过表达棉花葡萄糖醛酸激酶基因GbGlcAK促进拟南芥细胞伸长

doi:10.13304/j.nykjdb.2021.0806

摘要/Abstract

摘要：

纤维品质改良一直是棉花育种的重要目标。基于纤维不同发育时期的RNA-seq数据，发现1个在海岛棉和陆地棉间差异表达的葡萄糖醛酸激酶基因（glucuronic acid kinase/glucuronokinase，GlcAK）。从海岛棉（Gossypium barbadense）Pima90-53纤维中克隆了GbGlcAK，其ORF为1 101 bp，编码366个氨基酸。GbGlcAK具有GHMP 激酶 N和C结构域，为GHMP超家族成员，属于可溶性非分泌蛋白，与拟南芥GlcAK亲缘关系最近。亚细胞定位结果显示GbGlcAK分布在细胞质中。超表达GbGlcAK的拟南芥叶表皮毛、下胚轴、下胚轴细胞和根的长度较野生型均极显著增加，UDP-D-GlcA代谢相关基因表达量增加，果胶含量显著增加，说明过表达GbGlcAK能够上调UDP-D-GlcA代谢通路相关基因的表达从而提高果胶含量，进而促进细胞伸长，这为进一步研究GbGlcAK在棉纤维发育中的功能提供了参考。

关键词: 棉花, 葡萄糖醛酸激酶, 纤维发育, 细胞伸长

Abstract:

Fiber quality improvement is an important objective for cotton breeding. A differentially expressed gene GlcAK （glucuronic acid kinase/glucuronokinase） between sea island and upland cotton was identified based on RNA-seq data from different stages of fiber development. In this study， GbGlcAK （Gossypium barbadense GlcAK） with 1 101 bp of ORF was cloned from the fibers of sea island cotton Pima 90-53， which encoded 366 amino acids. GbGlcAK had the structure domain of GHMP kinases N and C domain and belonged to the GHMP superfamily. GbGlcAK was a soluble and non-secretory protein. In terms of phyletic evolution， GbGlcAK was most closely related to GlcAK from A.thaliana. Subcellular localization results showed that GbGlcAK was distributed in the cytoplasm. Compared with the wild type， the transgenic A.thaliana lines possessed significantly longer leaf trichomes， hypocotyls， hypocotyl cells and roots. The expression of uridine diphosphate-D-glucuronic acid （UDP-D-GlcA） metabolic related genes were upregulated， and the pectin content was also significantly increased in the transgenic lines. These results suggested that overexpression of GbGlcAK in A. thaliana up-regulated UDP-D-GlcA metabolic pathway， thereby increased pectin content and promoted cell elongation. Our study provided references for further exposing the function of GbGlcAK in cotton fiber development.

Key words: cotton, glucuronokinase, fiber development, cell elongation

中图分类号:

S562

吴楠, 杨君, 张艳, 孙正文, 张冬梅, 李丽花, 吴金华, 马峙英, 王省芬. 过表达棉花葡萄糖醛酸激酶基因GbGlcAK促进拟南芥细胞伸长[J]. 中国农业科技导报, 2022, 24(6): 36-46.

Nan WU, Jun YANG, Yan ZHANG, Zhengwen SUN, Dongmei ZHANG, Lihua LI, Jinhua WU, Zhiying MA, Xingfen WANG. Overexpression of a Cotton Glucuronokinase Gene GbGlcAK Promotes Cell Elongation in Arabidopsis thaliana[J]. Journal of Agricultural Science and Technology, 2022, 24(6): 36-46.

