Abstract：To enrich studies of NAC transcription factor in upland cotton (Gossypium hirsutum) and find related NAC genes involved in stresses induced senescence, we cloned and analyzed GhNAC63, a homologous gene of JUNGBRUNNEN1, and its promoter. By qRT-PCR analysis, we explored the expression patterns of GhNAC63 in different cotton tissues, different cotyledon developmental stages, and in responses to different abiotic stresses in various culture conditions. To explore the function of GhNAC63, we constructed 35S::GhNAC63 expression vector, and transferred it into Arabidopsis through Agrobacterium LBA4404 by flower dipping method, and observed its phenotype in homozygous T4 stage. In addition, pYL156::GhNAC63 vector was constructed with pYL156-pYL192 system, and transferred into cotton to knock down GhNAC63 expression by VIGS. Gene sequence analysis showed that GhNAC63 harbored the conserved NAM domain, belonging to the NAC transcription factors, and it was conserved in the N-terminal, but variant in the C-terminal, which was consist with most of the NAC transcription factors. The full length of GhNAC63 was 870 bp, and its predicted protein contained 289 amino acid residues, with a molecular weight about 33.12 kD. Based on the genome sequence of upland cotton, GhNAC63 was located in scaffold246.1, and about 2.0 kb of its 5' UTR was cloned and analyzed. Besides basal elements, TATA-Box and CAAT- Box, a lot of stresses-related Cis-elements were contained in its promoter, such as drought-related, hot-related, phytohormone-related, and light responsive-related elements. Expression patterns analysis showed that GhNAC63 dominantly expressed in fiber, flower, stem, leaf and cotyledon, and it had the highest expression level in the middle stage of cotyledon development. Under closed culture condition, GhNAC63 was upregulated under treatments of MeJA, SA, drought and ABA in leaf, while the expression level of GhNAC63 was down-regulated under other treatments both in leaf and root, and GhNAC63 showed the most sensitive to ethylene treatment. Under open culture condition, GhNAC63 showed different expression patterns under drought, salt, abscisic acid (ABA), methyl jasmonate (MeJA) or ethylene treatments, and its expression level was highly induced by ethylene treatment. Overexpression of GhNAC63 in Arabidopsis made plants more susceptive to ethylene treatment as leaves of transgenic Arabidopsis became yellow after ethylene treatment, in addition, leaves of the transgenic plants also became wilting after drought treatment, which might indicate the function of GhNAC63 in response to ethylene or drought. On the contrary, VIGS induced gene silence led to a low expression level of GhNAC63 in cotton, with about 0.4 times decrease compared with wild type or empty vector control plants. GhNAC63 silenced seedlings can flowering just as wild type seedlings, while empty vector control plants did not bloom, thus low expression level of GhNAC63 could make cotton seedlings being stronger. In conclusion, GhNAC63 was sensitive to ethylene treatment, and it might negatively regulate ethylene or drought treatments in Arabidopsis, and it negatively regulated plant development in cotton. Promoter of GhNAC63 contained various stresses-related Cis-elements, indicating GhNAC63 might response to different stimulates. This study helps to create new breeding material which was related with stresses induced senescence, and enriches the functional studies of NAC transcription factor in upland cotton.
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