Molecular Cloning And Expression Pattern Of A LFY Homologous Gene From Juglans Regia L.

Walnut (Juglans regia L.), was the main economic tree in China. The area and tree number cultivated in China ranked top around the world. The requirement of high qualitied walnut was increased year by year with the development of living standard. However, the existence of a long juvenile phase has impacted seriously on the early-stage economic benefits of the walnuts. To provide the theoretic base for the fine variety breeding of shortening the juvenile phase,we cloned the complete coding sequence of key gene on the flower development, detected its expression pattern and constructed the expression vector. The main results are as follows:1. A 674 bp cDNA fragment was cloned firstly according to conserved sequence of the LFY homologous genes. Then the full-length cDNA was isolated from Zhonglin No. 5 cultivar for the first time based on the fragment. It was 1,496 bp long including 54 bp of 5′UTR, 271 bp of 3′UTR, 13bp of polyA and 1,158 bp of ORF. The gene was named as JrLFY (Genebank:GU194836)2. The full-length genomic DNA (Genebank:HQ019159) of JrLFY was also obtained on the cDNA sequence. It contained 3 exons and 2 introns. The exons were 433 bp, 362 bp, 363 bp in turn and the introns were 545bp, 1,116 bp, respectively.3. We deduced a 43.15 kDa protein (PI=6.78) with 385 amino acid residues according to the JrLFY gene. It was very close to the LFY protein of hickory according to homology analysis and the similarity identity reached 99%. By bioinformation analysis the amino acid sequence contained some conserved domains, such as proline-rich region and leucine zipper motif in the N-terminal domain. These domains are the same as the structure of LFY homologous protein in the most dicotyledon. So the protein was considered to be a transcript factor.4. Tissue specific expression of JrLFY gene was analysed by RT-PCR in which the reference gene was 18S rRNA. The expression of walnut bud in the period of female flower physiology differentiation was higher than that of male. In the expression of different tissues, the buds, female inflorescence and male inflorescence are dominant, shoot took the second place and the expression of JrLFY was found in leaf. In the expression of different years trees, there was no significant didfference in the bud, one-year branch and leaf. However, the bud of different years trees in the period of female flower physiology differentiation was higher than that of male.5. An efficient expression vector was constructed by In-Fusion technique. It was transferred into arabidopsis using agrobacterium. 20-40 transgenic plants were obtained in every petri dish when screened with antibiotics of rifampicin and kanamycin, and the rate of transformation was up to 1%-2%.