Cloning and Functional Verification of GhD6PKL2 from Gossypium hirsutum

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

Abstract

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

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.

Figures/Tables 9

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

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

Fig.2 Pridiction of phosphorylation sites in GhD6PKL2

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

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

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

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

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

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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