The Study On Gene Transformation And Detection Mediated By Nanoparticles In Plant

As the gene and drug carriers, nanoparticles have been applied to gene transformation and drug release in mammalian cells. This is one of the major achievements that have been gained in nano-biotechnology field. But there are fewer researches have been made in plant science, so there laid unexplored mineral resources. This paper has described the systematic researches of biocompatible poly-L-lysine starch nanoparticles (PLL-StNPs) as plant transgenic vehicles for the first time, and a series of experiments using nanoparticles were carried out for realtime detecting transgenic plants in vivo. Based on those researches, a novel method of gene transformation and detection mediated by nanoparticles in plant was established. ?. Synthesis of PLL-StNPs and their properties Synthesis of nanoparticle gene vehicles: Anion starch nanoparticles (StNPs) were synthesized in water-in-oil microemulsion, with soluble starch as raw materials. PLL-StNPs were prepared by linking poly-L-lysine (PLL) on the surface of StNPs. Observed under automatic force microscope, PLL-StNPs distributed evenly, and their size was about 50 nm. PLL-StNPs carried positive charges in pH 7-8. Analysis of nanoparticles conjugated with DNA: When DNA was added to PLL-StNPs solution, gel electrophoresis illuminated that PLL-StNPs could integrate DNA effectively, and PLL-StNPs could integrate various DNA at one time. DNA could be released from the DNA-PLL-StNPs compounds; the strips of DNA did not change.This was to say that PLL-StNPs could conjugate different foreign genes. Furthermore, PLL-StNPs modified by RuBPY could also combine with DNA effectively. Function of nanoparticles protecting DNA from DNase I cleavage and ultrasound damage: DNA-PLL-StNPs complexes were treated with ultrasound for different time. Gel electrophoresis showed that DNA still bound to PLL-StNPs and strips of released DNA were not change, indicating that PLL-StNPs could prevent DNA from ultrasound damage. The PLL-StNPs-DNA complexes were digested with DNase I, and then DNA was eluted from the compound, the strips of DNA remained the same. The conclusion was that PLL-StNPs might prevent DNA not only from DNase I cleavage but also from ultrasound damage. Biological compatibility of nanoparticles with plant cells: The PLL-StNPs modified by RuBPY were transferred into the inspired suspension cells, observed under laser confocus microscope and compared with the control ones. It was found that fluorescence nanoparticles could enter plant cells. These cells were cultured for 3~5 days, decreased fluorescence could be observed in the offspring cells. The results implied that the nanoparticls did not influence the division and growth of the cells after entering the cells, the particles and plant cells had good biological compatibility, because the nanoparticls were digested by enzyme in the cytoplasm as time went on. П. Plant trans-gene mediated by nanoparticles The single gene transferred with nanoparticle gene carriers: pEGAD plasmid DNA, which comprised green fluorescence protein gene (GFP), was combined with PLL-StNPs to form gene carriers. Mediated by ultrasound, the DNA-PLL-StNPs were transferred into plant cells. PLL-StNPs that did not combine with DNA were added to plant cells as control, and they were observed under microscope. Some cells gave out green fluorescence. However, no control ones emitted green fluorescence in the cultured course. It suggested that the nanoparticles as gene carriers could transfer the GFP gene to the plant cells and expressed green fluorescence protein; the transformation efficiency was almost 30.7 %. Multi genes transferred into plant cells by nanoparticle gene carriers: pEGAD plasmid DNA and Ri plasmid DNA were exstracted, and were combined with PLL-StNPs at the same time to construct multi-gene carriers, and then DNA-PLL-StNPs were transferred into vigorous micro-tubers mediated by ultrasound. Hairy roots emerged on the micro-tubers after two weeks. These hairy roots sent out strong green fluorescence while those ones transformed by agrobacteria rhyzogenesis without GFP gene sent out faint back ground green fluorescence. The results demonstrated that genes of Ri T-DNA and GFP could be transferred into the plant genome at the same time and the nanoparticles could be used as multi-gene carriers. Transgenic hairy roots acted as bioreactor to accumulate diosgenin: The roots were sub-cultured in hormone free 1/2 MS liquid medium under 28 ℃ and 350 lux dim light condition. The root clones grew rapidly, RP-HPLC analysis showed that the content of diosgenin accumulated by hairy roots was 5.70, 6.25 and 2.88 times as that of micro-tubers, calli and plantlets respectively. Ш. Detection of transgenic plant with nanoparticles The ro1B gene in transgenic plant was detected with molecular beacons (MBs): molecular beacons and primers, which were designed according to ro1B gene of Ri T-DNA, were put into PCR tube. Real-time PCR was then carred out in Prism 7000.The increasing fluorescence was obviously observed in the PCR tube which contained DNA from hairy roots, but faint background fluorescence was observed in the control. Real-time PCR was time-saved and ultra-sensitive, eliminating carryover contamination to avoid fake result, and exactly quantifying gene copies. But it could not be used in live cells for real-time and in situ hybridized detection. Nanoparticles conjugated with molecular beacons detected target gene in vitro: Molecular beacons (MBs) were conjugated with PLL-StNPs to form MBs-PLL-StNPs, and then cDNA fragments which matched to MBs were added, cDNA-MBs-PLL-StNPs compounds emitted dramatically increasing fluorescence, similar to the course of MBs-PLL-StNPs hybridized with mRNA which was extracted from hairy roots. Those results illuminated that nanoparticles which combined molecular beacons could be used for the detection of target gene. Nanoparticles conjugated with molecular beacons detected target gene in vivo: Calli were induced from the hairy roots of Dioscrea Zigiberensis G.H. Wright, and then inspired cells were prepared. MBs-PLL-StNPs were transferred into the inspired cells when co-cultured with them. If there were mRNA that transcripted from ro1B gene in the cells, MBs-PLL-StNPs would hybridize with corresponding matched position in mRNA and would send out increasing flurosence. The fact was that parts of the cells send out obvious increasing fluorescence under fluorescence microcope, while control ones exhibited dim background fluorescence, which demonstrated that nanoparticles combined with molecular beacons, could be used in the live cells of transgenic plant for real-time and in situ detecting target genes. A new method to detect transgenic plant was established. In summary, linked by nanoparticles, this paper formed the outline of plant genetic transformation and detection including three parts: Firstly, bionconjugated poly-L-lysine starch nanoparticles (PLL-StNPs) were synthesized and were used to trace plant cells for the first time, those particles were also found to protect DNA from ultrasound damage as well as from Dnase I cleavage. Secondly, mediated by ultrasound, those particles trafficked through the nature barrier of plant cell wall and were used as novel gene carriers for single or multi genes transformation in plant cells. Thirdly, nanoparticles bound with molecular beacons were used to detect target gene not only in vitro but also in vivo cells of transgenic plants. Furthermore, those transgenic plants were found to accumulate increased secondary metabolites.