JIA-2019-11

2506 LI Li-shu et al. Journal of Integrative Agriculture 2019, 18(11): 2505–2513 are involved in responses to abiotic stresses, such as high temperature, osmotic pressure, drought, salt damage, and nutrient stress tolerance; moreover, eIF4E and eIF4G have significant effects against virus infection (Dong and Zhang 2006; Dutt et al. 2015). eIF1A plays a critical role in protein synthesis initiation. It is necessary for eIF1A to form the 43S preinitiation complex by the binding of eIF2/GTP/Met-tRNAiMet and 40S ribosomal subunits. eIF1A can promote mRNAbinding and prevent the 40S ribosomal subunit from associating with the 60S ribosomal subunit and stabilize eIF5B binding to small ribosomal subunits (Battiste et al . 2000; Kwon et al . 2007; Passmore et al . 2007; Yu et al. 2009; Grunwald et al . 2013; Nanda et al . 2013). eIF1 ( 1A ) is not only closely related to protein synthesis (Chaudhuri and Maitra 1997; Li et al. 2014) but also responds to various abiotic stresses, such as high temperature, salt, drought, oxidation, and nutrition in plants (Latha et al. 2004; Diédhiou et al. 2008; Rangan et al. 2009; Sun and Hong 2013; Sun et al. 2015). For example, overexpression of sugar beet eIF1A ( BveIF1A ) significantly enhanced sodium and lithium salt tolerance in yeast and Arabidopsis thaliana , showing that BveIF1A has a strong effect on sodium stress tolerance (Rausell et al. 2003). Tamarix hispida eIF1A ( TheIF1A ) can improve plant salt and osmotic tolerances and alleviate cell damage by regulating the activities of enzymes and scavenging harmful reactive oxygen species (Yang et al. 2017). The expression of OseIF1 in rice decreased the Na + and Cl – concentrations, regulating the H + -ATPase level and maintaining photosynthesis in the leaves, which significantly enhanced the salt tolerance of rice (Latha et al. 2004). Overexpression of LceIF1A increased the salt tolerance of Leymus chinensis (Sun and Hong 2013). In addition, eIF1 , eIF1A and eIF1B can strengthen the induction of salt damage in rice and up-regulate the effects of treatment with abscisic acid (ABA) and mannitol (Latha et al. 2004; Rangan et al. 2009), indicating that the eIF1 family could represent a standard mechanism in stress tolerance in plants (Sun et al. 2015). Therefore, eIF1 (1A) is an important factor in stress resistance engineering in plants. Mango ( Mangifera indica L.), known as the king of tropical fruits, is one of the major economic fruits in the tropical and subtropical regions of the world. However, during growth and development, mango may encounter a variety of abiotic stresses, such as low temperature, high temperature, drought, salt, and heavy metals, which affect its quality and productivity or even result in no productivity (Rao et al. 2016). Some studies of abiotic stresses in mango have been reported. For instance, under abiotic stresses, the expression patterns of nine mango stress related genes were analyzed by qRT-PCR (Luo et al. 2014). MiRab5 and MiASR responded to adverse stresses (Liu et al. 2014; Luo et al. 2014). However, few studies on eIF1A in mango have been reported. In a previous study, we obtained two MieIF1A genes from Oligo-dT-anchored cDNA-start codon-targeted marker (SCoT) differential display under abiotic stress (Luo et al. 2014). In the present study, we further analyzed biological information and expression of MieIF1A-b gene in different tissues and under abiotic stress treatment. We also performed function analysis by the overexpression of MieIF1A-b in transgenic A . thaliana . Our results indicated that the MieIF1A-b may be correlated with the fruit development and salt adaptation in mango. 2. Materials and methods 2.1. Plant materials and stress treatments The samples of the 12-year-old M . indica cv . Siji, including young and old leaves, young and old stems, flowers, and fruits (10, 20, 30, 40, 50, 60, 70, and 80 d after flowering), were collected in the orchard at Guangxi University, Nanning City, Guangxi Zhuang Autonomous Region, China. The plants of 1 year old (grafted M . indica L. cv . Siji plants ) were conducted by 300 mol L –1 NaCl, 300 g L –1 PEG–6000 and 4°C. Then the leaves were collected after 0, 12, 24, 36, 48, 72, and 96 h; The collected sample were sprayed on 0.1 mmol L –1 ABA, 1 mmol L –1 SA, and 1 mmol L –1 H 2 O 2 after 0, 1, 2, 4, 8, 12, and 24 h treatments. The above treatments were repeated 3 times. The samples were immediately frozen in liquid nitrogen and stored at –80°C for RNA isolation. 2.2. RNA isolation and cDNA synthesis Total RNA from mango samples was isolated according to the RNA Rapid Extraction Kit for polysaccharide polyphenol plants (Huayueyang Biotechnology, Beijing, China). RNA integrity was determined by 1% agarose gel electrophoresis and spectrophotometry. Well-integrated total RNA was treated with RNase-free DNase to remove genomic DNA. High-quality total RNA from the samples was used to synthesize first-strand cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase and the reverse transcription primer ancient ubiquitous protein 1 (AUP1) according to the manufacturer’s instructions (TaKaRa, Dalian, China). 2.3. MieIF1A-b gene cloning and sequence analysis The upstream and downstream primers MieIF1A-b-F and MieIF1A-b-R, respectively (Table 1), were designed to clone the MieIF1A-b full-length sequence and to verify the reliability of the sequence. Each 25-μLPCR reaction mixture contained 2.5 μL 10× buffer (with Mg 2+ plus), 0.5 μL dNTP