Cloning And Function Analysis Of A Type 2 Metallothionein Gene (LpMT2) In Lilium Pumilum

Lilium pumilumis a perennial bulbous herbaceous flower of the genus Lilium in Liliaceae,with large flower and beautiful color.It not only has high ornamental value,but also has strong resistance,so it can be used as an important parent of lily resistance breeding.However,studies on the resistance genes of L.pumilum were rarely reported.Metallothionein?MT?is a kind of non-enzymatic protein with low molecular mass,rich cysteine and metalion,which is related to the mechanism of resistance to salt and alkali stress,antioxidant stress and free radical scavenging.In this study,a 234bp long MT2 gene encoding 77 amino acids was cloned by PCR using cDNA of L pumilum leaves as template,and its expression was analyzed by quantitative real-time PCR?QRT-PCR?.The plant expression vector pBI121-LpMT2 was constructed and transformed into L.pumilum the resistance of LpMT2 gene overexpressing plants was further analyzed.The main results are as follows:?1?The MT2 gene isolated from L.pumilum has a Metallothio₂ domain and belongs to the Metallothio₂ superfamily.The results of amino acid comparison showed that LpMT2 had high homology with Dendrobium catenatum DcMT2,Actinidia chinensis var.chinensis AcMT2,Glycine max GmMT2 and Arabidopsis thaliana AtMT2a,it is speculated that its functions in response to stress and other aspects are similar to them.The results of subcellular localization prediction showed that LpMT2 mainly existed in cytoplasm.?2?QRT-PCR was used to analyze the expression pattern of LpMT2 gene under different organs and stress:the LpMT2 gene has the highest expression in flowers,followed by mature leaves,young leaves,seeds,bulbs and lowest in roots.The expression of LpMT2 gene was significantly induced under salt stress and oxidative stress.?3?The plant expression vector pBI121-LpMT2 was constructed and transferred into the bulbs of L.pumilum by Agrobacterium-mediated.The resistant plants were screened by kanamycin?Kana?and identified by Northern blot,the results showed that the gene overexpressing plants were successfully obtained.?4?The wild type and LpMT2 gene overexpressing L.pumilum were treated with different concentrations of NaCl,NaHCO3 and H2O2 for 48 h.The determination of physiological indexes showed that the LpAMT2 gene overexpressing plants had higher chlorophyll and proline content,lower malondialdehyde content,smaller cell membrane permeability and higher superoxide dismutase activity,which indicated that the LpMT2 gene enhanced plant resistance to stress.?5?The wild type and overexpressing LpMT2 gene L.pumilum were treated with different concentrations of NaCl and NaHCO3 for 48h,and the Na+and K+ion content were compared.The results showed that had lower Na+content and higher K+content.Treated with 200 mM NaCl and 20 mM NaHCO3 for 48h,the Na+efflux rate of the LpMT2 gene overexpressing plants was higher while the K+efflux rate was lower.It is indicated that under salt stress,the LpMT2 gene overexpressing plants can adapt to salt stress better by increasing the efflux rate of Na+and decreasing the efflux rate of K+to maintain a lower Na+/K+ratio.