Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (1): 86-94.doi: 10.3864/j.issn.0578-1752.2021.01.007

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning and Expression Analysis of Light Harvesting Chlorophyll a/b Protein Gene CcLhca-J9 in Conyza canadensis

LI ZuRen1(),LUO DingFeng1,BAI HaoDong1,XU JingJing1,HAN JinCai1,XU Qiang2,WANG RuoZhong2,BAI LianYang1,2()   

  1. 1Hunan Provincial Key Laboratory for Biology and Control of Weeds, Hunan Academy of Agricultural Sciences, Changsha 410125
    2Hunan Provincial Key Laboratory for Phytohormones, Hunan Agricultural University, Changsha 410128
  • Received:2020-04-12 Accepted:2020-06-28 Online:2021-01-01 Published:2021-01-13
  • Contact: LianYang BAI E-mail:lizuren88214@sina.com;bailianyang2005@aliyun.com

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Abstract:

【Background】Conyza canadensis, a pernicious weed, is harmful and lack of the effective management strategy. The caprylic acid can efficiently inhibit the photosynthesis of C. canadensis, which is a potential bio-herbicidal compound. Light harvesting chlorophyll a/b-binding protein (LHC) is the key complex protein in PSI, and plays an important role in photosynthesis.【Objective】The objective of this study is to explore the LHC gene in response to caprylic acid, and to provide a theoretical basis for the development of bio-herbicide.【Method】The open reading frame (ORF) of CcLhca-J9 was cloned from C. canadensis leaf by homologous PCR and RACE. The nucleotide sequence was analyzed by DNAMAN. The phylogenetic tree was constructed with the high similarity nucleotide from NCBI by neighbor-joining method and MEGA software. The molecular weight, isoelectric point and protein structure were analyzed using SWISS-MODEL and ExPaSy websites. Using homologous modeling results as a model, the affinity of caprylic acid and CcLhca-J9 protein was analyzed using AutoDock 4.2 software. RT-qPCR was used to analyze the difference of CcLhca-J9 expression level of caprylic acid, water and ferulic acid treatments.【Result】The CcLhca-J9 ORF is 744 bp, encoding 247 amino acid residues with a molecular weight of 26.766 kD and theoretical pI of 6.43, which belongs to Chloroa b-bind family protein. The phylogenetic analysis showed that the CcLhca-J9 had the most closed relationship with the Lhca protein of Tanacetum cinerariifolium (GEW73959.1) and Artemisia annua (PWA35049.1), and was in the same branch of Asteraceae group, with consistency of more than 85%, indicating that the gene family was conserved. The second structure of CcLhca-J9 protein had alpha helix, random coil, extended strand, beta turn. The homologous three-dimensional model of CcLhca-J9 protein was compared by 4y28.1.O (2.80Á), the tertiary structure was a monomolecular object with six chlorophyll a ligands, which was a typical light harvesting chlorophyll a/b-binding protein. Molecular docking result showed that hydrogen and p-π bond interaction were produced between caprylic acid and CcLhca-J9 amino acid residues Gly68, Phe67, Phe69 and Arg197. The result of RT-qPCR showed that the expression level of CcLhca-J9 was significantly decreased after 0-8 h caprylic acid treatment. Compared with ferulic acid control and water treatment, caprylic acid treatment inhibited the expression of CcLhca-J9.【Conclusion】CcLhca-J9 has the typical function of light harvesting chlorophyll a/b-binding protein, which may be involved in the process of inhibiting the growth of C. canadensis leaves and is a herbicidal target with the potential to develop herbicides.

Key words: Conyza canadensis, light harvesting protein, gene clone, molecular docking, caprylic acid

Table 1

The primers used for CcLhca-J9 cloning and RT-qPCR"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 引物用途 Use of primers
CcJ9-1 Forward: GAGYTGCGGYATCBCCGCCGT
Reverse: GTTGTGCCATGGRTCAGCCAA		 核心片段扩增 Fragment cloning
CcJ9-F2 Forward: CGGATTTGACCCACTTCGTCT 5′ RACE扩增 5′ cDNA end amplification
CcJ9-R2 Reverse: TATTGCCAGTATGGTGGGTAGGGT 5′ RACE扩增 5′ cDNA end amplification
CcJ9-R3 Reverse: GGCAGCCCATTCTTGTGCTTTTAC 5′ RACE扩增 5′ cDNA end amplification
CcJ9-F3 Forward: GGCAACTGGGTAAAAGCACAAGAAT 3′ RACE扩增 3′ cDNA end amplification
CcJ9-F4 Forward: AAGACCCAAAAACATTCGCAGAGTA 3′ RACE扩增 3′ cDNA end amplification
qR-J9 Forward: CATTCTTGTGCTTTTACCCAG
Reverse: GAGGTTTAAAGAGTCCGAGCT		 实时荧光定量PCR RT-qPCR
ACTIN Forward: CCGATCCAGACGCTGTATTT
Reverse: TGCTGATCGTATGAGCAAGG		 实时荧光定量PCR RT-qPCR

Fig. 1

Amplification of CcLhca-J9"

Fig. 2

CcLhca-J9 gene nucleotide and the encoded amino acid sequences"

Fig. 3

Multiple sequence alignment of CcLhca-J9 and homologous gene coding region"

Fig. 4

Phylogenetic tree of CcLhca-J9 protein with homologous proteins from other species (neighbor-joining method, 1 000 replicates)"

Fig. 4

Phylogenetic tree of CcLhca-J9 protein with homologous proteins from other species (neighbor-joining method, 1 000 replicates)"

Fig. 5

Secondary structure of CcLhca-J9"

Fig. 5

Secondary structure of CcLhca-J9"

Fig. 6

Homology model structure of CcLhca-J9 6x CLA (chlorophyll a) ligands were shown with different colors"

Fig. 6

Homology model structure of CcLhca-J9 6x CLA (chlorophyll a) ligands were shown with different colors"

Fig. 7

Molecular docking schema chart between caprylic acid and CcLha-J9"

Fig. 7

Molecular docking schema chart between caprylic acid and CcLha-J9"

Fig. 8

Relative expression level of CcLhca-J9 after caprylic acid treatment"

Fig. 8

Relative expression level of CcLhca-J9 after caprylic acid treatment"

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