Cloning of early light-induced protein gene ELIP and its interaction and co-expression analysis with targeting miRNA in Jatropha curcas under low temperature

Abstract

[ Objective ] As a kind of thermophilic and chilling-sensitive plants , low temperature severely af- fected the growth , development , geographical distribution , and yield of Jatropha curcas L.. Previous studies showed that chill-hardening at 12 ℃ significantly enhanced the chilling resistance of J . curcas . The early light-inducible protein ( ELIP ) gene in J . curcas was a highly responsive gene to low tempera- ture. The study aimed to explore the role of JcELIP in response to low temperature in J . curcas , to com- prehensively understand the structure , regulatory mechanisms , evolutionary relationships of JcELIP and its interaction with miRNAs , and to provide an important candidate gene resource for subsequent molecu- lar breeding of cold resistance J . curcas . [ Methods ] This study cloned the JcELIP gene from J . curcas by RT-PCR and conducted a comprehensive bioinformatics analysis. The expression of JcELIP gene in the roots , stems , and leaves , as well as during the chill-hardening at 12 ℃ , were analyzed by RT-qPCR. The miRNAs interacting with JcELIP was identified , and a co-expression analysis was conducted during the chill-hardening at 12 ℃. [ Results ] The results showed that the complete open reading frame ( ORF ) of JcELIP was 585 bp , encoding 194 amino acids. The size of the protein was 2.04 kD with a theoretical iso- electric point of 9.59. It was a stable hydrophobic alkaline protein , with 3 hydrophobic transmembrane helices. The tertiary structure mainly consisted of α -helices and irregular coils and possessed chlorophyll a / b binding sites. Cis -acting element prediction showed that JcELIP had hormone response elements such as abscisic acid. Evolutionary analysis showed that the JcELIP from J . curcas had the highest homology with MeELIP from Manihot esculenta . RT-qPCR analysis revealed that under normal growth conditions , there was no significant difference in the expression of JcELIP in the roots , stems , and leaves of J . cur- cas . During chill-hardening at 12 ℃ , the expression of JcELIP in the leaves was quickly up-regulated , reaching 64.8 times of the control at 48 h , indicating that JcELIP was involved in the response and adap- tation to cold stress. Based on the degradome data of J . curcas , eight miRNAs , including miR390-x , miR6476-x , and novel-m0090-3p , were identified as having regulatory effects on the expression of JcELIP . Co-expression analysis showed that the expression of JcELIP was significantly negatively regu- lated by miR390-x and novel-m0090-3p during the chill-hardening at 12 ℃. [ Conclusion ] JcELIP highly responded to low temperatures stress and interacted with miRNAs , showing that it played an important role in response to low temperature stress in J . curcas .

Keywords

• jatropha curcas
• early light-inducible protein ( elip ) gene
• chill-hardening
• expression anal- ysis
• mirna interaction