棉花<i>GhCRK26</i>基因克隆、亚细胞定位及表达分析

doi:10.7668/hbnxb.20195920

摘要/Abstract

摘要： 富含半胱氨酸类受体激酶作为受体激酶重要亚家族,在植物生长发育中发挥关键作用。旨在解析GhCRK26基因与棉花纤维发育的关系,为棉花纤维品质改良提供理论基础。选用陆地棉系9和海岛棉新海16,采集纤维发育不同时期样本及根、茎、叶组织。通过qRT-PCR分析GhCRK26基因表达模式;利用PCR技术克隆GhCRK26基因;借助生物信息学工具构建系统发育树,预测蛋白理化性质、跨膜结构及信号肽;运用PlantCare数据库分析启动子顺式作用元件;通过亚细胞定位试验明确蛋白定位。结果表明,GhCRK26基因在2个品种开花后10,20,30 d的表达量呈现极显著差异;在系9中叶片的表达水平最高,在新海16中,茎与叶的表达量未表现出显著差异。成功克隆获得2 049 bp的CDS序列,编码682个氨基酸。进化树显示,GhCRK26蛋白与野西瓜苗亲缘关系最远,与夏威夷棉、海岛棉最近;该蛋白为亲水不稳定蛋白,含跨膜结构和信号肽;启动子区鉴定到茉莉酸甲酯和脱落酸响应元件;亚细胞定位证实其定位于细胞膜。以上研究为进一步深入解析GhCRK26基因在纤维发育中的功能提供了依据。

关键词: 棉花, 纤维, GhCRK26, 基因克隆, 表达分析, 亚细胞定位

Abstract:

As an important subfamily of receptor kinases,cysteine-rich receptor kinases play a key role in plant growth and development.The aim of this study was to analyze the relationship between the GhCRK26 gene and the development of cotton fiber,and to provide a theoretical basis for the improvement of cotton fiber quality.This study selected upland cotton Line 9 and sea island cotton Xinhai 16,and collected samples at different periods of fiber development and root,stem and leaf tissues.The expression pattern of GhCRK26 was analyzed by qRT-PCR;the gene was cloned by PCR;a phylogenetic tree was constructed with the help of bioinformatics tools to predict the physicochemical properties of the protein,its transmembrane structure and signal peptide;the promoter cis-acting elements were analyzed by the PlantCare database;and the localization of the protein was clarified by the subcellular localization assay.The results showed that the expression of GhCRK26 gene showed highly significant differences at 10,20 and 30 days after flowering in two varieties;the highest expression levels were found in the leaves of Line 9,while in Xinhai 16,the expression of stem and leaves did not show significant differences.A 2 049 bp CDS sequence encoding 682 amino acids was successfully cloned.The evolutionary tree showed that GhCRK26 protein was the most distantly related to Hibiscus trionum,and the closest to Gossypium tomentosum and Gossypium barbadense;the protein was hydrophilic and unstable,containing a transmembrane structure and a signal peptide;methyl jasmonate and abscisic acid response elements were identified in the promoter region;and the subcellular localization confirmed that it was localized in the cell membrane.The above studies provide a basis for further in-depth analysis of the function of GhCRK26 gene in fiber development.

Key words: Cotton, Fiber, GhCRK26, Gene cloning, Expression analysis, Subcellular localization

中图分类号:

李静, 庞博, 张茹, 扎尔加玛丽·阿不都别克, 王正瑞, 史顺宇, 范珍珍, 马晶晶, 陈佳林, 宋武, 李生梅, 高文伟. 棉花GhCRK26基因克隆、亚细胞定位及表达分析[J]. 华北农学报, 2025, 40(6): 62-70. doi: 10.7668/hbnxb.20195920.

LI Jing, PANG Bo, ZHANG Ru, ZHAERJIAMALI Abudubieke, WANG Zhengrui, SHI Shunyu, FAN Zhenzhen, MA Jingjing, CHEN Jialin, SONG Wu, LI Shengmei, GAO Wenwei. Cloning,Subcellular Localization and Expression Analysis of GhCRK26 Gene in Cotton[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 62-70. doi: 10.7668/hbnxb.20195920.

