果树学报2017，Vol.34Issue(7)：817-827,11.DOI:10.13925/j.cnki.gsxb.20160393
荔枝ACS1基因的分离及其与幼果脱落的关系
Isolation of ACS1 gene and the relationship between its expression and fruitlet abscission in litchi
摘要
Abstract
[Objective]ACC synthase (ACS) and ACC oxidase (ACO) are the key enzymes for ethylene synthesis in plants.One ACO gene from litchi (Litchi chinensis Sonn.) was isolated in our previous study.ACS gene was isolated and the relationship between the gene and fruitlet abscission was analysed in this research.[Methods] Litchi trees were selected in the orchard of South China Agricultural University,Guangzhou,China (2009).Six 10-year-old ‘Nuomici’ trees were chosen for shading treatment at 30 d after anthesis (DAA).Three of them were treated by shading (shaded to 18％ of full sun with a neutral density black-polypropylene shade cloth),and the other three were used for control,and each tree was a biological replicate.Ten fruit-bearing shoots located in different directions of each tree were tagged for calculating relative fruitlet abscission rate (RFAR) and collecting samples.Three 10-year-old ‘Kulin’ litchi trees were treated by girdling plus defoliation at 25 DAA,and each tree was a biological replicate.Twenty fruit-bearing shoots located in different directions of each tree were tagged for calculating RFAR and collecting samples.Ten of them were treated by girdling (removing bark by about 0.5 cm in width) plus defoliation (removing all leaves above the girdling site),and the other ten were used as control.Two 10-year-old ‘Heiye’ litchi trees were chosen for NAA treatment at 30 DAA.One was sprayed with 100 mg· L-1 NAA,and another one was used as control.Fifteen fruit-bearing shoots located in different directions of each tree were tagged for calculating RFAR and collecting samples,and each five fruit-bearing shoots was a biological replicate.Fruit number on the tagged shoots was counted on each sampling date in order to determine fruit abscission rate.After the samples were transferred to the laboratory,fruitlets,abscission zone and pericarp were collected immediately.Abscission zones were excised by cutting about 1 mm at each side of the abscission fracture plane.After separation,all tissues were quickly frozen in liquid nitrogen and stored at-80 ℃ for future analysis.Total RNA was extracted from the frozen samples,DNase I and RNAse-free columns were used to remove any genomic DNA contamination and purify total RNA,respectively.For reverse transcription,2 μg of total RNA and oligo-dT primers were used with the M-MLV cDNA synthesis kit following the manufacturer's instructions (Promega).RT-PCR and rapid amplification of cDNA ends (RACE) were used for Lc-ACS1 gene isolation.Firstly,the degenerating primers were designed based on available sequence information in Genbank.The amplification conditions were as follows:94 ℃ for 3 min;35 cycles at 94 ℃ for 30 s,57 ℃ for 30 s,72 ℃ for 1 min;and 72 ℃ for 5 min.The amplified fragments were cloned into a pGEM-T Easy vector (Promega,USA),sequenced,and compared with Genbank sequences using the Blast program.Extension of partial cDNA segments was carried out using the 3'-and 5'-RACE kit (TaKaRa,Dalian Division,China) according to the manufacturer' s instructions.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-ACS1 gene expression and the fruitlet drop in litchi,qRT-PCR was conducted on an Applied Biosystems 7 500 Real-Time PCR System using SYBR Green qPCR SuperMix-UDG Kit (Invitrogen,USA).Each reaction mix contained 1 and 10 μL of diluted cDNAs and SYBR Green qPCR SuperMix,respectively,and 250 nmol· L-1 of each primer to a final volume of 20 μL.The amplification conditions were as follows:50 ℃ for 2 min;95 ℃ for 10 min;40 cycles at 95 ℃ for 15 s;55 ℃ for 30 s;and 72 ℃ for 30 s in 96-well optical reaction plates.The dissociation curve was analysed at 60-95 ℃ over 40 cycles to determine primer specificity.Each assay included three technical and biological replicates.[Results] One ACS gene named Lc-ACS1 was cloned from litchi.The full-length cDNA sequence was 1 844 bp with 179 bp 5'-UTR and 192 bp 3'-UTR,coded 491 amino acids,and covering seven conserved regions and eleven conserved amino residues ofACS gene.A higher homology was found between Lc-ACS1 amino acid sequence and that of the 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 at 5 d after the treatment (DAT),the RFAR also peaked at 5 DAT compared with that of the control.Under girdling plus defoliation treatment,the Lc-ACS1 gene expression in the abscission zone peaked at 1 DAT and 2 DAT in pericarp,the peak time of ethylene release rate in the fruitlets was 2 DAT.Meanwhile,the RFAR peaked at 4 DAT compared with that of the control.For 100 mg· L-1 NAA treatment,the Lc-ACS1 gene expression in the abscission zone and fruitlet peaked at 7 DAT and 3 DAT,respectively.The RFAR peaked 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 of Lc-ACS1 gene were higher in the fruitlets with treatments than that of the control.[Conclusion] One ACS gene named Lc-ACS1 was cloned from litchi.The treatments for promoting fruitlet abscission including shading,girdling plus defoliation,and 100 mg· L-1 NAA spraying significantly increased the Lc-ACS1 mRNA abundance.Meanwhile,the peak time of Lc-ACS1 gene expressions in abscission zone of the fruitlets with the three treaments were prior to the peak of the RFAR.Thus,Lc-ACS1 gene might be the key gene invovled in fruitlet abscission in litchi.关键词
荔枝/ACC合成酶/基因克隆/基因表达/落果Key words
Litchi/ACC synthase/Gene clone/Gene expression/Fruit abscission分类
农业科技引用本文复制引用
吴建阳,李彩琴,李建国..荔枝ACS1基因的分离及其与幼果脱落的关系[J].果树学报,2017,34(7):817-827,11.基金项目
农业部农业科研杰出人才及“荔枝花果发育理论与栽培技术创新团队”项目 （）
国家现代农业产业技术体系(CARS-33-11) （CARS-33-11）
