棉花GhD6PKL2的克隆及功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2020-1591

摘要/Abstract

摘要：

旨在探究丝氨酸苏氨酸蛋白激酶（serine/ threonine protein kinase,STPK）基因在棉纤维发育中的功能,解析棉纤维细胞的分化和发育机理。从陆地棉遗传标准系TM-1中克隆基因GhD6PKL2,并对其序列及结构特征进行生物信息学分析、表达量分析及过表达拟南芥的表型观察。GhD6PKL2含有典型的STPK保守序列位点。预测编码的蛋白相对分子质量为49.74 kD,等电点为6.17,含有多个丝氨酸苏氨酸磷酸化位点。表达模式结果表明,GhD6PKL2在与棉花纤维伸长阶段相吻合的、开花后20 d显著高表达。软件预测及在烟草叶片中的荧光蛋白定位结果均显示,GhD6PKL2编码的蛋白质定位在细胞膜上。诱饵自激活试验验证GhD6PKL2没有自激活活性及毒性。过表达拟南芥,能使转基因拟南芥表现出表皮毛数量增多,同时主根变短、侧根数目增多的表型。表明该丝氨酸苏氨酸蛋白激酶基因在拟南芥表皮毛及主侧根发育方面发挥一定作用,可能为一个棉纤维伸长发育阶段的潜在调控基因。

关键词: 陆地棉, 丝氨酸苏氨酸蛋白激酶（STPK）, 纤维发育

Abstract:

This work aims to investigate the functionality of gene of serine/threonine-protein kinase（STPK）during the development of cotton fiber,to provide an insight of development and differentiation mechanism of cotton fiber cells. The gene GhD6PKL2 from Gossypium hirsutum genetic standard line TM- 1 was cloned,and its sequence and structure were analyzed by bioinformatics,expressions and phenotypic observation in the overexpression of Arabidopsis GhD6PKL2,with a molecular mass of 49.74 kD,and a pI of 6.17,contained typical STPK conserved sequence sites and a number of serine and threonine phosphorylation sites. The analysis of expression pattern indicated that GhD6PKL2 significantly expressed at 20 d of flowering,which was consistent with fiber elongation stage. Both prediction by software and results of fluorescence protein localization in tobacco leaves showed that the protein encoded by GhD6PKL2 was localized on the plasma membrane. Bait self-activation assay confirmed that GhD6PKL2 was of no self-activation and no toxicity. The overexpression of GhD6PKL2 in Arabidopsis plants resulted in the increase of the number of trichome and lateral roots,while the length of main root shorting. These results imply that GhD6PKL2 may play a certain role in regulating the Arabidopsis’s trichome and lateral roots,and thus can be a potential candidate gene in cotton fiber elongation stage.

Key words: Gossypium hirsutum, serine/threonine- protein kinase（STPK）, development of cotton fiber

刘媛媛, 杨冬杰, 左东云, 程海亮, 张友平, 吕丽敏, 王巧连, 宋国立. 棉花GhD6PKL2的克隆及功能验证[J]. 生物技术通报, 2021, 37(8): 111-120.

LIU Yuan-yuan, YANG Dong-jie, ZUO Dong-yun, CHENG Hai-liang, ZHANG You-ping, LV Li-min, WANG Qiao-lian, SONG Guo-li. Cloning and Functional Verification of GhD6PKL2 from Gossypium hirsutum[J]. Biotechnology Bulletin, 2021, 37(8): 111-120.

图/表 9

表1 本研究所用到的引物列表

Table1 Primers used in this study

引物用途 Function of the primer	引物名称 Name of the primer	引物序列 Sequence of the primer（5'-3'）
pRI101载体通用引物 The primers in pRI101 vector	35ScexuPF	CCTTCGCAAGACCCTTCCTC
pRI101载体通用引物 The primers in pRI101 vector	pRI101- AN- cexuRV	CAGGAAACAGCTATGAC
cDNA扩增& 过表达载体构建引物 The primers for cDNA amplification and over-expression vector’s construction	D6PKPFCZ（Sal I）	TTGATACATATGCCCGTCGACATGGAGCCGTTTCTCGACGACT
	D6PKPR1CZ（EcoR I）	GAATTCCTAATGATGATGATGATGATGATAATACTCTACTGGGGTCTCT
	D6PKPR2CZ（EcoR I）	AGAGTTGTTGATTCAGAATTCCTAATGATGATGATGATGATG
定量引物 The primers for quantitative real-time PCR	A07GD6PKPF1	TGAGTTCCCTAAAGAACCCATTGT
定量引物 The primers for quantitative real-time PCR	A07GD6PKPR1	GGTGGTGCTTGATGGCTGAT
cDNA检测& 内参引物 The primers for cDNA quality’s verification and reference	AtactinePF	TGCTATTCTGCGTTTGGACCTTG
	AtactinePR	ATCCCTTACGATTTCACGCTCTG
	Histone3PF1	CCGTAAATCTGCCCCAACCA
	Histone3PR1	GACCCACAAGGTATGCCTCTGC
GFP& Linker片段扩增引物 The primers for GFP and Linker fragments amplification	GFPlinkPF1	TGGCTCTGGCGGTGGCGGATCGATGGGTAAAGGAGAAGAACTTT
	GFPPR1（EcoR I）	AGAGTTGTTGATTCAGAATTCTCATTTGTATAGTTCATCCATG
	LinkPF2（BamH I）	GGATCCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG
亚细胞定位中基因扩增引物 The primers for genes in subcellular location	AD6PKPF1（Sal I）	TTGATACATATGCCCGTCGACATGGAGCCGTTTCTCGACGACT
亚细胞定位中基因扩增引物 The primers for genes in subcellular location	AD6PKPR1（BamH I）	CTGAACCGCCTCCACCGGATCCATAATACTCTACTGGGGTCTCT
诱饵载体引物 The primers for bait vector construction	A0D6PKPF1GB（Sal）	ATGCGGCCGCTGCAGGTCGACATGGAGCCGTTTCTCGACGACT
诱饵载体引物 The primers for bait vector construction	A0D6PKPR1GB（EcoR）	ATGGCCATGGAGGCCGAATTCATAATACTCTACTGGGGTCTCT

