Function Analysis Of Two NAC Transcription Factors In Citrus

NAC transcription factors are unique to plants, and also one of the largest transcription factors families, which originally named from the consensus sequence of Petunia NAM, Arabidopsis ATAF1/2 and Arabidopsis CUC2. NAC transcription factors play an important role in the regulation of plant growth and development, including embryonic development, formation of apical meristem, and lateral root development, transition of auxin, biotic and abiotic stress responses, fruit ripening and tissues senescence.’Hong Anliu’ is a red-flesh bud sport of wild type ’Anliu’(Citrus sinensis[L.] Osbeck). Our previous studies on the fruit development subtractive cDNA library showed that two NAC gene-related ESTs’ expression abundance were significantly different between the two varieties’Hong Anliu’ and ’Anliu’. They were selected to clone two NAC transcription factors, aiming at the analysis of the genotype differences between two materials. We constructed plant vectors of over-expression and RNAi expression of citrus NAC, and then transformed into tomato and kumquat respectively, in order to explore the functions of citrus NAC transcription factor.The major resuLts are summarized as follows:1. citNAC1(FE659187) and citNAC2 (FE659307) genes were cloned from ’Anliu’ and ’Hong Anliu’ using homology-based method. We constructed fusion expression vector with green fluorescent protein (GFP) reporter, and transferred to the onion epidermal cells by agro bacterium-mediated genetic transformation. Fluorescence microscopy analysis showed that the expression product of ciNACl and citNAC2 located in the nuclear.2. citNACl and citNAC2 over-expression vector were constructed through the double digestion and connection methods, using PCAMBIA-1380 which were added with the tomato fruit-specific promoter E8.3. The citNAC1 and citNAC2 sequence were compared with other NAC transcription factor family genes, and selected two specific sequences of citNAC1 and citNAC2 to construct the RNAi vector via gateway technology, and after two rounds of PCR reaction, we connected the citNAC1 and citNAC2 to pHGRV vector respectively.4. Agrobacterium-mediated genetic transformation of citNAC1 and citNAC2 over-expression vector into tomato-resuLted in 25 positive renewable plant materials.5. Also, agrobacterium-mediated genetic transformation of citNACl and citNAC2 RNAi vector into kumquat has regenerated 22 positive buds.Of which,11 buds already grew out roots.