Isolation of ACS1 gene and the relationship between its expression and fruitlet abscission in litchi

Abstract:【Objective】ACC synthase (ACS) and ACC oxidase (ACO) are the key enzymes for ethylene syn⁃thesis in plants. One ACO gene from litchi (Litchi chinensis Sonn.) was isolated in our previous study. ACSgene was isolated and the relationship between the gene and fruitlet abscission was analysed in this re⁃search.【Methods】Litchi trees were selected in the orchard of South China Agricultural University, Guang⁃zhou, China (2009). Six 10-year-old‘Nuomici’trees were chosen for shading treatment at 30 d after an⁃thesis (DAA). Three of them were treated by shading (shaded to 18% of full sun with a neutral densityblack-polypropylene shade cloth), and the other three were used for control, and each tree was a biologi⁃cal replicate. Ten fruit-bearing shoots located in different directions of each tree were tagged for calculat⁃ing relative fruitlet abscission rate (RFAR) and collecting samples. Three 10-year-old‘Kulin’litchitrees were treated by girdling plus defoliation at 25 DAA, and each tree was a biological replicate. Twentyfruit-bearing shoots located in different directions of each tree were tagged for calculating RFAR and col⁃lecting samples. Ten of them were treated by girdling (removing bark by about 0.5 cm in width) plus defoli⁃ation (removing all leaves above the girdling site), and the other ten were used as control. Two 10-yearold‘Heiye’litchi trees were chosen for NAA treatment at 30 DAA. One was sprayed with 100 mg·L- 1NAA, and another one was used as control. Fifteen fruit-bearing shoots located in different directions ofeach tree were tagged for calculating RFAR and collecting samples, and each five fruit-bearing shootswas a biological replicate. Fruit number on the tagged shoots was counted on each sampling date in orderto determine fruit abscission rate. After the samples were transferred to the laboratory, fruitlets, abscissionzone and pericarp were collected immediately. Abscission zones were excised by cutting about 1 mm ateach side of the abscission fracture plane. After separation, all tissues were quickly frozen in liquid nitro⁃gen and stored at -80 °C for future analysis. Total RNA was extracted from the frozen samples, DNase Iand RNAse-free columns were used to remove any genomic DNA contamination and purify total RNA, re⁃spectively. For reverse transcription, 2 μg of total RNA and oligo-dT primers were used with the M-MLVcDNA synthesis kit following the manufacturer’s instructions (Promega). RT-PCR and rapid amplifica⁃tion of cDNA ends (RACE) were used for Lc-ACS1 gene isolation. Firstly, the degenerating primers weredesigned based on available sequence information in Genbank. The amplification conditions were as fol⁃lows: 94 °C for 3 min; 35 cycles at 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min; and 72 °C for 5 min.The amplified fragments were cloned into a pGEM-T Easy vector (Promega, USA), sequenced, and com⁃pared with Genbank sequences using the Blast program. Extension of partial cDNA segments was carriedout using the 3’- and 5’- RACE kit (TaKaRa, Dalian Division, China) according to the manufacturer’sinstructions. Full-length cDNA sequence of Lc-ACS1 was assembled through sequential ligation and cloning, and verified by DNA sequencing. qRT-PCR was used to analyze the relationship between Lc-ACS1gene expression and the fruitlet drop in litchi. qRT-PCR was conducted on an Applied Biosystems 7 500Real-Time PCR System using SYBR Green qPCR SuperMix-UDG Kit (Invitrogen, USA). Each reactionmix contained 1 and 10 μL of diluted cDNAs and SYBR Green qPCR SuperMix, respectively, and 250nmol·L^-1 of each primer to a final volume of 20 μL. The amplification conditions were as follows: 50 °C for 2 min; 95 °C for 10 min; 40 cycles at 95 °C for 15 s; 55 °C for 30 s; and 72 °C for 30 s in 96-well optical reaction plates. The dissociation curve was analysed at 60-95 °C over 40 cycles to determine primerspecificity. Each assay included three technical and biological replicates.【Results】One ACS gene namedLc-ACS1 was cloned from litchi.The full-length cDNA sequence was 1 844 bp with 179 bp 5’-UTR and192 bp 3’-UTR, coded 491 amino acids, and covering seven conserved regions and eleven conserved ami⁃no residues of ACS gene. A higher homology was found between Lc-ACS1 amino acid sequence and that ofthe other plants, and the highest homology was found with Citrus sinensis and Ricinus communis (76%).Under the treatment of shading, the Lc-ACS1 gene expression in the abscission zone and fruitlet peaked at5 d after the treatment (DAT), the RFAR also peaked at 5 DAT compared with that of the control. Undergirdling plus defoliation treatment, the Lc-ACS1 gene expression in the abscission zone peaked at 1 DATand 2 DAT in pericarp, the peak time of ethylene release rate in the fruitlets was 2 DAT. Meanwhile, theRFAR peaked at 4 DAT compared with that of the control. For 100 mg·L^-1 NAA treatment, the Lc-ACS1gene expression in the abscission zone and fruitlet peaked at 7 DAT and 3 DAT, respectively.The RFARpeaked at 10 DAT compared with that of the control. Moreover, Shading, girdling plus defoliation, and 100 mg·L^-1 NAA treatments could significantly increase the rate of fruitlet abscission. The expressions ofLc-ACS1 gene were higher in the fruitlets with treatments than that of the control.【Conclusion】One ACSgene named Lc- ACS1 was cloned from litchi. The treatments for promoting fruitlet abscission includingshading, girdling plus defoliation, and 100 mg·L^-1 NAA spraying significantly increased the Lc- ACS1mRNA abundance. Meanwhile, the peak time of Lc-ACS1 gene expressions in abscission zone of the fruit⁃lets with the three treaments were prior to the peak of the RFAR. Thus, Lc-ACS1 gene might be the keygene invovled in fruitlet abscission in litchi.