超表达苹果细胞分裂素响应基因MdCRF6影响花青苷积累和盐胁迫抗性

doi:10.3864/j.issn.0578-1752.2017.16.014

Abstract

Abstract: 【Objective】The objective of this study is to clone apple cytokinin response factor gene MdCRF6, to analyze its homology with Arabidopsis AtCRF6, its expression of exposing to cytokinin and salt stress as well as its binding to DRE motif (ACCGAC), to identify its role in regulating anthocyanins accumulation and salt stress, and to provide a theoretical basis for studying cytokinin signaling pathway and regulating fruit tree growth and development. 【Method】 MdCRF6 gene was cloned by homology sequence alignment and PCR technique. The phylogenetic tree of MdCRF6 and Arabidopsis CRFs was constructed using MEGA5.0. The conserved domains of MdCRF6 were analyzed using SMART software and DNAMAN software. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the gene expression of MdCRF6 exposing to cytokinin and salt stress. The electrophoretic mobility shift assay (EMSA) was conducted to verify whether MdCRF6 could bind to the DRE motif. A plant over-expression vector of MdCRF6 was constructed and used to transform apple calli by Agrobacterium-mediated method. The phenotypes of wild-type and transgenic apple calli on anthocyanins accumulation and salt stress tolerance were characterized to investigate the function of MdCRF6 in regulating anthocyanins accumulation and salt stress tolerance in apple. 【Result】 A cytokinin response factor gene named MdCRF6 (MDP0000783818) was cloned from Malus domestic ‘Gala’. Sequence analysis showed that its open reading frame (ORF) was 1 047 bp, which encoded 348 amino acids. The results of phylogenetic tree and amino acid sequence alignment indicated that MdCRF6 contained a CRF domain in its N-terminal side and an AP2/ERF domain in its C-terminal side. qRT-PCR analysis indicated that MdCRF6 was responsive to cytokinin and salt stress, and the expression levels peaked at 3 h and 6 h exposing to 10 μmol?L-1 BA and 100 mmol?L-1 NaCl, respectively. EMSA assay showed that MdCRF6 could bind to the DRE motif. The MdCRF6-overexpressing apple calli exhibited reduced anthocyanins content and decreased salt stress tolerance. The gene expression analysis showed that overexpression of MdCRF6 significantly repressed the expression levels of anthocyanins biosynthetic genes and salt response related genes. 【Conclusion】The apple MdCRF6 exhibited high similarity to AtCRF6, and it was involved in the response to cytokinin and salt stress. Overexpression of MdCRF6 in apple calli inhibited anthocyanins accumulation and decreased salt stress tolerance. It is speculated that MdCRF6 down-regulated anthocyanins accumulation and salt stress tolerance by repressing the expression of anthocyanins biosynthetic genes and salt response related genes.

Key words: apple, MdCRF6, anthocyanins accumulation, salt stres

AN JianPing, SONG LaiQing, ZHAO LingLing, YOU ChunXiang, WANG XiaoFei, HAO YuJin. Effects of Overexpression of Apple Cytokinin Response Factor Gene MdCRF6 on Anthocyanins Accumulation and Salt Stress Tolerance[J].Scientia Agricultura Sinica, 2017, 50(16): 3196-3204.

References

[1] 翟衡, 赵政阳, 王志强, 束怀瑞. 世界苹果产业发展趋势分析. 果树学报, 2005, 22(1): 44-50.

Zhai H, Zhao Z Y, Wang Z Q, Shu H R. Analysis of the development trend of the world apple industry. Journal of Fruit Science, 2005, 22(1): 44-50. (in Chinese)

[2] 闫国华, 甘立军, 孙瑞红, 张利华, 周燮. 赤霉素和细胞分裂素调控苹果果实早期生长发育机理的研究. 园艺学报, 2000, 27(1): 11-16.

Yan G H, Gan L J, Sun R H, Zhang L H, Zhou X. The study of mechanism about gibberellin and cytokinin regulation apple fruit early growth. Acta Horticulturae Sinica, 2000, 27(1): 11-16. (in Chinese)

[3] 邹养军, 王永熙. 内源激素对苹果果实生长发育的调控作用研究进展. 陕西农业科学, 2002(10): 13-15.

ZOU Y J, WANG Y X. The readjustment of endogenous hormones on apple fruit growth and development. Shaanxi Journal of Agricultural Sciences, 2002(10): 13-15. (in Chinese)

[4] MOK W, MOK M C. Cytokinin metabolism and action. Annual Review of Plant Biology, 2001, 52(1): 89-118.

[5] WERNER T, SCHMULLING T. Cytokinin action in plant development. Current Opinion in Plant Biology, 2009, 12(5), 527-538.

[6] HEYL A, WULFETANGE K, PILS B, NIELSEN N, ROMANOV G A, SCHMULLING T. Evolutionary proteomics identifies amino acids essential for ligand-binding of the cytokinin receptor CHASE domain.BMC Evolutionary Biology, 2007, 7(1): 62.

[7] 王三根. 细胞分裂素在植物抗逆和延衰中的作用. 植物学通报, 2000, 17(2): 121-126.

WANG S G. Roles of cytokinin on stress-resistance and delaying senescence in plants. Chinese Bulletin of Botany, 2000, 17(2): 121-126. (in Chinese)

[8] Werner T, Motyka V, Strnad M, Schmülling T. Regulation of plant growth by cytokinin.Proceedings of the National Academy of Sciences, 2001, 98(18): 10487-10492.

[9] 周蕾, 魏琦超, 高峰. 细胞分裂素在果实及种子发育中的作用. 植物生理学通讯, 2006, 42(3): 549-553.

