| Festuca arundinacea, which belongs to the family Gramineae, originated in West Europe and North Africa. Not only does it possess the characteristics of drought- resistance, barrenness-tolerance and high adaptability, but also it can be used to beautify urban environment and cover playgrounds. However, since it also has disadvantages such as coarse leaf, no creeping stem, slow growth in summer and sensitiveness to insect and weed invasion, it needs improving urgently with modern biotechnology.It is well-known that the establishment of high-frequency regeneration system is a key factor to transgenic manipulation of plant. In this study, high-frequency regeneration system was established with mature seeds of Festuca arundinacea as explants and MS medium as basic medium at first. Meanwhile, the optimization of the ratio of medium components was performed for embryonic callus induction, subculture, differentiation and rooting. It showed that MS medium containing 9mg/L 2,4-D was optimal for callus induction. Shoots were regenerated on MS medium supplemented with 5.0mg/L 2,4-D, 400mg/L CH, 60g/L sucrose and 12g/L agar and the ratio of callus differentiation reached 78.9%. Regenerated shoots were rooted on half strength MS medium supplemented with 0.5mg/L NAA. Furthermore, it revealed that hyperosmotic subculture medium with high concentration of agar and sugar, supplement of certain amount of CH(casein hydnolysate) were beneficial to callus differentiation and formation of high-frequency regeneration system.Microprojectile bombardment technique is often used in genetic transformation of monocotyledon. In this study, after precultured on osmotic medium for about 6h, embryonic calluses of Festuca arundinacea were bombarded by PDS1000/He) DNA delivery system with pARN6 containing rice chitinase gene(RC24) and pDB1 containing reporter gene (gus) and selective marker gene (bar), and were then selected gradiently on differentiation media supplemented with 2.0mg/L, 3.0mg/L, 5.0mg/L PPT respectively. Results showed that all calluses bombarded were alive on medium with 2.0mg/L PPT, dead on medium with 5.0mg/L, while small part of calluses kept growing on medium with 3.0mg/L. It suggested that 3.0mg/L PPT was optimal selective concentration. Ninety-three regenerated plantlets were sprayed with a solutioncontaining 0.2% Tween-20, 200mg/L PPT, but only two regenerated plants kept alive among them.These two plants were preliminarily identified as transgenic plant with the target gene (RC 24) integrated.In this study, a pair of primers(Forward Primer: 5'-TGCCCCAACTGCCTC- TGCT-3', Reverse Primer: 5'-CCTCTGG (T/C) TGTAGCAGTC (C/G) A-3') were designed according to homology comparison of nucleotide and amino acid sequence of Class I chitinase of several Gramineae plants. Total RNA was extracted from leaf which was treated in solution containing 0.2% HgCl2 for 24h, while genomic DNA was extracted from fresh leaf. Two nucleotide sequences, named fescue-1 (849bp) and fescue-2 (843bp), were amplified with PCR and RT- PCR respectively, and two amino acid sequences, named gym-pro-1 and gym-pro-2 respectively, were deduced. The comparison with Megalign software indicated that the similarity between two nucleotide sequences was 97%, while that of two amino acid sequences was 99%. The BLAST homology search in NCBI website showed that there existed higher homology between the two nucleotide sequences and Class I chitinase gene of other Gramineae plants, and between the two amino acid sequences and Class I chitinase of other Gramineae plants. For example, homology between gym-pro-1 and its counterparts from Poa pratensis, Secale cereale and Hordeum vulgare subsp. reached 85%, 87% and 84% respectively.On the other hand, the minor difference between two nucleotide sequences indicated that they did not result from the amplification of the same gene. That is to say, mRNA template amplifying fescue-2 in RT-PCR was not the transcript of DNA template amplifying fescue-1 in PCR. This showed that there probably exists a Class I chitinase multi-gene family in Festucca arundinacea, among which nucleotide sequences and amino acid sequences of different members were homologyical. |
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