Effects Of Phytochelatin-like Gene On The Endurence And Enrichment Of Cd²⁺ In Tobacco

With the aggravation of environmental pollution caused by human production activities,the heavy metal pollution is increasing the damage to animals and plants.The treatment of heavy metal pollution in the environment is an urgent problem that needs to be solved.In China's agricultural production activities,due to the use of various inappropriate,unreasonable and unsustainable methods to increase the output of plants,agricultural soils all over the country have been contaminated by heavy metals of varying degrees,which restricts the development of agricultural economy.In the process of remediation of heavy metal pollution in soil,it mainly relies on physical,chemical and biological remediation technologies to solve the problem of remediation of heavy metal pollution in soil.Compared with physical and chemical remediation technologies,bioremediation technology has low cost,easy operation,and good repair effect.The advantages are very safe and effective ways to repair heavy metal pollution in soil.In recent years,bioremediation technology,as a new technology,has played an important role in the restoration of heavy metal pollution in soil.In the treatment of soil pollution,the use of genetic engineering techniques to cultivate high-enriched heavy metal-transgenic plants can achieve better effects than ordinary hyperaccumulators in removing soil heavy metal pollution.It is of great value to the treatment of soil pollution and improvement of agricultural production.Metallothionein?MT?can respond to heavy metal stress and combine with heavy metal ions to exert metal enrichment and thus reduce the toxic effects of heavy metals.Phytochelatins?PCs?are the class III metallothioneins in plants.They are low molecular weight polypeptides,and they are rich in cysteine residues.They can bind to metal ions and affect the physiological metabolism in plants.process.Compared with other types of metallothioneins,the biosynthesis of phytochelatins is not caused by gene coding but catalyzed by phytochelatin synthase?PCS?and synthesized using glutathione?GSH?as a substrate,including multiple enzymatic reactions.The chemical formula of the phytochelatin is a mixture of??-Glu-Cys?n-Gly?n=2-11?and therefore is influenced by many factors during synthesis.Phytochelatin-like?PCL?is a peptide designed by our laboratory and encoded by a gene encoding the amino acid sequence of natural phytochelatin??-Glu-Cys?₁₁-Gly.That is,Met-??-Glu-Cys?₁₁-Gly.In this study,the gene encoding this phytochelatin was controlled by CaMV 35S,a promoter of 35S coat protein gene of cauliflower mosaic virus,and transformed into tobacco stemless tobacco by Agrobacterium-mediated transformation.The accumulation of heavy metal cadmium(Cd²⁺)in order to obtain highly enriched Cd²⁺transgenic tobacco plants will lay a foundation for the future bioremediation of heavy metal contaminated soils such as Cd²⁺.The main results are as follows:1.Construction of vector pVCT2383 containing 35S-?::PCLThe pBK-35S-?-HsMT1L and pGFP-PCL vectors were digested respectively to recover the target fragment,ligated with T4 ligase,and the ligation product was transformed into E.coli competent cells to obtain the vector pVCT2383 having35S-?::PCL.2.Transgenic tobacco homozygous lines with high expression of PCL geneThe vector was introduced into tobacco by Agrobacterium-mediated method to obtain a Kan-resistant plant,and a transgenic independent strain integrated with the PCL gene was identified by PCR.The results of qRT-PCR analysis showed that PCL gene expression was different in different transgenic lines.Three independent strains with relatively high PCL gene expression were screened out and homozygous for the transgene was obtained from the T₂ generation.3.Transgenic tobacco with PCL gene has high tolerance to Cd²⁺stressAfter transplating the PCL gene and wild-type tobacco seedlings in Cd²⁺medium,it was found that the effect of cadmium on the growth of roots was especially obvious.The roots of wild-type tobacco were more inhibited than the roots of transgenic tobacco.Under the condition of hydroponic cultivation with Cd²⁺,the integrity of leaves of transgenic tobacco seedlings was better,and healthy new leaves could be grown.The overall growth was better than that of wild type tobacco.These results indicate that transgenic tobaccos have higher tolerance to Cd²⁺stress than wild-type tobaccos.4.PCL gene enhances the ability of tobacco to enrich Cd²⁺Under the Cd²⁺stress,the cadmium uptake of transgenic tobacco plants was much higher than that of wild tobacco plants,and the root enrichment was very significant.5.The PCL gene enhances the antioxidant capacity of Cd²⁺stress in tobaccoTobacco plants were treated with different concentrations of Cd²⁺for 7 days.The physiological parameters such as MDA content,CAT activity,SOD activity,POD activity,hydrogen peroxide content and DAB chemical tissue staining were determined.It was confirmed that PCL gene expression in transgenic tobacco could increase the antioxidant ability under heavy metal toxicity stress.In summary,the expression of PCL gene enhances the tolerance and enrichment ability of tobacco to heavy metal cadmium,and can be used as a transgenic plant for phytoremediation of soil heavy metal pollution in the removal of heavy metal Cd²⁺.