EcHPPD Gene of Barnyard Grass: Cloning and Bioinformatics Analysis

doi:10.11924/j.issn.1000-6850.casb2022-0789

Abstract

Abstract:

In order to clone the gene of hydroxyphenyl pyruvate dioxygenase (HPPD) in Echinochloa crus-galli for exploring its genetic function and better developing herbicide, the homologous cloning and rapid amplification of cDNA ends (RACE) techniques were used to clone the EcHPPD gene sequence using E. crus-galli leaves as materials. The characteristics of EcHPPD were analyzed using bioinformatics software. The coding sequence of EcHPPD gene is 1317 bp in length, encoding a protein consisted of 439 amino acids with a relative molecular weight of 46.659 kD and an isoelectric point of 5.30. E. crus-galli is most evolutionarily close to Dichanthelium oligosanthes (OEL23409.1: 8-249) in the Poaceae family based on HPPD protein. The tertiary protein structure of EcHPPD is composed of two chains, with two Fe²⁺ binding sites, and can interact with the pyrazolide active center skeleton through hydrogen bonds. EcHPPD may interact with 11 proteins such as HGO, HPT1, TAT3, AT5G36160 and TAT7. This study reported the EcHPPD gene sequence and conducted bioinformatics analysis, providing a theoretical basis for creating new HPPD inhibitors.

Key words: barnyard grass, HPPD gene, gene cloning, bioinformatics, protein interaction

HAN Jincai, LUO Dingfeng, BAI Haodong, LI Zuren. EcHPPD Gene of Barnyard Grass: Cloning and Bioinformatics Analysis[J]. Chinese Agricultural Science Bulletin, 2024, 40(3): 87-94.

Figures/Tables 8

References 33

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Primer name	Primer sequence(5’-3’)	Use of primers
HPPD4-ZJ	Forward: TTCCACCACGTSGAGYTCTG Reverse: TCRTCCTTCTCCATGCACCC	Fragmen cloning
HPPD4-5GSP1	CGACGATGTGGTCGAACCGCCTG	5’RACE the first round of amplification
HPPD4-5GSP2	GTAGCTGACGTAGCGGAGCACGAC	5’RACE Second round of amplification
HPPD4-3GSP1	TGCTCGCCAACAACTCCGAGACC	3’RACE the first round of amplification
HPPD4-3GSP2	GCCGCAGCCAGATTCAGACGTACC	3’RACE Second round of amplification
R=A/G Degenerate bases were underlined, degenerate primers Y=C/T; R=A/G;

Primer name	Primer sequence(5’-3’)	Use of primers
HPPD4-ZJ	Forward: TTCCACCACGTSGAGYTCTG Reverse: TCRTCCTTCTCCATGCACCC	Fragmen cloning
HPPD4-5GSP1	CGACGATGTGGTCGAACCGCCTG	5’RACE the first round of amplification
HPPD4-5GSP2	GTAGCTGACGTAGCGGAGCACGAC	5’RACE Second round of amplification
HPPD4-3GSP1	TGCTCGCCAACAACTCCGAGACC	3’RACE the first round of amplification
HPPD4-3GSP2	GCCGCAGCCAGATTCAGACGTACC	3’RACE Second round of amplification
R=A/G Degenerate bases were underlined, degenerate primers Y=C/T; R=A/G;

ddH₂O	17 μL
2×Phanta Max Buffer	25 μL
dNTP Mix (10 mmol/L each)	1 μL
Template DNA	2 μL
primer1 (10 μmol/L)	2 μL
primer2 (10 μmol/L)	2 μL
Phanta Max Super-Fidelity DNA Polymease(1 U/μL)	1 μL

ddH₂O	17 μL
2×Phanta Max Buffer	25 μL
dNTP Mix (10 mmol/L each)	1 μL
Template DNA	2 μL
primer1 (10 μmol/L)	2 μL
primer2 (10 μmol/L)	2 μL
Phanta Max Super-Fidelity DNA Polymease(1 U/μL)	1 μL