Characterization Of MsZFN Gene From Alfalfa (medicago Sativa L.) And HST Gene Of Arabidopsis Thaliana

In this study, we characterized the MsZFN gene from Medicago sativa (alfalfa) and HST gene fromArabidopsis thaliana by gene genetic engineering, transgenic technology and phenotype analysis. Theresults are showed below:1. A6,829-bp genomic DNA sequence (Access No.: JX131368) was obtained for MsZFN by PCR.Seven exons making up the1,667bp cDNA sequence of MsZFN were interrupted by six introns of820-bp,494-bp,1825-bp,286-bp,641-bp and1093-bp in length when the cDNA and the gDNAsequences were compared.2. To determine the expression pattern of the MsZFN gene in alfalfa, RT-PCR and qRT-PCR wereperformed. Using RT-PCR, the MsZFN transcript was detected in all alfalfa organs tested, includingroots, stems, leaves, and in florets before and after flower opening. Transcript levels were strongestin leaves compared with stems and roots and was weakest in floral buds and florets. Expressionwas also increased during growth under continuous dark conditions, but not affected by20μMABA,20μM GA3,3%sugar treatments or cold conditions.3. For transgenic expression experiments, the full-MsZFN coding domain sequence was inserted intothe pCAMBIA1302to generate binary fusion construct35S::MsZFN. To generate transgenicArabidopsis plants expressing MsZFN, Agrobacterium GV3101transformed with plasmid35S::MsZFN was used to infect Arabidopsis and tobacco. The results showed that overexpressionof MsZFN gene delayed flowering time in transgenic plants.4. We found one Arabidopsis mutant, and the mutant lines are albino and had shorter roots, fewer roothairs, fewer and smaller leaves with shorter petioles, and reduced trichome density. TEM analysisshowed that the mutants had lost the ability to develop into mature chloroplasts. SEM resultsshowed that mutant trichomes were shorter in length and some trichomes had two branches insteadof three. Stomata had bigger openings, suggesting that stomata may close abnormally.5. The TAIL-PCR and sequencing showed that the mutant phenotypes were caused by a T-DNAinsertion and the insertion was located at the second intron of the Arabidopsis HST gene(At3g11945). When expressed HST gene in mutants, the HST gene could completely rescue themutant phenotype. Transgenic Arabidopsis with low expressed levels of HST gene by RNAitechnology were obtained.6. To analyze expression pattern of HST gene, qRT-PCR and promoter expression methods wereperformed. The results showed that HST gene was highly expressed in green tissues and the levelsof HST transcript were highest in non-senescent leaves, and the transcript levels graduallydecreased as leaves began to senesce.7. Enzyme-linked immunosorbent assays (ELISA) were performed to determine concentrations ofdifferent hormones in WT and mutant plants. A substantial decrease in ABA, GA3and ZRconcentrations occurred in mutant plants compared with WT. This disruption of the HST gene also led to a substantial increase (by41.6%) in the IAA content in mutant.