How does the IgWRKY2 gene respond to Pb stress induction in German iris?
Foreword: Heavy metals in the soil are non-degradable, and when the heavy metal lead is concentrated at low concentrations, it will cause damage to some tissues and functions of plants, which will affect the growth and development, biochemical and molecular levels of plants, and can also directly or indirectly cause harm to the human body through the enrichment of the food chain, so it is very important to control the problem of lead pollution in the external environment.
German iris is a perennial root flower of the iris family, which has good ornamentality, strong resistance to stress, and strong resistance to toxic gases, and can be used as a greening plant in industrial areas.
As a DNA-binding protein widely present in plants, WRKY transcription factors not only play an important role in the growth and development of plants, but also participate in the regulation of plant resistance to biotic and abiotic stresses.
The expression levels of 55 ZjWRKY genes in jujube changed under high temperature stress, the GhWRKY4 and GhWRKY5 genes of cotton were involved in the stress responses such as drought and salt stress, and the CiWRKY75-1 gene of Caragana mesophyllum decreased the tolerance of plants under drought stress.
In this paper, the cDNA of the leaves of the German iris "Sakura Garden" was used as a template for PCR amplification, and the length of the amplified fragment was about 570 bp by agarose gel electrophoresis.
The sequencing results showed that it was completely consistent with the transcriptome sequencing sequence, and the CDS sequence of the amplified IgWRKY2 gene was analyzed by open reading frame, and the complete open reading frame of IgWRKY2 was up to 534 bp, encoding 117 amino acids, and the WRKY family-specific sequence WRKYGQK sequence was found at 41 amino acid sequences.
The results showed that the molecular formula of the protein encoded by IgWRKY2 gene was C579H882N172O186S3, the theoretical relative molecular weight was 13324.59 Da, and the theoretical isoelectric point was 6.42.
The protein had the largest number of proline (Pro) and serine (Ser), the sum of negatively charged residues (Asp+Glu) was 17, the sum of positively charged residues (Arg+Lys) was 15, and the unstable parameters were 46.32 and greater than 40, which was an unstable protein.
The lipid solubility index of the protein was 47.88, and the average hydrophilicity was -1.239, which was a hydrophilic protein, and the subcellular localization prediction showed that IgWRKY2 was located in the nucleus, and the transmembrane structure prediction showed that IgWRKY2 did not have a transmembrane structure and was an extracellular protein.
The results of secondary protein structure prediction showed that IgWRKY2 was composed of 5.93% α-helix, 6.78% β-turn, 15.25% extended chain and 72.03% irregular coil, and the BLAST alignment analysis results showed that IgWRKY2 had high homology with 11 plant proteins, including EgWRKY2 from oil palm and PdWRKY2 from jujube.
The amino acid sequences encoded by IgWRKY2 in German iris were homologous compared with the 11 amino acid sequences screened, and the results showed that the gene sequences of PdWRKY2 from jujube and EgWRKY2 from oil palm were highly similar, and the lowest gene sequence consistency was CeWRKY2 from Coffea oides.
The genes from these plants were structurally similar to IgWRKY2 and contained a WRKY domain, which further indicated that the IgWRKY2 gene belonged to the WRKY transcription factor family.
The results showed that IgWRKY2 was the same branch as InWRKY2 of morning glory, ItWRKY2 of trifid potato and NtWRKY2 of tobaccous fluffy tobacco was closely related.
In order to study the expression of IgWRKY2 gene in German iris under Pb stress, plant materials were subjected to Pb2+ solution for 0h, 6h, 12h, 24h and 48h, and RNA was extracted from leaves and rhizome tissues at different stress time points, and real-time PCR analysis was performed after reverse transcription.
The results showed that IgWRKY2 gene was expressed in both rhizomes and leaves of "Sakura Garden", and the expression level in rhizome tissues was more obvious than that in leaf tissues.
When the leaf tissue was stressed, the expression level of IgWRKY2 gene was the highest at 6 h, which was 1.42 times that of the control, and the expression level began to decrease at 12 h, and increased briefly at 24 h, but the expression level was still lower than the control value, indicating that the gene was inhibited in the subsequent expression.
When the rhizome tissue was stressed, the expression level of IgWRKY2 gene first decreased and then increased, indicating that the response degree of IgWRKY2 gene in rhizome tissue was higher than that in leaf tissue.
Under Pb stress, the IgWRKY2 gene responded to different degrees in leaves and rhizomes, and the response of rhizome tissues was more obvious, and it was speculated that the ability of rhizomes to respond to Pb was stronger than that of leaves when they were subjected to Pb stress.
Conclusion:
The expression of IgWRKY2 gene in German iris was induced by Pb stress, and the expression level in rhizomes was more obvious than that in leaf tissues, which may be related to the fact that rhizomes are in direct contact with the stress environment, indicating that this gene plays a certain regulatory role in the process of plant resistance to heavy metal stress, but the specific regulatory mechanism needs to be further studied.