图/表 9

表1 试验中用到的PCR和qPCR引物

Table 1 PCR and qPCR primers used in this study

引物名称 Primer names	登录号 Accession No.	引物序列 Primer sequences （5’-3’）	用途 Purpose
GlcAK-F	—	TGGTATTGGCTTGGTGCAGT	基因克隆 Gene cloning
GlcAK-R	—	CAAAAACAATCCCTAAAATGCTCTT	基因克隆 Gene cloning
sGbGlcAK-R	—	GGTGGTACCCTGCTTAGACAATGT	亚细胞定位Subcellular location
GbGlcAK-F	—	GGCGTCTAGAATGGATCAAAATATG	亚细胞定位和载体构建 Subcellular location and vector construction
GbGlcAK-R	—	GGTACCCGCCTACTTAGACAATG	载体构建 Vector construction
qGbGlcAK-F	—	CATCATCAACCCTCACCCCATTC	拟南芥实时定量PCR Real-time PCR for A. thaliana
qGbGlcAK-R	—	GCCGCATACAATAGCACTGGACC
qAtGlcAK-F	AT3G01640	GGATAGAGCATCGGTCCTTCGC
qAtGlcAK-R	AT3G01640	CGCCATTAGCAACCTTACCCCA
qAtMIPS-F	AT4G39800	AAGGAGAAGAATAAGGTGGATAAGGTT
qAtMIPS-R	AT4G39800	GCAATCGCATAAAGTGTTGAAGG
qAtIMP-F	AT3G02870	GACAGAGACTGATAAAGGATGTGAAGA
qAtIMP-R	AT3G02870	AGGGAACCCGTGAACGAAAT
qAtMIOX-F	AT4G26260	CGAAGCCATCCGCAAAGATTACC
qAtMIOX-R	AT4G26260	CCAACAACAGCCCATTGAGGAAGTC
qAtPGM-F	AT1G23190	GCTACCTACGGTCGTCACTATTACACTCG
qAtPGM-R	AT1G23190	GTAACGGATTCCCTGGTGCTTCG
qAtUGP-F	AT5G17310	GCCCAGCAAGGGAAAGACCG
qAtUGP-R	AT5G17310	CGATGGCACCCAAGTTGTCTGAAT
qAtUGD-F	AT5G15490	GATTGCGGTTCTCGGCTTCG
qAtUGD-R	AT5G15490	TGCTTCACAGTGGTGGGGCTC
qAtUXS-F	AT5G59290	TGAGAAGAATGAGGTGGTTGTTGC
qAtUXS-R	AT5G59290	GGTTGTATTTGTAGAAGATAGGAGAGGC
qAtGAE-F	AT3G23820	GCGACGGCGGATACAAGCA
qAtGAE-R	AT3G23820	GATGGCGAAGATGAGGACGAGG
qAtUB5-F	AT3G62250	CCTCGCCGACTACAACATCCAG
qAtUB5-R	AT3G62250	CTTCTTCCTCTTCTTAGCACCACCA

图1 GbGlcAK蛋白特征A：ProtScale分析GbGlcAK肽链亲/疏水性分布曲线；B、C：TOPpred2和TMHMM 2.0工具对GbGlcAK氨基酸序列的跨膜区预测；D：GbGlcAK蛋白信号肽分析

Fig. 1 Characterization of GbGlcAK proteinA： Hydrophilic/hydrophobic distribution curve of the GbGlcAK peptide analyzed by ProtScale； B， C： Transmembrane region prediction of the GbGlcAK amino acid sequence by TOPpred2 and TMHMM 2.0； D： Signal peptide analysis of the GbGlcAK protein

图2 GbGlcAK系统进化分析注：Rc—蓖麻；Pt—毛果杨；Vv—葡萄；At—拟南芥；Gm—大豆；Os—水稻；Zm—玉米；Sb—高粱。

Fig.2 Evolutionary analysis of GbGlcAKNote： Rc—Ricinus communis； Pt—Populus trichocarpa； Vv—Vitis vinifera； At—Arabidopsis thaliana； Gm—Glycine max； Os—Oryza sativa； Zm—Zea mays； Sb—Sorghum bicolor.

图3 GbGlcAK的亚细胞定位A、B、C：蓝光、可见光叠加蓝光、可见光下的转pCam：：GFP细胞；D、E、F：质壁分离后蓝光、可见光叠加蓝光、可见光下的转pCam：：GbGlcAK-GFP细胞

Fig. 3 Subcellular localization of GbGlcAKA， B， C： Cell transfected with pCam：：GFP under Blu-ray， visible light superimposed Blu-ray， and visible light； D， E， F： Cell transfected with pCam：：GbGlcAK-GFP under Blu-ray， visible light superimposed Blu-ray， and visible light after plasma-wall separation

图4 转基因拟南芥叶表皮毛长度分析注：**表示在P<0.01水平差异显著（双尾t检验）。

Fig. 4 Analysis of leaf trichome length in transgenic A. thalianaNote：** indicates significant difference at the P<0.01 level （2-tailed t-test）.

图5 转基因拟南芥下胚轴长度分析A、B：超表达GbGlcAK拟南芥与野生型拟南芥下胚轴长度比较；C：显微镜下观察超表达GbGlcAK拟南芥与野生型拟南芥叶下胚轴细胞。**表示P<0.01水平的显著性（双尾t检验）；白色箭头所示细胞边界

Fig. 5 Analysis of hypocotyl length in transgenic A. thalianaA， B： Comparison of hypocotyl length between transgenic and wild-type Arabidopsis； C： Microscopic observation of hypocotyl cells； ** indicates significant difference at the P<0.01 level （2-tailed t-test）；white arrows show the ends of cells

图6 转基因拟南芥根长分析注：**表示P<0.01水平的显著性（双尾t检验）。

Fig. 6 Analysis of root length in transgenic A. thalianaNote：** indicates significant difference at the P<0.01 level （2-tailed t-test）.

