCRPK1在拟南芥中异源表达后可促进表皮毛的伸长;拟南芥中同源基因突变后,表皮毛变短。酵母双杂交实验发现,GhCRPK1与热激蛋白Gh_D08G0057相互作用。【结论】GhCRPK1在棉花纤维发育起始时期优势表达,定位在细胞膜上,在拟南芥中异源表达后调控拟南芥表皮毛的伸长。


    [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.

    棉花HDAC基因家族鉴定及其在黄萎病菌侵染下的表达分析
    王艳情, 郑杰, 许艳超, 蔡小彦, 周忠丽, 侯宇清, 王坤波, 王玉红, 陈浩东, 刘方, 李志坤
    棉花学报. 2021, 33(6):  469-481.  doi:10.11963/cs20210010
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    【目的】组蛋白去乙酰化酶(Histone deacetylases,HDAC)在植物发育及抗病应答中发挥重要作用,本研究旨在鉴定棉花HDAC基因家族成员并对其在黄萎病抗性中的作用进行分析。【方法】利用生物信息学方法对棉花基因组中HDAC基因家族成员进行鉴定,并对其理化性质、系统发育关系、基因结构进行系统分析。利用实时荧光定量聚合酶链式反应分析了黄萎病菌诱导下瑟伯氏棉HADC基因的表达模式。【结果】陆地棉、亚洲棉和瑟伯氏棉基因组中分别包括30、15和13个HDAC基因。棉花HDAC分为RPD3/HDA1、HD2和SIR2三个亚族,同一亚族成员间具有相似的基因结构和保守基序。棉花HDACs基因启动子中含有大量的激素应答、胁迫响应以及植物生长发育相关的顺式作用元件。瑟伯氏棉GthHDAC基因响应乙烯、水杨酸和茉莉酸的诱导;在黄萎病菌胁迫下,多数GthHDAC基因上调表达,表明该基因家族可能通过乙烯/茉莉酸和水杨酸等信号通路调控棉花黄萎病抗性。【结论】本研究从全基因组水平上鉴定了亚洲棉、陆地棉和瑟伯氏棉中58个HDAC家族基因,并明确了瑟伯氏棉HDAC家族成员在黄萎病菌胁迫下的表达模式。黄萎病菌处理后,大部分GthHDAC基因在叶片中上调表达,并且受到乙烯、水杨酸和茉莉酸的诱导。


    [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.

    基于广泛靶向代谢组学的不同颜色棉花花瓣中类黄酮成分差异分析
    李秋琳,李燕,陈伟,姚金波,朱守鸿,袁黎,张永山
    棉花学报. 2021, 33(6):  482-492.  doi:10.11963/cs20210036
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    【目的】本研究旨在对棉花黄色、乳白色和白色花瓣中类黄酮类代谢物质成分及差异进行分析,为进一步研究棉花花瓣颜色的形成机制和花瓣色素的利用提供一定的理论基础。【方法】用超高效液相色谱-串联质谱检测平台和Analyst 1.6.3软件对海7124(黄色花)、TM-1(乳白色花)和石系亚1号(白色花)3种花瓣中的类黄酮物质进行检测和分析;然后使用多元统计分析方法对检测到的类黄酮类代谢物数据进行分析,筛选差异代谢物;最后利用KEGG网站结合K-means分析对差异代谢物参与的合成途径进行分析。【结果】3种棉花花瓣材料中共检测到184种类黄酮类代谢物,其中黄酮醇类代谢物(76种,占41.30%)和黄酮类代谢物(51种,占27.72%)居多;差异代谢物171种,显著富集在黄酮和黄酮醇的生物合成途径及类黄酮的生物合成途径。通过对黄色、乳白色、白色花瓣进行类黄酮类代谢物分析,发现差异代谢物含量呈升高趋势和降低趋势的物质各15种,其中被注释到KEGG通路上的有5种:在黄酮和黄酮醇的生物合成途径上,金圣草黄素、紫云英苷和芦丁含量均升高;在类黄酮的生物合成途径上,香橙素和表儿茶素含量升高,异杞柳苷含量降低。【结论】通过分析发现3种棉花材料中存在的类黄酮类差异代谢物主要参与黄酮和黄酮醇的生物合成途径及类黄酮的生物合成途径;属于查耳酮类的异杞柳苷在黄色、乳白色、白色花瓣中的含量逐渐减少,推测该物质与棉花花瓣的黄色相关。


    [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.