http://www.hbnxb.net/CN/Y2025/V40/I6/62

图/表 13

表1 生物信息学预测网站

Tab.1 Bioinformatics prediction websites

数据库/软件 Databases/softwares	预测功能 Predictive functions	网址 Website
Expasy	蛋白质理化性质预测	https://web.expasy.org/protparam/
ProtScale	氨基酸亲疏水性分析	https://prosite.expasy.org/
TMHMM 2.0	跨膜结构域预测	https://services.healthtech.dtu.dk/services/TMHMM-2.0/
SignalP-5	信号肽序列预测及剪切位点分析	https://services.healthtech.dtu.dk/services/SignalP-5.0/
NetPhos 3.1	磷酸化位点预测	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
SOPMA	蛋白质二级结构预测	https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html
SWISS-MODEL	三级结构	https://www.swissmodel.expasy.org/
NCBI CD Search	保守结构域注释	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
NCBI BLAST	同源序列比对与检索	https://blast.ncbi.nlm.nih.gov/Blast.cgi
CLUSTALW	多序列比对分析	https://www.genome.jp/tools-bin/clustalw^[23]
ESPript 3.0	多序列比对结果可视化美化	https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi
PlantCare	启动子顺式作用元件预测	https://bioinformatics.psb.ugent.be/webtools/plantcare/html/
EvolView	系统发育树可视化与美化	https://evolgenius.info//evolview-v2/#login
MEGA 11	进化树构建	—
TBtools	启动子序列提取	—

图1 GhCRK26在亲本中的表达量不同大写字母表示差异在P<0.01具有显著性。图2同。

Fig.1 Expression of GhCRK26 in parents Different letters show significant difference at P<0.01 level.The same as Fig.2.

图2 GhCRK26 在系9和新海16根、茎、叶组织的表达

Fig.2 Expression of GhCRK26 in root,stem and leaf tissues of Line 9 and Xinhai 16

图3 GhCRK26基因克隆 M.DL2000 Marker;1.GhCRK26基因PCR 扩增片段。

Fig.3 Cloning of the GhCRK26 gene M.DL2000 Marker;1.PCR amplification fragment of GhCRK26 gene.

图4 不同物种中GhCRK26蛋白序列比对

Fig.4 Sequence comparison of GhCRK26 protein in different species

图5 不同物种GhCRK26蛋白序列的系统发育树分析

Fig.5 Phylogenetic tree analysis of GhCRK26 protein sequences from different species

图6 GhCRK26基因编码的蛋白质特征 A.GhCRK26编码蛋白的氨基酸亲、疏水预测;B.GhCRK26编码蛋白的跨膜区预测。

Fig.6 Protein characteristics encoded by GhCRK26 gene A.Prediction of amino acid affinity and hydrophobicity of the GhCRK26-encoded protein; B.Prediction of the transmembrane region of the GhCRK26-encoded protein.

图7 GhCRK26基因编码蛋白的信号肽预测和磷酸化位点预测 A.GhCRK26基因编码蛋白的信号肽预测;B.GhCRK26基因编码蛋白的磷酸化位点预测。

Fig.7 Signal peptide prediction and phosphorylation site prediction of the protein encoded by the GhCRK26 gene A.Signal peptide prediction of GhCRK26;B.Phosphorylation site prediction of GhCRK26.

图8 GhCRK26基因编码蛋白的二级结构和三级结构 A.GhCRK26二级结构;B.GhCRK26三级结构。

Fig.8 Secondary structure and tertiary structure of the protein encoded by the GhCRK26 gene A.Secondary structure of GhCRK26;B.Tertiary structure of GhCRK26.

图9 GhCRK26的保守结构域分析

Fig.9 Analysis of conserved domains in GhCRK26

图10 GhCRK26基因启动子区域的顺式作用元件分析

Fig.10 Cis-acting element analysis of the promoter region of the GhCRK26 gene

图11 PHG∷GhCRK26菌液PCR检测 A.PHG∷GhCRK26菌液克隆引物PCR检测:M.DL2000 Marker,1.PHG∷GhCRK26菌液PCR结果;B.PHG∷GhCRK26菌液特异性引物PCR检测:M.DL2000 Marker,1.PHG∷GhCRK26菌液PCR结果。

Fig.11 PHG∷GhCRK26 bacteriophage PCR detection A.PCR detection of PHG∷GhCRK26 bacterial solution by cloning primers:M.DL2000 Marker,1.PCR results of PHG∷GhCRK26 bacterial solution;B.PCR detection of specific primers for GhCRK26 bacterial solution:M.DL2000 Marker,1.PCR results of PHG∷GhCRK26 bacterial solution.

图12 GhCRK26蛋白的亚细胞定位第一行为空载体转化的烟草叶肉细胞,第二行为融合载体转化的细胞。GFP对应荧光视野观察结果,Bright field 代表明场视野, Merge 显示的是荧光与明场的叠加融合视野。

Fig.12 Subcellular localization of GhCRK26 protein The first line is the transformed tobacco cells by empty load and the second line is the cells transformed by the fusion vector.GFP corresponds to the fluorescence field of view,Bright field represents the bright field of view,and Merge shows the superimposed fusion field of fluorescence and bright field.

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棉花GhCRK26基因克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of GhCRK26 Gene in Cotton

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