Cloning and Expression Analysis of St4CL Gene in Solanum tuberosum

doi:10.13560/j.cnki.biotech.bull.1985.2019-0505

Abstract

Abstract: 4-coumarate：CoA ligase（4CL）is one of the most important enzymes in phenylpropane metabolism,thus cloning 4CL from Solanum tuberosum and analyzing its expression will lay foundation for studying its function. A gene named St4CL was cloned from Qingshu 9,bioinformatics for it was conducted,and qRT-PCR was used to detect the expressions of St4CL in the different tissues and varieties. The results demonstrated that the CDS of St4CL gene was 1 710 bp,and it encoded 569 amino acids. The molecular weight of protein was 61.83 kD,and the isoelectric point was 5.53. Bioinformatics analysis showed that St4CL was a stable hydrophobic protein with random coil（42.36%）,α-helix（28.47%）,extended strand（21%）,and β-turn（8.08%）. Subcellular localization prediction showed that St4CL was located on the thylakoid membrane of chloroplast. Phylogenetic analysis revealed that St4CL protein had the highest sequence similarity（about 97%）with and the closest genetic relationship to Sl4CL in Solanum lycopersicum. The promoter of St4CL gene contained many cis-acting elements related to stress response,such as abscisic acid responsive element,gibberellin-responsive element,MYB binding site involved in drought and flavonoid biosynthesis,etc. qRT-PCR analysis showed that St4CL expressed in all tissues,the highest in leaves followed by flowers,and the lowest in roots. The expression of St4CL varied in different varieties,with the highest expression in Qingshu10 and the lowest expression level in Xiazhai 65.

Key words: Solanum tuberosum, 4-coumarate：CoA ligase, expression analysis, late blight

XIE Hai-juan, FAN Xi-de, YE Guang-ji, ZHOU Yun, WANG Jian, YANG Yong-zhi. Cloning and Expression Analysis of St4CL Gene in Solanum tuberosum[J]. Biotechnology Bulletin, 2019, 35(11): 1-8.