    化学调控
    甲哌鎓通过调节棉花叶片水分平衡和光合性能提高苗期耐旱性的生理机制
    王玉贤, 董莹莹, 李芳军, 杜明伟, 田晓莉, 李召虎
    棉花学报. 2021, 33(6):  493-503.  doi:10.11963/cs20210054
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    【目的】研究植物生长延缓剂甲哌鎓 (Mepiquat chloride,MC/DPC)提高棉花幼苗耐旱性的调控机制。【方法】本研究以陆地棉品种中棉所41为供试材料,设CK-Ctrl(正常供水+喷施清水)、CK-DPC(正常供水+喷施30 mg·L-1 DPC)、DS-Ctrl(干旱+喷施清水)和DS-DPC(干旱+喷施30 mg·L-1 DPC)共4个处理。于2叶期叶面喷施DPC或清水,5 d后进行控水干旱处理,以正常供水为对照。【结果】正常供水条件下,叶面喷施DPC对叶片相对含水量、总叶绿素含量、净光合速率和水分利用率均无显著影响。干旱胁迫条件下,叶面喷施DPC可显著增加叶片中脯氨酸的积累、叶片相对含水量;DPC处理的叶片气孔导度和蒸腾速率虽呈增加趋势,但因净光合速率的显著提高最终增加了水分利用率。此外,DPC处理还可促进叶片可溶性蛋白的积累以抵御干旱胁迫。【结论】叶面喷施DPC可显著提高干旱胁迫条件下棉花幼苗的相对含水量、净光合速率和水分利用率,通过调节水分平衡和光合性能增强棉花抵御干旱胁迫的能力。


    [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.

    研究简报
    多倍体同源区段二代测序生物信息学分析关键参数优化
    武建楠,陈肯,王欢,庞铂实,周宇荀,肖君华,李凯
    棉花学报. 2021, 33(6):  504-512.  doi:10.11963/cs20200085
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    【目的】多倍体植物同源区段单核苷酸多态性(Single nucleotide polymorphism,SNP)标记分型挑战颇大。本研究以四倍体陆地棉同源区段聚合酶链式反应(Polymerase chain reaction,PCR)靶向扩增子数据集为例,观测同源区段的影响并优化生物信息学分析方案。【方法】首先扩增并测序获得136个陆地棉样本DNA的包含潜在变异的3个区段,其次使用不同参数进行比对与SNP检出,最后比较优化不同方案分析结果的异同。【结果】常规分析发现,区段1的3个SNP位点和区段2的1个SNP位点在样本中均鉴定为野生型或纯合突变型,而几乎所有样本的区段3鉴定为杂合突变型。Blast分析表明,位于A12染色体的区段3与其同源序列(位于D12染色体)相似性为96.28%。仅将区段3的同源序列作为参考序列分析,潜在SNP位点的基因型鉴定结果无变化;而将区段3与其同源序列同时作为参考序列分析,比对到区段3与其同源序列的读长的比例分别为48%、52%,因此存在较多同源区段3的读长是导致分型错误的主要原因,并确定了区段3 潜在SNP位点的基因型应为TT。此外,通过对比GATK结果在区段3发现了2个新SNP且排除了3个因部分同源序列变异造成的假阳性SNP。【结论】本研究验证了同源序列的存在会严重影响多倍体SNP鉴定与生物信息学分析;对关键参数优化特别是将多倍体同源序列同时作为参考序列,能够提高SNP分型的准确度。


    [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.

    总目次
    2021年第33卷总目次
    棉花学报. 2021, 33(6):  513-513. 
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