图7 转基因拟南芥中UDP-D-GlcA代谢相关基因的表达分析

Fig.7 Expression analyses of UDP-D-GlcA metabolism related genes in transgenic Arabidopsis

图8 转基因拟南芥果胶含量注：*表示P<0.05水平的显著性（双尾t检验）。

Fig. 8 Pectin content in transgenic ArabidopsisNote：* indicates significant difference at P<0.05 level （2-tailed t-test）.

参考文献 43

1	喻树迅,魏晓文,赵新华.中国棉花生产与科技发展 [J].棉花学报, 2000, 12(6): 327-329.
	YU S X, WEI X W, ZHAO X H. Cotton production and technical development in China [J]. Acta Gossypii Sin., 2000, 12(6): 327-329.
2	MA Z, ZHANG Y, WU L, et al.. High-quality genome assembly and resequencing of modern cotton cultivars provide resources for crop improvement [J]. Nat. Genet., 2021, 53(9): 1385-1391.
3	BASRA A S, MALIK C P. Development of the cotton fiber [J]. Int. Rev. Cytol., 1984, 89(6): 65-113.
4	HAIGLER C H, BETANCUR L, STIFF M R, et al.. Cotton fiber: a powerful single-cell model for cell wall and cellulose research [J/OL]. Front. Plant Sci., 2012, 3: 104 [2021-11-09]. .
5	QIN Y M, ZHU Y X. How cotton fibers elongate: a tale of linear cell-growth mode [J]. Curr. Opin. Plant Biol., 2011, 14(1): 106-111.
6	SINGH B, AVCI U, EICHLER INWOOD S E, et al.. A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles [J]. Plant Physiol., 2009, 150(2): 684-699.
7	ZABLACKIS E, JING H, MüLLER B, et al.. Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves [J]. Plant Physiol., 1995, 107(4): 1129-1138.
8	KANTER U, USADEL B, GUERINEAU F, et al.. The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides [J]. Planta, 2005, 221(2): 243-254.
9	LOEWUS F A, KELLY S, NEUFELD E F. Metabolism of myo-inositol in plants: conversion to pectin, hemicellulose, D-xylose, and sugar acids [J]. Proc. Natl. Acad. Sci. USA, 1962, 48(3): 421-425.
10	NEUFELD E F, FEINGOLD D S, HASSID W Z. Enzymic phosphorylation of D-glucuronic acid by extracts from seedlings of Phaseolus aureus [J]. Arch. Biochem. Biophys., 1959, 83(1): 96-100.
11	LEIBOWITZ M D, DICKINSON D B, LOEWUS F A, et al.. Partial purification and study of pollen glucuronokinase [J]. Arch. Biochem. Biophys., 1977, 179(2): 559-564.
12	PIESLINGER A M, HOEPFLINGER M C, TENHAKEN R. Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum [J]. Anal. Biochem., 2009, 388(2): 254-259.
13	PIESLINGER A M, HOEPFLINGER M C, TENHAKEN R. Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars [J]. J. Biol. Chem., 2010, 285(5): 2902-2910.
14	HOLDEN H M, THODEN J B, TIMSON D J, et al.. Galactokinase: structure, function and role in type Ⅱ galactosemia [J]. Cell. Mol. Life Sci., 2004, 61(19-20): 2471-2484.
15	XIAO W, HU S, ZHOU X, et al.. A glucuronokinase gene in Arabidopsis, AtGlcAK, is involved in drought tolerance by modulating sugar metabolism [J]. Plant Mol. Biol. Rep., 2017, 35(2): 1-14.
16	IVANOV KAVKOVA E, BLöCHL C, TENHAKEN R. The Myo-inositol pathway does not contribute to ascorbic acid synthesis [J]. Plant Biol., 2019, 21 (): 95-102.
17	LIU Z, WANG X, SUN Z, et al.. Evolution, expression and functional analysis of cultivated allotetraploid cotton DIR genes [J/OL]. BMC Plant Biol., 2021, 21(1): 89[2022-03-07]. .
18	WANG Z, YANG Z, LI F. Updates on molecular mechanisms in the development of branched trichome in Arabidopsis and nonbranched in cotton [J]. Plant Biotechnol. J., 2019, 17(9): 1706-1722.
19	吴立柱,王省芬,李喜焕,等.通用型植物表达载体pCamE的构建及功能验证[J].农业生物技术学报, 2014, 22(6): 661-671.
	WU L Z, WANG X F, LI X H, et al.. Construction and function identification of universal plant expression vector pCamE [J]. J. Agric. Biotechnol., 2014, 22(6): 661-671.
20	WANG M, TU L, YUAN D, et al.. Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense [J]. Nat. Genet., 2019, 51(2): 224-229.
