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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 402-413.doi: 10.3724/SP.J.1006.2023.24031

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Role of calmodulin gene (HcCaM7) and its protein acetylation is involved in kenaf response to abiotic stress

HUANG Zhen1(), WU Qi-Jing1, CHEN Can-Ni1, WU Xia1, CAO Shan1, ZHANG Hui1, YUE Jiao1, HU Ya-Li1, LUO Deng-Jie1, LI Yun1, LIAO Chang-Jun3, LI Ru2, CHEN Peng1,*()   

  1. 1College of Agriculture, Guangxi University / Key Laboratory of Plant Genetics and Breeding, Nanning 530004, Guangxi, China
    2College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, China
    3Guangxi Bossco Environmental Protection Technology, Nanning 530007, Guangxi, China
  • Received:2022-01-25 Accepted:2022-06-07 Online:2022-07-04 Published:2022-07-04
  • Contact: CHEN Peng E-mail:549935163@qq.com;hustwell@gmail.com
  • Supported by:
    National Natural Science Foundation of China(31960368);China Agriculture Research System of MOF and MARA(CARS-16-E14);Guangxi Bossco Environmental Protection Technology Co., Ltd.(GXU-BFY-2020-015)

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Abstract:

Calmodulin (CaM) is a kind of calcium-dependent regulatory proteins involved in plant growth and development, stress tolerance, and other biological processes. In the previous study in kenaf acetylome, our team found that the protein acetylation modification calmodulin protein7 (CaM7) was involved in the regulation pollen development in kenaf. In order to explore its specific mechanism, we cloned the calmodulin gene HcCaM7 from kenaf P3B binuclear anther by using the PCR cloning way. Its maximum open reading frame (ORF) was 450 bp, encoding a protein containing 149 amino acids with a molecular weight of 16.85 kD. Subcellular localization revealed that HcCaM7 was mainly located in cytoplasm and cell membrane. Silencing HcCaM7 by virus induced gene silencing technique caused growth inhibition in kenaf. Furthermore, the protein HcCaM7mut with acetylation modification site was successfully obtained in vitro and the expression of HcCaM7 without acetylation modification was successfully induced. Acetylation of HcCaM7 protein significantly promoted NADK (NAD kinase) activity, indicating HcCaM7 acetylation involved in its functional regulation. The recombinant bacteria containing HcCaM7 protein and HcCaM7mut protein were detected in NaCl (400 mmol L-1 and 500 mmol L-1 NaCl), drought (400 mmol L-1 and 600 mmol L-1 mannitol) and heavy metals (30 μmol L-1 and 50 μmol L-1) by dot plate method. The results showed that the survival rate of recombinant bacteria containing HcCaM7 protein was significantly higher than that of empty control bacteria, in addition, the survival rate of recombinant bacteria containing acetylated HcCaM7mut protein was further improved. The results indicated that HcCaM7 protein could enhance the abiotic stress resistance of E.coli, and the effect of acetylation modification was better. Therefore, HcCaM7 gene regulated the growth and development of kenaf and abiotic stress resistance, and acetylation modification could promote the role of HcCaM7 protein.

Key words: kenaf, calmodulin protein 7, protein acetylation modification, virus induced gene silencing, gene codon extension technology, abiotic stress

Table 1

Primers used in the study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
HcCaM7-F ATGGCCGATCAGCTCACC
HcCaM7-R CTTGGCCATCATCACTTTGA
HcCaM7-GFP-F CAGGATATCCAGATCCAGTGGGATCCATGGCCGATCAGCTCACC
HcCaM7-GFP-R TAAGCTTGGTACCGAGCTCACCCGGGGATCCCTTGGCCATCATCACTTTGA
HcCaM7- pTRV2-F GATTCTGTGAGTAAGGTTACCGAATTCGGCGGAACTCCAAGATATGA
HcCaM7- pTRV2-R CCCCATGGAGGCCTTCTAGAGAATTCACCATCAACATCGGCTTCAC
HcCaM7mut-F GGTGTTTGACtAGGATCAGAATGGTTTCATATCTGC
HcCaM7mut-R GATCCTaGTCAAACACCCTGAATGCCTCTTTA
HcCaM7-pET32α-F AAGCTTGTCGACGGAGCTC GAATTCCTTGGCCATCATCACTTTGA
HcCaM7-pET32α-R CATGGCTGATATCGGATCC GAATTCATGGCCGATCAGCTCACC
HcCaM7-qPCR-F GATGCTGATGGAAACGGG
HcCaM7-qPCR-R CCATCACCATCAACATCGG

Fig. 1

Cloning and relative expression analysis of HcCaM7 A: PCR amplification product of HcCaM7; M: DL2000 marker; 1: HcCaM7 ORF amplification product. B: expression analysis of HcCaM7 gene in different tissues of kenaf P3A and P3B. The error bar represents ± SD of three biological replication. Different lowercase letters are significantly different at the 0.05 probability level."

Fig. 2

Bioinformatics of HcCaM7 A: the prediction of conserved domains of HcCaM7; B: the predicted 3D structure of HcCaM7 protein; C: the phylogenetic tree of HcCaM7 proteins."

Fig. 3

Subcellular localization of HcCaM7 protein A: Mcherry channel; B: GFP channel; C: bright channel; D: merge channel."

Fig. 4

VIGS silencing of HcCaM7 A: the qRT-PCR of transgenic plants. The control plant of empty vector is pTRV2. 1-9 represents gene silencing plants of HcCaM7. B: phenotypic characters of HcCaM7 silencing plants; C: the fresh weight of kenaf CaM7-pTRV2; D: plant height of kenaf CaM7-pTRV2. Values are means ± SDs of three biological replication. Different lowercase letters are significantly different at the 0.05 probability level."

Fig. 5

Quantitative expression analysis of HcCaM7 and its protein in kenaf anther of P3A and P3B A: the relative expression levels of HcCaM7 genes in kenaf P3A and P3B anthers; B: the relative expression level of HcCaM7 protein in the anthers of P3A and P3B of kenaf, 1 and 2 represent the anther of P3A and P3B, respectively. Values are means ± SDs with three biological replication. Different lowercase letters are significantly different at the 0.05 probability level."

Fig. 6

Site-directed mutation of HcCaM7 protein acetylation modification site"

Fig. 7

Purification results of HcCaM7 protein and its point mutant protein and detection of His label and acetylation modification A: the purification results of HcCaM7mut protein; B: the purification results of HcCaM7 protein; C: His-Tag Western-blot detection results, 1 is HcCaM7mut protein, 2 is HcCaM7 protein; D is the acetylation modification test result, 1 is HcCaM7mut protein and 2 is HcCaM7 protein."

Fig. 8

Effect of HcCaM7 acetylation modification on NADK activity Values are means ± SDs with three biological replication. Different lowercase letters are significantly different at the 0.05 probability level."

Fig. 9

Detection of HcCaM7 resistance to abiotic stress in Ecoli A: salt stress; B: mannitol stress; C: CdCl2 stress; D: cold stress; a, b, c, d: the colony survival number diluted 104 times after cold stress. Values are means ± SDs with three biological replication. Different lowercase letters are significantly different at the 0.05 probability level."

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