图1 GhD6PKL2序列分析及蛋白比对 A：GhD6PKL2结构示意图;B：GhD6PKL2与其他几个物种（AtD6PKL2-1：拟南芥;NtD6PKL2-11：烟草;OsD6PKL2-1：水稻;ZmHPK-1：玉米）氨基酸序列的多重比对。图中标注罗马数字Ⅰ-Ⅺ中处为STPK家族中11个典型的活性位点

Fig.1 Analysis of the GhD6PKL2 sequence and protein sequence alignment A: The structure of the GhD6PKL2. B: Multiple alignment of amino acid sequences between GhD6PKL2 and other’s species(AtD6PKL2- 1: Arabidopsis; NtD6PKL2- 11: Nicotiana tabacum; OsD6PKL2-1: Oryza sativa; ZmHPK-1: Zea mays). The number Ⅰ to Ⅺ indicate 11 typical active sites of STPK family

图2 GhD6PKL2蛋白的磷酸位点预测

Fig.2 Pridiction of phosphorylation sites in GhD6PKL2

图3 GhD6PKL2在棉纤维各发育时期的相对表达分析

Fig.3 Relative expression level of GhD6PKL2 in different periods at cotton fiber development

图4 GhD6PKL2的亚细胞定位 A-C：对照;D-F：GhD6PKL2-GFP融合蛋白;bar=100 µm

Fig.4 Subcellular localization of GhD6PKL2 A- C：GFP only; D- F: the fusion protein: GhD6PKL2- GFP; bar: 100 m

图5 GhD6PKL2诱饵载体自激活的检测 A：阳性对照pGBKT7-53和pGADT7-T在SD-trp-leu/X-α-gal培养基上的生长情况;B：阴性对照pGBKT7-Lam和pGADT7-T在SD-trp-leu/X-α-gal培养基上的生长情况;C：空白对照PGADT7-T在SD-trp培养基上的生长情况;D：pGBKT7-GhD6PKL2在SD-trp培养基上的生长情况;E：pGBKT7-GhD6PKL2在SD-trp/X-α-gal培养基上的生长情况;F：pGBKT7-GhD6PKL2在SD-trp/X-α-gal/AbA培养基上的生长情况

Fig.5 Detection of transcriptional activity of GhD6PKL2 A: Positive control: pGBKT7-53 and pGADT7-T, growth on SD-trp-leu/X-α-gal;B: nagetive control: pGBKT7-Lam and pGADT7-T growth on SD-trp-leu/X-α-gal; C: blank control: PGADT7-T growth on SD-trp; D: pGBKT7-GhD6PKL2 growth on SD-trp; E: pGBKT7-GhD6PKL2 growth on SD-trp/X-α-gal; F: pGBKT7-GhD6PKL2 growth on SD-trp/X-α-gal/AbA

图6 转GhD6PKL2拟南芥的分子鉴定

Fig.6 PCR and RT- PCR analysis of the GhD6PKL2 transgenic plants

图7 转GhD6PKL2拟南芥叶片表皮毛的表型 A：转GhD6PKL2拟南芥和WT表皮毛观察;B：转GhD6PKL2拟南芥和WT表皮毛数量平均值。***表示在P=0.001水平差异显著。下同

Fig.7 Phynotype of Arabidopsis trichome between GhD6PKL2 trans-genic plants and wide type A: Arabidopsis trichome observation between transgenic plants and wide type;B: average arabidopsis trichome in transgenic plants and wide type. ***indicate that there is a significant difference at P= 0.001. The same below

图8 转GhD6PKL2拟南芥根的表型 A：转GhD6PKL2拟南芥和WT根生长情况;B：转GhD6PKL2拟南芥和WT主根长度

Fig.8 Phenotype of Arabidopsis main root between GhD6PKL2 trans-genic plants and wide type A: Arabidopsis root growths of GhD6PKL2 transgenic plants and WT type; B: Main root length in GhD6PKL2 transgenic plants and WT type.

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