STUDIES OF ANNEXIN GENE FAMILY AND RECOMBINANT ANNEXIN-1 PROTEIN OF GUAR

Abstract

Guar is an annual legume that contains guar gum in its seeds, a water-soluble polymer that acts as a stabilizing and emulsifying agent in various applications. Although guar is regarded to be a drought-resistant crop since it requires less water and can endure drought, high temperatures, and salinity stress, certain studies have shown that drought stress can damage morphology and productivity in some guar varieties, resulting in reduced yields. To improve the guar production and yield there is a need to identify more drought stress-responsive genes in guar at the molecular level to develop guar varieties with features for tolerance to drought stress. Plant annexins are Ca2+-dependent membrane-binding proteins, are involved in the growth and development and are responsible for multiple biotic and abiotic stress tolerance in various plant species. Plant annexins have calcium ion binding domains that access the detection of increased cytosolic Ca2+, allowing them to participate in various calcium and phospholipid-based stresstolerant signaling mechanisms. Plant annexins have a lower ROS scavenging capacity than traditional antioxidants, affecting the amount of ROS in mitochondria and chloroplast and acting as ROS centers in the cell. In the current study, a total of five annexin unigenes (designated as AnnCt1-AnnCt5) were identified from the transcriptome database of economic important crop guar. Out of the five guar annexin unigenes, the CDS of two guar annexin unigenes (AnnCt1 and AnnCt5) were completely cloned into the pJET1.2 blunt vector. The multiple sequence alignment of the primary amino acid sequence of AnnCt1-AnnCt5 showed similarity from a minimum 24.44 % score (between AnnCt4 & AnnCt5) to a maximum of 71.74 % score (between AnnCt1 & AnnCt2) among each other. The predicted molecular weight of detected guar annexins amino acids was from 35.22 to 37.03 kDa, with theoretical pIs ranging from 5.97 to 9.15. The analysis of guar annexin AnnCts polypeptides (except AnnCt4) revealed four annexin repeats conserving endonexin domains at first and fourth domains. Based on their phylogenetic analysis, the guar annexins have split into four groups indicates their close relationship with annexin from Glycine max and evolutionary ties with higher plants annexins. Differential expression patterns of guar annexin isoforms (AnnCts) in various organs such as leaves, roots, stems, flowers, developing seeds (30 DAF) of guar at both early and mature stages were analyzed. AnnCts predominantly expressed in the green organs such as mature leaves, shoots, and developing seeds (30DAF) suggested their possible roles such as redox regulationt. All AnnCts responded differentially towards various abiotic stress treatments such as drought (15 % (m/v) PEG-6000), salt (200 mM sodium chloride), cold (4 ͦ C), and 100 μM ABA hormone to the two weeks old guar seedlings at different time points. These findings indicate that members of AnnCts belong to a typical plant annexin gene family. The up-regulation of AnnCts transcripts under various abiotic stresses and ABA hormone in late hours suggested that they may involve in downstream stress signaling regulation. In-silico analysis of soybean annexin (evolutionary close to guar annexin) predicted interactions with 10 proteins (PHT1-12, PHT1-13, CLV1B, GlYMA05G02590.1 and GLYMA17G09270.1, GLYMA19G38800.2, GLYMA03G36140.5, GLYMA15G15171.1, GLYMA02G44951.1, and GLYMA14G03810.1 proteins) that are possibly involved in inorganic phosphate transmembrane transport activity, regulation of root and shoot growth, root nodule organogenesis and cellular processes such as RNA metabolism. To study the role of the Annexin-1 gene in guar the complete CDS, designated as AnnCt1, was isolated from the developing seeds (30DAF) tissue of guar. The primary amino acid sequence of AnnCt1 contained 951-bp nucleotides encodes a 316-amino-acids polypeptide with a predicted molecular mass of 35.74 kDa and a theoretical pI of 6.07. AnnCt1 polypeptide has shown an evolutionary relationship with sequences of annexin-1 gene from higher plants and offers a close relationship to legumes. The AnnCt1 gene transcript has increased at different times after being exposed to H2O2. The homology model, of AnnCt1, showed stable structure. The heterologous expression and purification of recombinant His-tag AnnCt1 protein in E.coli cells was performed. The fluorescence spectra of recombinant AnnCt1 protein have decreased gradually as the concentration of calcium ions was increased. The far UV-CD spectra of recombinant AnnCt1 protein have shown a prominent α-helical structure with the thermal stability of 60.02 ± 5°C temperatures. The far-UV CD spectra of recombinant AnnCt1 protein have shown more α dominant structure. We observed a very slight increase in the α-helical content of recombinant AnnCt1 protein after the addition of Ca2+ in the reaction, it is suggested that rAnnCt1 could bind to Ca2+ in-vitro. Purified rAnnCt1 protein has radical scavenging activity. The survival rates of recombinant bacterial cells expressing rAnnCt1 protein have high survival rate as compared to the control bacterial cells under TBOOH treatment. In addition, purified recombinant AnnCt1 protein has prevented the oxidation of guar gDNA caused by OH• produced by Fenton's reagent. Our study is the first study on guar annexin-1 protein, which suggests that the stress-responsive AnnCt1 gene encodes for Annexin-1 protein, could have antioxidant and Ca2+-binding characteristics in-vitro that may play an essential role in the regulation of ROS during oxidative stress responses in guar.