紫羊茅重金属抗性和敏感品种中类金属硫蛋白基因的鉴定及表达初探

PRELIMINARY STUDIES OF THE IDENTIFICATION AND EXPRESSION OF METALLOTHIONEIN-LIKE GENE IN FESTUCA RUBRA

重金属对生命机体的作用具有双重性。一方面,作为多数辅酶的辅助因子对细胞的正常代谢必不可少;另一方面,当重金属超过一定的浓度时对细胞有较大的毒性。在长期的进化过程中,生物可能形成一种调节细胞内重金属浓度的机制。这种机制在动物和真菌中被认为同金属硫蛋白(metallothionein,MT)的作用密切相关。植物中也存在类似的与重金属结合的低分子量蛋白(heavy metal binding pep-tide)。最近对拟南芥菜和水稻中类金属硫蛋白(MT-like)基因的研究证实其作用与动物MT相似。紫羊茅品种“Merlin”是一种从锌铅矿区的重金属污染地采集的单子叶草种,对镉和铜的抗性都较高,分别达到50mg/L和30mg/L,而5mg/L Cd^(2+)或2mg/L Cu^(2+)便可抑制敏感品种“S59”的正常生长。

Metallothionein (MT) is a cysteine rich protein related to heavy metal sequestering in animal and fungal cell systems. Festuca rubra cv. 'Merlin' is a monocotyledonous grass species isolated from a zinc and lead mine area. Laboratory studies had demonstrated that 'Merlin' could tolerate cadmium and copper up to 50 mg/L and 30 mg/L, respectively. On the contrary, just 5 mg/L Cd2+ and 2 mg/L Cu2+ already inhibited the growth of the sensitive cultivar, Festuca rubra 'S59' . Molecular understanding on the metal tolerant ability of 'Merlin' was studied. Based on the conserved region of plant MT-like genes, a pair of primers was synthesized as follows: 5'-GGAT-CAAGCTGCGGCTGCGGCTCAA-3' and 5'-GCAGTTGCAAGGGTCGCACTTGCAG-3'. Using PCR, a fragment of 418 bp was amplified with this pair of primers from both the 'Merlin' and 'S59' ge-nomic DNA. Sequence analysis showed that the MT-like gene of the two cultivars differed by only 5 base pairs, sharing a 98 .8 % homology. Amino acid sequence deduced from the amplified fragment also demonstrated a high homology (60% -75%) to the other known higher plant MT-like proteins and shared the similar structural characteristics of cys-Xaa-cys regions at both end of the sequence. Northern blot analysis on the total RNA showed that the expression of the MT-like gene in the shoots of 'Merlin' was enhanced when treated with both 20 mg/L Cu2+ and 20 mg/L Cd2+ . In shoots of 'S59' the expression of the MT-like gene was enhanced by 20 mg/L Cu2+ but no effect was observed on the expression upon Cd2+ treatment. These evidences suggested that MT-like gene was present in both 'Merlin' and 'S59' cultivars of Festuca rubra; the expression of the MT-like gene was induced by heavy metal stress, and the expression pattern was different between the tolerant ('Merlin') and the sensitive ('S59') cultivars. Currently, the cloning of the whole gene from the cDNA and genomic library has been under-going. This would probably give a more profound and better understanding of the molecular mechanism on metal tolerance in Festuca .