Study On The Function Of PCS Genes And Transgenic Technology In Mulberry Plants

Phytochelatin is a kind of peptide compound containing cysteine. It exists in most higher plants, some animals and fungus. Because containing Cys, it can form complex through the thiol with metal, which enhances the tolerance of heavy metals by plants, relieves or reduces the toxicity of heavy metals to plants and maintains the relative stability of metal ion concentration in cell’s internal and external environment.The structure of phytochelatin usually take gamma-Glu-Cys dipeptide as repeat unit and ends with Gly in its C-terminal. Its basic structure is (gamma-Glu-Cys) n-Gly, in which n usually changes betwwen 2 and 5, and is up to 11. Phytochelatin is synthesized using glutathione (GSH) as substrate under the catalysis of phytochelatin synthase.Along with the increase of heavy metal pollution around the globe, alleviating heavy metal pollution through phytoremediation attracts more attention.So far, phytochelatin synthase (PCS) genes have been found in various plants, animals and fungus.The study shows that PCS plays an important role in improving the tolerance and accumulation of heavy metal. Although the studies in PCS gradually increase, but they are mainly concentrated in the herbaceous and grasses plant. The research in woody plants which has strong tolerance of heavy metals has not yet been reported.Mulberry (Morus L.) is a kind of important forest tree species in our country, It has strong adaptability to the harsh natural environment and has many characteristics, such as dry resistance, salt alkali tolerance, barren resistance, strong vitality, fast-growing, and so on. Studies have found that the uptakes of mulberry for cadmium and lead in the atmosphere were more and the mulberry leaves affected by heavy metals could continue to feed silkworm and had no effect on the individual indicators of silkworm and silk. As you see that mulberry exist great potential in solving and alleviating heavy metal pollution of soil and atmospheric. However, studies on PCS in mulberry and even woody plants were still not reported. Study on mulberry PCS can not only perfect the application of the PCS gene in improving the tolerance and accumulation of heavy metalfor further exploring and improving the detoxification mechanism of phytochelatins, but also can establish a good foundation for germplasm resource selection and ecological economic value in mulberry, which provide important basis for mulberry as a kind of greening tree species that can effective solve and alleviate heavy metal pollution.Transgenic technology has attracted much attention in research of gene function and material innovation etc.. It only succeed in several mulberry varieties,such as Indian mulberry,which have strong ability of regeneration and Morus alba Linne).Because of its poor repeatability, currently only a few research groups have made a breakthrough. It is difficult to effectively apply tofunctional genomics research and material innovation in mulberry. Therefore it is in urgent need of an effective transgenic technology in mulberry for related research.In this research, we identified phytochelatin synthase genes in M. notabilis data base by using bioinformatic methods and made related bioinformatics analysis. At the same time, we investigated the expression pattern of Gui you 62 mulberry by qRT-PCR under heavy metals stress. Then we made the corresponding functional study of PCS gene in the model plant tobacco. Through the method of mulberry buds injection, we explored the mulberry transgenic technology.The main results of this research were as follows:1. Identification and bioinformatic analysis of PCS genes in Morus notabilisThrough the bioinformatics methods, we identified 2 phytochelatin synthase gene in Morus notabilis genome, which is the same number with Arabidopsis thaliana. Then we cloned the two genes respectively using the cDNA of 6 mulberry tissues as template.And the sequence information is same with Morus notabilis database through sequencing validation. The protein molecular weight of MnPCSl was predicted to be 55.8kDa and the protein molecular weight of MnPCS2 was predicted to be 55.2kDa, which fits the protein molecular weight of PCS gene that was between 40kDa and 70kDa. We search the PCS genes expression in different tissue in the database. Among them, MnPCSl gene expression is the highest in bark and is the lowest in leaf. MnPCS2 gene expression is the highest in flower and is the lowest in bud.Multiple genetic structure analysis showed that the two PCS genes had multiple exons and the number of exons were all 8. Domain prediction results showed that they were same with the discovered PCS genes which have highly conserved N-terminal catalytic domain and variable C-terminal and they contained more cysteine residues in its C-terminal and mostly exsisted in the form of pairs or approximate in pairs. The C-terminal of MnPCSl contained 12 cysteine residues, and that of MnPCS2 contained 9 cysteine residues.Among them, The number of Cys residues was more than that of PCS genes in Arabidopsis thaliana. It was noted that arragement of Cys residues in C-terminal of MnPCS2 was greatly different with PCS genes in other plant. Phylogenetic analysis results showed that the MnPCS1 and the PbPCSl of Pyrus betulaefolia,which belonged to Rosales, were clustered into one branch and this was fit with the result that mulberry should be divided into Rosales.MnPCS2 and Suaeda salsa were together clustered into one branch.2. Stress induced expression analysis of PCS genes in mulberryWe cloned the PCS genes of Gui you 62 mulberry varieties based on the PCS gene sequences of Morus notabilis database to design the cloning primers using its cDNA as template. After sequencing,we compared with PCS genes of Morus notabilis by multiple sequence alignment. The