21	MISTRY J, CHUGURANSKY S, WILLIAMS L, et al.. Pfam: The protein families database in 2021 [J]. Nucleic Acids Res., 2021, 49(D1): D412-D419.
22	ARTIMO P, JONNALAGEDDA M, ARNOLD K, et al.. ExPASy: SIB bioinformatics resource portal [J]. Nucleic Acids Res., 2012, 40(W1): W597-W603.
23	CUTHBERTSON J M, DOYLE D A, SANSOM M S. Transmembrane helix prediction: a comparative evaluation and analysis [J]. Protein Eng. Des. Sel., 2005, 18(6): 295-308.
24	KROGH A, LARSSON B, GVON HEIJNE, et al.. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes [J]. J. Mol. Biol., 2001, 305(3): 567-580.
25	MÖLLER S, CRONING M D, APWEILER R. Evaluation of methods for the prediction of membrane spanning regions [J]. Bioinformatics, 2001, 17(7): 646-653.
26	ALMAGRO ARMENTEROS J J, TSIRIGOS K D, SøNDERBY C K, et al.. SignalP 5.0 improves signal peptide predictions using deep neural networks [J]. Nat. Biotechnol., 2019, 37(4): 420-423.
27	KUMAR S, STECHER G, TAMURA K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Mol. Biol. Evol., 2016, 33(7): 1870-1874.
28	YANG J, JI L, WANG X, et al.. Overexpression of 3-deoxy-7-phosphoheptulonate synthase gene from Gossypium hirsutum enhances Arabidopsis resistance to Verticillium wilt [J]. Plant Cell Rep., 2015, 34(8): 1429-1441.
29	CHEN H, NELSON R S, SHERWOOD J L. Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection [J]. Biotechniques, 1994, 16(4): 664-668, 670.
30	CLOUGH S J, BENT A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [J]. Plant J., 1998, 16(6): 735-743.
31	An C, Wang C, Mou Z. The Arabidopsis Elongator complex is required for nonhost resistance against the bacterial pathogens Xanthomonas citri subsp. citri and Pseudomonas syringae pv. phaseolicola NPS3121 [J]. New Phytol., 2017, 214(3): 1245-1259.
32	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔ ^CT method [J]. Methods, 2001, 25(4): 402-408.
33	JEAN-MARC L, TRUNG-BIEU N, BRIGITTE C, et al.. QTL analysis of cotton fiber quality using multiple Gossypium hirsutum × Gossypium barbadense backcross generations [J]. BMC Plant Biol., 2005, 45(1): 123-140.
34	SERNA L, MARTIN C. Trichomes: different regulatory networks lead to convergent structures [J]. Trends Plant Sci., 2006, 11(6): 274-280.
35	GUAN X, YU N, SHANGGUAN X, et al.. Arabidopsis trichome research sheds light on cotton fiber development mechanisms [J]. Chin Sci. Bull., 2007, 52(13): 1734-1741.
36	GUAN X, PANG M, NAH G, et al.. miR828 and miR858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development [J/OL]. Nat. Commun., 2014, 5: 3050[2022-03-07]. .
37	MA Z, HE S, WANG X, et al.. Resequencing a core collection of upland cotton identifies genomic variation and loci influencing fiber quality and yield [J]. Nat. Genet., 2018, 50(6): 803-813.
38	GENDREAU E, TRAAS J, DESNOS T, et al.. Cellular basis of hypocotyl growth in Arabidopsis thaliana [J]. Plant Physiol., 1997, 114(1): 295-305.
39	BORON A K, VISSENBERG K. The Arabidopsis thaliana hypocotyl, a model to identify and study control mechanisms of cellular expansion [J]. Plant Cell Rep., 2014, 33(5): 697-706.
40	武耀廷,刘进元.棉纤维细胞发育过程中非纤维素多糖的生物合成 [J].棉花学报, 2004, 16(3): 189-192.
	WU Y, LIU J. Noncellulosic polysaccharides biosynthesis in cotton fiber developing [J]. Cotton Sci., 2004, 16(3): 189-193.
41	喻树迅,朱玉贤,陈晓亚.棉花纤维发育生物学 [M].北京:科学出版社, 2016: 169.
42	KROH M, MIKI-HIROSIGE H, ROSEN W, et al.. Incorporation of label into pollen tube walls from myoinositol-labeled Lilium longiflorum pistils [J]. Plant Physiol., 1970, 45(1): 92-94.
43	SEITZ B, KLOS C, WURM M, et al.. Matrix polysaccharide precursors in Arabidopsis cell walls are synthesized by alternate pathways with organ-specific expression patterns [J]. Plant J., 2000, 21(6): 537-546.

