Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (8): 111-120.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1591
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LIU Yuan-yuan(), YANG Dong-jie, ZUO Dong-yun, CHENG Hai-liang, ZHANG You-ping, LV Li-min, WANG Qiao-lian, SONG Guo-li()
Received:
2020-12-31
Online:
2021-08-26
Published:
2021-09-10
Contact:
SONG Guo-li
E-mail:yuanyl4956@163.com;sglzms@163.com
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.
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- 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 |
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 |
AD6PKPR1(BamH I) | CTGAACCGCCTCCACCGGATCCATAATACTCTACTGGGGTCTCT | |
诱饵载体引物 The primers for bait vector construction | A0D6PKPF1GB(Sal) | ATGCGGCCGCTGCAGGTCGACATGGAGCCGTTTCTCGACGACT |
A0D6PKPR1GB(EcoR) | ATGGCCATGGAGGCCGAATTCATAATACTCTACTGGGGTCTCT |
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- 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 |
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 |
AD6PKPR1(BamH I) | CTGAACCGCCTCCACCGGATCCATAATACTCTACTGGGGTCTCT | |
诱饵载体引物 The primers for bait vector construction | A0D6PKPF1GB(Sal) | ATGCGGCCGCTGCAGGTCGACATGGAGCCGTTTCTCGACGACT |
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.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.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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