result showed that the sequences of the two PCS proteins in the two mulberry varieties are highly conservative and the similarity is up to 99.8% and 96.4%. Then we designed the quantitative primers based on PCS gene sequences of Gui you 62 and treated Gui you 62 mulberry with Zn2+ stress. The results of qRT-PCR showed that after the PCS genes of Gui you 62 mulberry were treated with Zn2+ stress, its expression patterns within 24 h was decreased first and then increased and decreased last. The relative expression level of MaPCSl in the eighth hour in root and stem was highest, while the highest relative expression level in leaf was in the 12th hour.The relative expression level of MaPCS2 in leaf and stem in the 2th hour reached the maximum while the highest relative expression level in root was in the 8th hour. The relative expression of MaPCSl in root was higher than that in stem and leaf, which was consistent with the expression of PCS that had been reported in different tissues., but the relative expression of MaPCS2 in leaf was highest than others. When compared with the transcriptome data of Morus notabilis, we found that both the two genes in relative expression and trends are similar with that of Morus notabilis. The results showed that Gui you 62 PCS genes could quickly response and work together after induced by Zn2+ stress, but in different tissues their functions might be different.3. Study on the function of PCS gene in mulberrySeparately using the floral dip method and leaf disc transformation method, we obtained MnPCSl and MnPCS2 transgenic Arabidopsis and transgenic tobacco lines. Through the genomic PCR and qRT-PCR authentication, we selected the genes that had highest and lowest expression level for further function researches.The research results showed that, under the heavy metals stress, the mean root lengths of MnPCS1 transgenic Arabidopsis strains were 1.47cm and 1.65cm respectively according to the expression level of target genes, and they were longer than that of wild, which was 0.97cm.The mean fresh weight of every 10 seedings were 209mg and 220mg respectively and they were heavily than that of wild, which was 174.4mg. The mean root lengths of MnPCS2 transgenic Arabidopsis strains were 1.58cm and 1.57cm respectively according to the expression level of target genes, and they were longer than that of wild, which was 1cm.The mean fresh weight of every 10 seedings were 244mg and 288mg respectively than 174.4mg of wild. The accumulation of zinc ion in MnPCS1 transgenic strains were 0.223mg/kg and 0.122mg/kg and that in MnPCSl transgenic strains were 0.261mg/kg and 0.248mg/kg according to the expression level of target genes. Both of them were one order higher than that of wild, which was 0.027mg/kg. The mean root lengths of MnPCSl transgenic tobacco strains were 1.98cm and 1.52cm respectively according to the expression level of target genes, and they were longer than that of wild, which was 0.98cm.The mean fresh weight of every 3 seedings were 302mg and 243mg respectively and they were heavily than that of wild, which was 147mg. The mean root lengths of MnPCS2 transgenic tobacco strains were 2.25cm and 1.93cm respectively according to the expression level of target genes, and they were longer than that of wild, which was 0.98cm.The mean fresh weight of every 3 seedings were 260mg and 207mg respectively and they were heavily than that of wild, which was 147mg.In conclusion, genes in mulberry could help to improve plant resistance and accumulation under plant aerial portion to heavy metals.4. Mulberry transgenic technologyWe directly injected Agrobacterium tumefaciens into the buds of zhenzhubai and hongguo No.1 mulberry varieties, and identified their offspring to screen out the positive branch. Then through the method of artificial pollination, we reaped the T1 seeds, and carried on the genomic PCR detection in order to determine the transgenic lines. The results showed that the method could remove cumbersome steps, such as tissue culture, and aseptic operating environment requirements, We could directly operated on plants in the natural environment, and it had many advantages, such as low cost, simple operation,facility to grasp technical essentials, high conversion efficiency, ablity to quickly get appropriate local wild natural conditions of transgenic plants, seedlings and seeds. It is suitable for a variety of mulberry varieties.In this study, we identified 2 phytochelatin synthase gene in mulberry using the bioinformatics methods. By compared with PCS genes in other plants, we discovered that structure domains of these genes were highly conserved, suggesting the consistency of its function. Through the way that heavy metal stressed Gui you 62 mulberry varieties and qRT-PCR technology, we proved up the expression pattern of PCS gene in mulberryand assume that the functions of the 2 genes in different tissues might be different. Transgenic experiments showed that overexpression of the PCS genes could greatly increase the resistance of Arabidopsis thaliana and tobacco and accumulation under plant aerial portion to heavy metals. This mulberry transgenic technology based on the method to inject Agrobacterium tumefaciens into the buds is simple, can get rid of restrictions in tissue culture and regeneration system and can go forward in the natural environment in a large-scale. This was the first time to study phytochelatin synthase gene in woody plants, and it further proofed and perfected the important role of PCS genes in improving heavy metal resistance and accumulation aspectswhich laid the foundation for screening and solving the pollution of heavy metals to mulberry resources. At the same time, the mulberry transgenic method through winter bud injection in mulberry laid a good foundation for setting up and improving an efficient and practical transgenic technology system.