[1]	贾浩, 王洪这, 孙正文, 谷淇深, 张冬梅, 王星懿, 张艳, 卢怀玉, 马峙英, 王省芬. 棉花VOZ基因家族鉴定及GhVOZ1耐盐功能研究[J]. 中国农业科技导报, 2025, 27(9): 58-68.
[2]	陈宜新, 杨秀波, 田士军, 王聪, 白志英, 李存东, 张科. 陆地棉GhCOMT28对干旱胁迫的响应[J]. 中国农业科技导报, 2025, 27(4): 45-56.
[3]	董志多, 付秋萍, 黄建, 祁通, 付彦博, 开赛尔·库尔班. 新疆棉花萌发期的耐盐能力分析[J]. 中国农业科技导报, 2025, 27(4): 57-67.
[4]	彭梓程, 杜洪力, 王铭, 张凤华, 杨海昌. 丛枝菌根真菌调控盐碱胁迫下棉花生长及离子平衡的研究[J]. 中国农业科技导报, 2025, 27(2): 33-41.
[5]	翁慧婷, 刘海洋, 郭惠明, 程红梅, 李君, 张超, 苏晓峰. 棉花抗黄萎病相关基因GhERF020功能的初步分析[J]. 中国农业科技导报, 2024, 26(9): 112-121.
[6]	李紫琴, 王家强, 李贞, 邹德秋, 张小功, 罗霄玉, 柳维扬. 基于光谱指数的棉花叶片叶绿素密度估算研究[J]. 中国农业科技导报, 2024, 26(8): 103-111.
[7]	庞博, 李生梅, 李彦霖, 杨涛, 梁维维, 张茹, 黄雅婕, 任丹, 崔进鑫, 李静, 马晶晶, 高文伟. 192份陆地棉杂交种的遗传多样性分析[J]. 中国农业科技导报, 2024, 26(8): 34-50.
[8]	秦宇坤, 陈俊英, 张丽娟. 赣北棉区棉花干物质积累特征和产量对减氮措施的响应[J]. 中国农业科技导报, 2024, 26(6): 191-199.
[9]	李江博, 高文举, 运晓东, 赵杰银, 耿世伟, 韩春斌, 陈全家, 陈琴. 不同水分胁迫处理对陆地棉核心种质资源的影响[J]. 中国农业科技导报, 2024, 26(3): 26-39.
[10]	李丽花, 孙正文, 柯会锋, 谷淇深, 吴立强, 张艳, 张桂寅, 王省芬. 陆地棉纤维强度KASP-SNP标记的开发及效应评价[J]. 中国农业科技导报, 2024, 26(2): 46-55.
[11]	翟梦华, 孙明辉, 李雪瑞, 徐新龙, 高海洲, 张巨松. 不同株行距配置下缩节胺对棉花株型塑造的影响[J]. 中国农业科技导报, 2024, 26(12): 145-156.
[12]	程珍, 牛建龙, 马玉婷, 柳维扬, 蒋学玮, 梁雪齐, 董红强. 1990—2020年南疆阿拉尔垦区棉花物候期的动态变化[J]. 中国农业科技导报, 2024, 26(10): 206-214.
[13]	郑德有, 左东云, 王巧莲, 吕丽敏, 程海亮, 顾爱星, 宋国立. 氟节胺与杀菌剂复配防治棉花枯萎病的增效药剂筛选[J]. 中国农业科技导报, 2024, 26(1): 119-124.
[14]	王为, 赵强, 穆妮热·阿卜杜艾尼, 阿里木·阿木力null, 李欣欣, 田阳青. 烯效唑复配不同外源物质对棉花化学封顶及产量品质的影响[J]. 中国农业科技导报, 2023, 25(9): 57-68.
[15]	孙正冉, 张翠萍, 张晋丽, 吴昊, 刘秀艳, 王振凯, 杨玉珍, 贺道华. 喷施化学打顶剂对关中棉区棉花植株生长的影响[J]. 中国农业科技导报, 2023, 25(4): 167-177.