关键词: 甘蔗, 金属硫蛋白, 重金属, 实时荧光定量PCR

Abstract:

Metallothioneins (MTs) are cysteine-rich, low-molecular-weight proteins. Plant MTs play important roles in detoxi?cation and cellular redox regulation. In this study, hydroponic experiments of sugarcane (Saccharum officinarum L. cv. Badila) were carried out to study the effects of CdCl₂, ZnSO₄, and CuCl₂ treatments on plantlet growth. And then three kinds of heavy metal content in both shoots and roots of sugarcane were detected, showing an enrichment and tolerance ability to Cd ²⁺, Zn ²⁺, and Cu ²⁺ in Badila seedlings. Three metallothionein genes, termed as ScMT1 (accession number: KJ504373), ScMT2-1-5 (accession number: MH191346), and ScMT3 (accession number: KJ5043704), were isolated from Badila. ScMT1 contained an open reading frame (ORF) of 228 bp encoding 75 amino acids residues. The first (52 bp) and second (176 bp) extrons of ScMT1 were separated by an intron (283 bp). ScMT2-1-5 contained an ORF of 246 bp encoding 81 amino acids residues. The first (64 bp), second (87 bp), and third (101 bp) extrons of ScMT2-1-5 were separated by two introns (483 bp and 853 bp). ScMT3 contained an ORF of 198 bp encoding 65 amino acids residues. The first (50 bp) and second (148 bp) extrons of ScMT3 were separated by an intron (259 bp). The deduce protein ScMT1, ScMT2-1-5, and ScMT3 were categorized into the subfamily of plant type1, type2, and type3 MTs, respectively. The expression profiles of the three genes under different heavy-metal stresses were investigated by real-time quantitative PCR (qRT-PCR) analysis. When treated with Cd ²⁺, the expression of ScMT2-1-5 was continuously and significantly up-regulated in both shoots and roots, while that of ScMT1 showed a delayed up-regulation pattern. The expression of ScMT3 showed a delayed up-regulation pattern in shoots and a trend of first raising and then suppressing in roots. The results suggested that ScMT2-1-5 might play a more active role in response to Cd ²⁺in sugarcane while ScMT3 might not, and ScMT1 is involved in the molecular responses of Cd ²⁺ stress at the later stage. When treated with Cu ²⁺, the expression of ScMT1 was continuously and significantly up-regulated, and that of ScMT2-1-5 and ScMT3 general up-regulated in shoots. As in the roots, the expression of both ScMT1 and ScMT2-1-5 showed a delayed up-regulation pattern which was significantly up-regulated at the later stage, while that of ScMT3 significantly up-regulated at the early stage. It revealed that ScMT1 cooperated with ScMT2-1-5 and ScMT3 is involved in the positive response to Cu ²⁺ stress in roots, and might play a more positive role than ScMT2-1-5 and ScMT3, and the three genes are successively involved in the molecular responses of Cu ²⁺ stress in roots. When treated with Zn ²⁺, the expression of ScMT1 and ScMT3 increased only in shoots and roots, respectively. The expression of ScMT2-1-5 was up-regulated at the early stage and then suppressed in both shoots and roots. It suggests that ScMT1 and ScMT3 mainly function in shoots and roots, respectively, when exposed to Zn ²⁺ stress. ScMT2-1-5 was involved in the molecular responses of Cd ²⁺ stress at the early stage. ScMT1, ScMT2-1-5, and ScMT3 showed similar or complementary expression patterns in different tissues of sugarcane when exposed to heavy metals, which revealed the functional diversity of sugarcane MTs in detoxi?cation and cellular redox regulation, and their spatiotemporal coordination in mitigating or even preventing tissue injury caused by excessive heavy metals. The results provide a basic information for further research on mechanisms in the synergistic enhancement of tolerance to heavy metals for MTs family genes in the highly polyploid sugarcane.

Key words: sugarcane (Saccharum officinarum L.), metallothionein, heavy metal, real-time qPCR