ZHOU L, WEI Q C, GAO F. The effect of cytokinins on fruit and seed development. Plant Physiology Communications, 2006, 42(3): 549-553. (in Chinese)

[10] 杨晓红, 陈晓阳, 刘克锋. 细胞分裂素对植物衰老的延缓作用. 热带亚热带植物学报, 2006, 14(3): 256-262.

YANG X H, CHEN X Y, LIU K F. The role of cytokinins in retarding of senescence in plants. Journal of Tropical and Subtropical Botany, 2006, 14(3): 256-262. (in Chinese)

[11] ZWACK P J, RASHOTTE A M. Interactions between cytokinin signalling and abiotic stress responses.Journal of Experimental Botany, 2015, 66(16): 4863-4871.

[12] JI X H, WANG Y T, ZHANG R, WU S J, AN M M, LI M, CHEN X S. Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tissue and Organ Culture, 2015, 120: 325-337.

[13] SHI M Z, XIE D Y. Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells. Planta, 2011, 233: 787-805.

[14] 蔺经, 盛宝龙, 常有宏. 赤霉素和细胞分裂素类植物生长调节剂在苹果生产中的应用. 北方果树, 2000(1): 1-3.

LIN J, SHENG B L, CHANG Y H. The application of gibberellin and cell division plant growth regulator in apple production. Northern Fruits, 2000(1): 1-3. (in Chinese)

[15] 辛艳伟, 丁春刚. 不同浓度植物细胞分裂素对苹果叶片衰老的影响. 山西农业科学, 2016, 44(4): 467-469, 490.

XIN Y W, DING C G. Effects of different concentrations plant cytokinin on senescence of apple leaves. Journal of Shanxi Agricultural Sciences, 2016, 44(4): 467-469, 490. (in Chinese)

[16] 辛艳伟, 牛颜冰, 李晓瑞. 不同植物细胞分裂素对‘红富士’苹果果实抗氧化活性及品质的影响. 中国农学通报, 2016, 32(4): 83-86.

XIN Y W, NIU Y B, LI X R. Effects of different plant cytokinin on antioxidant activity and quality of ‘Fuji’ apple fruit. Chinese Agricultural Science Bulletin, 2016, 32(4): 83-86. (in Chinese)

[17] FERREIRA F J, KIEBER J J. Cytokinin signaling.Current Opinion in Plant Biology, 2005, 8(5): 518-525.

[18] KANG J, LEE Y, SAKAKIBARA H, MARTINOIA E. Cytokinin transporters: GO and STOP in signaling.Trends in Plant Science, 2017, 6(22): 455-461.

[19] RASHOTTE A M, MASON M G, HUTCHISON C E, FERREIRA F J, SCHALLER G E, KIEBER J J. A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway. Proceedings of the National Academy of Sciences, 2006, 103(29): 11081-11085.

[20] RASHOTTE A M, GOERTZEN L R. The CRF domain defines cytokinin response factor proteins in plants. BMC Plant Biology, 2010, 10(1): 74.

[21] WEIRAUCH M T, YANG A, ALBU M, COTE A G, MONTENEGRO- MONTERO A, DREWE P, NAJAFABADI H S, LAMBERT S A, MANN I, COOK K. Determination and inference of eukaryotic transcription factor sequence speci?city. Cell,2014, 158: 1431-1443.

[22] ZWACK P J, DE CLERCQ I, HOWTON T C, HALLMARK H T, HURNY A, KESHISHIAN E A, RASHOTTE A M. Cytokinin response factor 6 represses cytokinin-associated genes during oxidative stress.Plant Physiology, 2016, 172(2): 1249-1258.

[23] LIANG Y S, ERMAWATI N, CHA J Y, JUNG M H, SUUDI M, KIM M G, SON D. Overexpression of an AP2/ERF-type transcription factor CRF5 confers pathogen resistance to Arabidopsis plants. Journal of the Korean Society for Applied Biological Chemistry, 2010, 53(2): 142-148.

[24] ZWACK P J, ROBINSON B R, RISLEY M G, RASHOTTE A M. Cytokinin response factor 6 negatively regulates leaf senescence and is induced in response to cytokinin and numerous abiotic stresses. Plant and Cell Physiology, 2013, 54(6): 971-981.

[25] RAMAIAH M, JAIN A, RAGHOTHAMA K G. Ethylene Response Factor070 regulates root development and phosphate starvation- mediated responses. Plant Physiology, 2014, 164(3): 1484-1498.

[26] SHI X, GUPTA S, RASHOTTE A M. Solanum lycopersicum cytokinin response factor (SlCRF) genes: Characterization of CRF domain-containing ERF genes in tomato. Journal of Experimental Botany, 2012, 63(2): 973-982.

[27] 杨昌. 甘蓝型油菜CRF家族的特征分析及BnaCRF8s的克隆和功能初步研究[D]. 武汉: 华中农业大学, 2016.

Yang C. Analysis of CRF family genes, cloning and preliminary function research of BnaCRF8s in Brassica napus [D]. Wuhan: Huazhong Agricultural University, 2016. (in Chinese)

[28] AN J P, Li H H, Song L Q, Su L, Liu X, You C X, Hao Y J. The molecular cloning and functional characterization of MdMYC2, a bHLH transcription factor in apple. Plant Physiology and Biochemistry, 2016, 108: 24-31.

[29] DEIKMAN J, HAMMER P E. Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana.Plant Physiology, 1995, 108(1): 47-57.

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Effects of Overexpression of Apple Cytokinin Response Factor Gene MdCRF6 on Anthocyanins Accumulation and Salt Stress Tolerance

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