Garlic, And The Stumbling Blades Of Grass In The Heavy Metal Resistance Molecular Mechanism Study

Feng Baomin (Plant Molecular Biology) Directed by Professor Ma MiThe increasingly severe heavy metal pollution constitutes a great threat to global environment and human health. Revealing the heavy metal tolerance mechanism at molecular level together with it's application on phytoremediation and green agriculture has become a growing point of the crossed fields of plant, environment and agriculture sciences. In order to understand the molecular mechanism of heavy metal tolerance, our work was focused on the isolation of heavy metal tolerance genes from tolerant plant Allium sativum L. and Cynodon dactylon. The expression pattern of these genes and their respective role in heavy metal tolerance were also of our interest.In higher plants, some evidences implied that both metallothionein like(MT Like),a kind of Cystein-rich, low molecular weight protein, and phytochelatins(PCs), which had the special structure of (γ-Glu-Cys ) n-Gly, took important roles in heavy metal tolerance. However, little was known about the relative role of these two chelates' function in one plant. At the same time, there was no reference reporting the expression pattern of MT Like and PCs synthase in one plant, such as the metal ion specificity and the space-time expression pattern.In this thesis, the MT Like cDNA(GenBank Accesion No.AY050510) from A. sativum L. was isolated using RACE method. PCR detection and Southern bolt analysis revealed that the MT Like genes were of a gene family which existed in garlic genome. Sequencing and homology analysis showed that the MT Like cDNA from A.sativum L.(AsMT) contained a complete open reading frame(ORF), which encoded a peptide of 73 amino acids. There were 12 Cys residues in this peptide(16.4% of the total residue number). The homology of this protein and other MT Like from wheat, rice, and festuca was significant: 89%. There were the typical motifs, Cys-Xaa-Cys, in this protein that seemed to belong to the Type 1 MT Like. The AsMT with this structure had the potential to bind metal ion. Under heavy metal stress, this gene was induced by both Cu2+ and Cd2+, which implied that AsMT took an important role in heavy metal tolerance. Now, we are carrying out research to make clear the heavy metal accumulation and distribution pattern in A. sativum L. with the electron microscopy. At the same time, in situ hybridization was used to detect the expression of AsMT at the tissue level.PCs were also important in heavy metal tolerance. However, they were not translated peptide but the products of an enzyme catalyzed pathway. This enzyme wasnamed as phytochelatins synthase(PCS). Now the genes encoding this enzyme have been isolated from Triticum aestivum, Arabidopsis and Schizosaccharomyces pombe. But it was difficult to clone this gene from A.sativm L. due to limited sequences and low sequence conservation among them. We designed a pair of degeneracy primers according to the PCS gene and amplified a cDNA(345bp) from A.sativm L.. Sequence analysis and the deduced amino acids sequence comparison showed that this cDNA encoded a peptide with very high homology to other PCS. So we concluded that this cDNA was the partial sequence of PCS gene from A.sativm L. (GenBank Accession No.AF384110). Now we are amplifying the complete sequence of this PCS gene and revealing the expression pattern of both AsMT and PCS in A.sativm L..On the other hand, we tried to isolate heavy metal tolerance genes with novel function in higher plant Cynodon dactylon.. The functional complementation of a yeast mutant strain M379/8, which was sensitive to heavy metal, Cd, was carried out. First of all, we constructed a plasmid cDNA library for expression in yeast. We tried different kinds of yeast transformation protocols to set up a high efficiency yeast transformation system. In the following works we will change the mutant strain as a receptor and improve the vector as well.