Ioslation And Exptession Analysis Of Floral Genes In Hazelnut

Hazelnut is one of the most cultivated dry-seed species in the world and has economic importance for food industry. Hazelnut is monoecious and unisexual flowers, the proportion of female and male is very low, which greatly affect its economic value. LFY, FT, AP1and SOC1gene play key role in floral inductive pathways, a number of these genes have now been cloned and extensively characterized from different species. However, the related research in hazelnut has not been reported. So, it is of great economically significance that to carry out study on regularity and mechanism of floral bud differentiation in hazelnut. The cDNAs of ChaLFY、ChaFT、ChaAP1and ChaSOCl wwre isolated from hazelnut using RACE method in this paper, then conducted the relevant bioinformatics analysis. Finally, we studied ChaLFY function by transgenic method in the model plant tobacco. Besides, we studied the period of hazel male flower bud physiological differentiation using band girdle and leaf removal metnod, which is the basis for applying time of GA3and PP333. We further analyzed the effect of GA3and PP333on flowering of woody fruit hazelnut and the expression pattern of related genes. Main conclusions as follows:1. The coding region of ChaLFY, ChaFT, ChaAP1and ChaSOC1gene was1200bp,528bp,729bp and729bp nucleotide, ecoded399,175,242and217putative amino acids, respectively. A comparison of the amino acid sequences of ChaLFY and other plant FLO/LFY shared the presence of two highly conserved regions, and ChaLFY showed highest amino acid identity (86%) to Juglans regia JrLFY. ChaFT was one of the PEBP proteins family members. Closest protein homolog of putative ChaFT was96%and95%identical Betula luminifera BIFT and Betula platyphylla BpFT, respectively. The putative protein ChaAP1and ChaSOC1belonging to the MADS-box family.The putative protein ChaAP1showed high amino acid identity (93%) to Betula platyphylla MADS3. ChaSOC1showed high amino acid identity (83%) to Prunus yedoensis PySOC1.2. The results of the band girdle and leaf removal showed that the stage of physiological differentiation of hazel male flower was roughly from April22th to May28th. The Gene expression pattern of ChaLFY, ChaFT, ChaSOC1and ChaAP1was quantified analysis by using the method of Real-time Quantitative PCR (RT-qPCR) which relies on an internal standards gene18S rRNA. The results showed that ChaLFY relative expression in mixed bud and leaf expression quantity was on the rise during the period of male flower physiological differentiation, expression of ChaFT, ChaSOC1and ChaAP1were gradually decreased. In morphological differentiationstage of male flower, expression quantity of ChaLFY, ChaFT, ChaSOC1, ChaAP1were on the rise trend. By comparing the analysis, throughout the period of male flower differentiation, relative expression of ChaSOCl gene was the highest in compare with others, followed by the ChaFT and CJuiLFY, ChaAP1was the lowest.3. GA3(100mg/L) significantly increased the number of male inflorescence compared to the control, but the number of female flower was not altered. The ratio of female to male was markedly reduced by GA3Whereas, PP333(1000mg/L) significantly increased the number of female flower, but the number of male inflorescence was not significantly altered by this treatment. We further analyzed the changes in relative expression of ChaLFY, ChaFT, ChaSOC1and ChaAP1in response to GA3and PP333RT-qPCR analysis showed that treatment of GA3significantly increased the relative expression of the ChaLFY and ChaAP1gene compared to the control, expression of ChaFT gene was inhibited by GA3, but the relative expression of ChaSOC1was no significant altered by GA3. PP333inhibited the relative expression of ChaLFY gene and promoted expression of ChaFT and ChaSOC1, but anyhow the relative expression of ChaAPl gene was no significant altered. The tissue expression analysis of ChaLFY showed that its transcripts were detected in different organs, but expressing quantity was different. ChaLFY transcripts were abundant in staminate inflorescence and female flower, whereas vegetative organs were very low.4. We constructed the plant over expression vector of ChaLFY gene, and transformed it into tobacco through the method of agrobacterium mediated. The result of PCR detection showed that we had got successfully positive transformed tobacco plants, and obtained the transgenic seeds.