Cloning And Biology Function Research Of Metallothionein Genes From Tamarix Androssowii And Puccinellia Tenuiflora

Heavy metal pollution in soil has been a serious problem and a matter of great urgency to resolve in our country. Studying the heavy metal tolerance mechanism of plant, cloning the related genes and improving plant ability of heavy metal tolerance by genetic engineering technique have become a valid path to decontaminate the polluted soil. Metallothionein (MT) is a kind of low molecular, cysteine-rich and heavy metal-binding protein. MT already caused the people to value highly in controlling the soil heavy metal pollution.Two MT genes were cloned from Tamarix androssowii cDNA library and PuccineUia tenuiflora cDNA library using bioinformatics technology. Results of sequence analysis revealed two MT genes were the member of plant MT family.The MT gene from T. androssowii was cloned into the plant express vector pROKâ…¡. The recombinant plasmid was transformed into tobacco by Agrobacterium mediated genetic transformation. 75 kanamycin-resistant buds were obtained in study, the transform efficiency was 78 percent. We selected 10 excellent lines to PCR test. All the lines were positive in PCR. In PCR-Southern blotting and Northern blotting hybridization, we checked 6 lines selected from 10 transgenic lines randomly. The results demonstrated that foreign target gene had been integrated into tobacco genome already and could be expressed in tobacco.The MT genes from T. androssowii and P. tenuiflora were cloned into the plant express vector pROKâ…¡. The recombinant plasmid was transformed into tobacco by Agrobacterium mediated genetic transformation. 83 kanamycin-resistant buds were obtained in study, the transform efficiency was 75 percent. We selected 10 excellent lines to PCR test. All the lines were positive in PCR. In PCR-Southern blotting hybridization, we checked 7 lines selected from 10 transgenic lines randomly. The result demonstrated that two MT genes had been integrated into tobacco genome already. In Northern blotting hybridization, we checked 5 lines selected from 7 transgenic lines randomly. The test showed that two MT genes could be expressed in tobacco.Rooting rate, the average gain and plant height were analyzed after the transgenic tobacco lines To generation and control line were subjected to Cd and Cu stress treatment: The results showed: the Cd and Cu tolerance of transgenic tobacco lines were higher than control. Under CdC12 stress the control line could live in the medium containing 100/zmol.L^-1 CdCl2; The transgenic tobacco lines with MT gene from T. androssowii could live in the medium containing 200/zmol·L^-1 CdCl2; But the transgenic tobacco lines with MT genes from T. androssowii and P. tenuiflora could live in the medium containing 300/μmol·L^-1 CdCl2. Under CuSO4 stress three kinds of tobacco lines could grow at the concentration of 75/μmol·L^-1; At the concentration of 150μmol·L^-, the control line couldn't live and the transgenic tobacco lines with MT genes from T. androssowii and P tenuiflora could grow better than the transgenic tobacco lines with MT gene from T. androssowii. At the concentration of 225μmol·L^-1, the transgenic tobacco lines with MT genes from T. androssowii and P. tenuiflora couldn't live but the growth had increased. The above results confirmed that the MT gene can enhance the Cd and Cu tolerance of transgenic tobacco lines. The tolerance of transgenic tobacco lines to Cd was higher to Cu, and the tolerance of transgenic tobacco lines with two MT genes to Cd and Cu was higher than that with one MT gene.We transplanted transgenic tobacco lines To generation in the greenhouse and obtained T1 generation seeds. We growed them on culture medium of MS at the concentration of 100 mg.L^-1 kanamycin. After 20 days, we count numbers of green plants and white plants. The results showed: the ratios of T₁-5, T₁-8, T₁'-2 and T₁'-3 were accord with (2²-1) : 1, the ratios of T₁-6 and T 1'-9 were accord with (2⁴-1) : 1.We selected 8 transgenic tobacco lines T₁ generation to PCR test, all the lines were positive in PCR. The results demonstrated that foreign target genes still had been integrated into tobacco genome.We studied the growth condition of 8 transgenic tobacco lines T₁ generation and control line under different Cd concentration. The results showed: the transgenic tobacco lines were better than control line in root length, root quantity, the average gain and leave quantity under Cd stress. The transgenic tobacco lines with MT gene from T. androssowii lived well at the concentration of 100μmol.L¹ CdCl2, weighed 3.07 times of the control, and were root length 1.96 times of the control. The transgenic tobacco lines with MT genes from T. androssowii and P. tenuiflora could live at the concentration of 200,μmol·L^-1 CdCl2, weighed 3.85 times of the control, and were root length 2.74 times of the control. While the control hardly rooted at the concentration of 100μmol·L^-1 CdCl2. All the results proved that the transgenic tobacco lines T₁ generation with MT genes have the genetic stability, and the expression of MT genes could enhance the resistence of the transgenic tobacco to Cd.