References

[1] 李明, 谢海娟, 叶广继, 等. 马铃薯光敏色素作用因子基因PIF4的克隆及表达分析[J]. 分子植物育种, 2018, 16(19):6193-6201.
[2] 王连平, 席与芳, 王汉荣, 等. 外源处理对机械损伤甘薯块根木质素合成的影响[J]. 江西农业学报, 2004, 16(1):20-24.
[3] 胡尚连, 曹颖, 黄胜雄, 等. 慈竹4CL基因的克隆及其生物信息学分析[J]. 西北农林科技大学学报:自然科学版, 2009, 37(8):204-210.
[4] Vogt T.Phenylpropanoid biosynthesis[J]. Molecular Plant, 2010, 3(1):2-20.
[5] Awasthi P, Mahajan V, Jamwal VL, et al.Characterization of the gene encoding 4-coumarate:CoA ligase in Coleus forskohlii[J]. J Plant Bioch Biotech, 2018, 28(2):203-210.
[6] Hakeem KR, Rehman RU, Tahir I.Plant signaling:Understanding the molecular crosstalk[M]. India:Springer New Delhi, 2014.
[7] Tang YH, Liu F, Mao KQ, et al.Cloning and characterization of the key 4-coumarate CoA ligase genes in Boehmeria nivea[J]. South African Journal of Botany, 2018, 116:123-130.
[8] Li Y, Kim JI, Pysh L, et al.Four isoforms of Arabidopsis thaliana 4-coumarate:CoA ligase(4CL)have overlapping yet distinct roles in phenylpropanoid metabolism[J]. Plant Physiology, 2015, 169(4):2409.
[9] Sun HY, LI Y, Feng SQ, et al.Analysis of five rice 4-coumarate:coenzyme Aligase enzyme activity and;stress response for potential roles in lignin and flavonoid biosynthesisin rice[J]. Biochemical & Biophysical Research Communications, 2013, 430(3):1151-1156.
[10] Hu WJ, Kawaoka A, Tsai CJ, et al.Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in Aspen(Populus tremuloides)[J]. Proceedings of the National Academy of Sciences of the USA, 1998, 95(9):5407-5412.
[11] 常胜合, 孙威, 许桂莺, 等. 利用4-香豆酸:CoA连接酶基因Mu4CL15提高香蕉植株的倒伏抗性[J]. 分子植物育种, 2017, 15(12):4905-4911.
[12] 姜勇, 胡尚连, 曹颖, 等. 青杨4CL基因的克隆及生物信息学分析[J]. 西南农业学报, 2016, 29(7):1547-1553.
[13] Stuible H, Kombrink E.Identification of the substrate specificity-conferring amino acid residues of 4-coumarate:Coenzyme A ligase allows the rational design of mutant enzymes with new catalytic properties[J]. Journal of Biological Chemistry, 2001, 276(29):26893-26897.
[14] Alberstein M, Eisenstein M, Abeliovich H.Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution[J]. Plant J, 2012, 69(1):57-69.
[15] Kiyoon K, Kyungjin L, Ishihara A, et al.Induced synthesis of caffeoylserotonin in pepper fruits upon infection by the anthracnose fungus, Colletotrichum gloeosporioides[J]. Scientia Horticulturae, 2010, 124(2):290-293.
[16] Lee D.Two divergent members of a tobacco 4-coumarate:coenzyme A ligase(4CL)gene family. cDNA structure, gene inheritance and expression, and properties of recombinant proteins[J]. Plant Physiology, 1996, 112(1):193-205.
[17] 倪志勇, 吕萌, 范玲. 棉花Gh4CL1基因克隆及表达[J]. 西北植物学报, 2010, 30(5):876-882.
[18] 李萌, 哈努拉·塔斯肯, 陈卓, 等. 白桦4CL基因启动子克隆及表达分析[J]. 中国农学通报, 2017, 33(5):29-34.
[19] 申晚霞, 王志彬, 薛杨, 等. 柑橘4CL 基因家族的结构及其功能分析[J]. 园艺学报, 2019, 46(6):1068-1078.
[20] 母洪娜, 孙陶泽, 徐晨, 等. 桂花(Osmanthus fragrans Lour. )4-香豆酸辅酶A连接酶(4CL)基因克隆与表达分析[J]. 分子植物育种, 2016, 14(3):536-541.
[21] Liu XY, Wang PP, Wu YF, et al.Cloning and functional characterization of two 4-coumarate:CoA ligase genes from Selaginella moellendorffii[J]. Molecules, 2018, 23(3):595-606.
[22] 陈夏晔, 彭宣祥. 普通烟草Nt4CL基因家族的生物信息学分析[J]. 贵州农业科学, 2018, 46(8):11-15.
[23] 贾彩红, 王宏芝, 杜克久, 等. 抑制4CL基因表达的转基因毛白杨中木质素含量与茎杆颜色的关系[J]. 农业生物技术学报, 2004, 12(6):621-624.
[24] 赵艳玲, 陆海, 陶霞娟, 等. GRP1. 8融合反义4CL1基因调控烟草木质素生物合成[J]. 北京林业大学学报, 2003, 25(4):16-20.
[25] Fritzemeier KH, Cretin C, Kombrink E, et al.Transient induction of phenylala-nine ammonia-lyase and 4-coumarate:CoA ligase mRNAs in po-tato leaves infected with virulent or avirulent races of Phytophthora infestans[J]. Plant Physiol, 1987, 85(1):34-41.
[26] 张蕾, 林晓, 罗赟, 等. RNAi沉默Fa4CL基因对草莓果实花色苷代谢的影响[J]. 果树学报, 2015, 32(3):434-439.
[27] 尹军良. 西北地区马铃薯主栽品种的抗晚疫病性评价及致病疫霉菌候选核心RXLR效应基因的鉴定[D]. 杨凌:西北农林科技大学, 2018.
[28] 张珊珊, 翟伟卜, 郭慧敏, 等. 香豆素合成途径关键酶基因Ghpal、Ghc4h和Gh4cl在棉花抗链格孢菌中的作用[J]. 植物病理学报, 2017(2):73-82.