Cloning And Sequence Analysis Of Ginkgo Biloba Var. Epiphylla Mak. Involved In Epiphyllous Female Reproductive Organ(efro) Development

Ginkgo biloba L. is a type of the relict spermatophyte found in China that has survived since the 4th Ice Age. There were obvious morphological variaton occured in seeds and leaves of Ginkgo biloba along the long evolutionary process. In 1891, Shirai discovered the first Ginkgo biloba var. epiphylla Mak. tree of the wolrd in Japan. The Ginkgo biloba var. epiphylla which is a special germplasm of Ginkgo family has great ornamental and scientific values. The taxonomic position and poylogeny of Ginkgo biloba var. epiphylla have studied in our laboratory. However, the occurrence mechanism of Ginkgo biloba var. epiphylla is still lacking.The LEAFY gene and GbMADS5 gene were cloned and analyzed for Ginkgo biloba var. epiphylla and Ginkgo biloba . The results obtained as follows.1. Genomic DNA of the Ginkgo biloba var. epiphylla and Ginkgo biloba were extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method.The results identified by agarose gel electrophoresis showed that the total DNA was about 23 kb long, the bands were neat and distinct and did not have obvious tail, which demonstrated that we have obtained high purity DNA. RNA of the Ginkgo biloba var. epiphylla were extracted using CTAB large method. The results identified by agarose gel electrophoresis showed that the 28SRNA and 18SRNA bands were neat and distinct and did not have obvious tail, and the brightness of 28SRNA was higher than the 18SRNA. All thoses showed that the integrity of RNA was well and did not degradation. It could use for the following experiments such as molecule cloning. 2. The length of the GinLFY gene cDNA translated region was 1209bp by RT-PCR. The result of sequence analysis showed that this gene encoding 402 amino acid residues by using DNAman software. The nucleotide sequence data reported in this paper will appear in the GeneBank databases under accession number GU563897. Sequence comparisons by performing Blast Search in GeneBank revealed that GinLFY gene has a high identify of 85% with PRFLL gene of Pinus radiata and 82% with LEAFY gene of Podocarpus matudae. This sequence also has a high identify of 99% with GinLFY gene of Ginkgo biloba in the GenBank on nucleotide and amino acid. The genome sequence of Ginkgo biloba var. epiphylla was 3455bp in length, it contained two introns and three extons, first was 479bp, second was 404 bp, third was 326bp.The length of the GinNdly gene cDNA translated region was 1209bp. The result of sequence analysis showed that this gene encoding 402 amino acid residues by using DNAman software. The nucleotide sequence data reported in this paper will appear in the GeneBank databases under accession number GU563898. Sequence comparisons by performing Blast Search in GeneBank revealed that GinNdly gene has a high identify of 86% with NEEDLY-like gene of Taxus globosa and 83% withCjNdly gene of Cryptomeria japonica and 84% with NEEDLY-like gene of Podocarpus matudae. This sequence also has a high identify of 99% with GinNdly gene of Ginkgo biloba in the GenBank on nucleotide and amino acid. The genome sequence of Ginkgo biloba var. epiphylla was 1504bp in length, it contained two introns and three extons, first was 449bp, second was 428 bp, third was 332bp. 3. The length of the GbMADS5 gene translated region was 666bp. The nucleotide sequence data reported in this paper will appear in the GeneBank databases under accession number GU563899. Sequence analysis indicated that this gene with an open reading frame encoding a 221 amino acids protein, which displayed the structure of typical MIKC type plant MADS-box gene including MADS domain,â… domain, K domain and C terminus. The results of sequence alignment showed that GbMADS5 gene has a high identify of 91.7% with AG gene of Cycas revolute and 83.33% with DAL2 of Picea abies and 60.87% with AGL11 of Arabidopsis thaliana. The phylogenetic tree analysis revealed that GbMADS5 gene belonged to AG-like gene family and had a near relationship with Cycas revolute.The homologous gene of LEAFY and MADS-box gene of Ginkgo biloba var. epiphylla were discussed in this paper. This research is of great importance to the preservation, utilization and exploitation of this ornamental resource.