Cloning, Functional Identification and Expression Analysis of FAH, a Key Gene for Tyrosine Metabolism in Brassica napus L.

doi:10.13560/j.cnki.biotech.bull.1985.2023-0270

Abstract

Abstract:

The objective of this work is to clone the tyrosine metabolism key gene FAH in Brassica napus L., identify its function and analyze its expression, so as to provide theoretical evidence for further understanding the role and function of FAH in B. napus L. Two FAH genes BnaA06g38260D（BnaA06FAH）and BnaC05g49430D（BnaC05FAH）, which was the highest protein homology to AtFAH in Arabidopsis, were cloned from the B. napus L. variety‘westar’, the phylogenetic relationship was analyzed by bioinformatics, and the overexpression vector was constructed and transformed into Arabidopsis mutant sscd1 for function verification; the promoter sequences of two FAH genes BnaA06FAH and BnaC05FAH were cloned. PlantCARE was used to analyze the promoter cis-acting elements, and the fusion expression vector of gene promoter and GUS was constructed to analyze its expression patterns. As results, the amino acid sequence similarity of BnaA06FAH, BnaC05FAH and AtFAH was 93.11% and 92.40% respectively, the overexpressions of BnaA06FAH and BnaC05FAH completely inhibited the mimic lesion in sscd1 under short-day condition, suggesting both BnaA06FAH and BnaC05FAH had high structural and functional similarity with AtFAH. BnaA06FAH and BnaC05FAH promoters had the core elements of eukaryotic promoter TATA-box and CAAT-box, and also contained several cis-acting elements related to stress, hormone, stress response and a variety of cis-acting elements related to disease resistance, however, BnaC05FAH promoter shared more cis-acting elements with Arabidopsis AtFAH than with BnaA06FAH. GUS activity assays indicated BnaC05FAH promoter drove the expression of GUS gene stronger than BnaA06FAH, and the site of GUS gene expression drove by two different promoters were not completely consistent. Conclusively, there were differences in the sites and intensity where BnaA06FAH and BnaC05FAH promoters functioned. The results showed that both BnaA06FAH and BnaC05FAH played a role in regulating lesion mimic in the sscd1 mutant, but the expression of gene downstream and the expression site drove by BnaA06FAH and BnaC05FAH promoters was different.

Key words: Brassica napus L., fumarylacetoacetate hydrolase, functional identification

ZHI Tian-tian, ZHOU Zhou, CHEN Ji-peng, HAN Cheng-yun. Cloning, Functional Identification and Expression Analysis of FAH, a Key Gene for Tyrosine Metabolism in Brassica napus L.[J]. Biotechnology Bulletin, 2023, 39(10): 115-127.

Figures/Tables 10

References 48

[1]	Lindblad B, Lindstedt S, Steen G. On the enzymic defects in hereditary tyrosinemia[J]. Proc Natl Acad Sci USA, 1977, 74(10): 4641-4645. doi: 10.1073/pnas.74.10.4641 pmid: 270706
[2]	Ruppert S, Kelsey G, Schedl A, et al. Deficiency of an enzyme of tyrosine metabolism underlies altered gene expression in newborn liver of lethal albino mice[J]. Genes Dev, 1992, 6(8): 1430-1443. doi: 10.1101/gad.6.8.1430 URL
[3]	Grompe M al-Dhalimy M, Finegold M, et al. Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice[J]. Genes Dev, 1993, 7(12a): 2298-2307. doi: 10.1101/gad.7.12a.2298 URL
[4]	Lock EA, Gaskin P, Ellis MK, et al. Tissue distribution of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1-3-dione(NTBC): effect on enzymes involved in tyrosine catabolism and relevance to ocular toxicity in the rat[J]. Toxicol Appl Pharmacol, 1996, 141(2): 439-447. doi: 10.1006/taap.1996.0310 URL
[5]	Sparnins VL, Chapman PJ. Catabolism of L-tyrosine by the homoprotocatechuate pathway in gram-positive bacteria[J]. J Bacteriol, 1976, 127(1): 362-266. doi: 10.1128/jb.127.1.362-366.1976 pmid: 931949
[6]	Dixon DP, Edwards R. Enzymes of tyrosine catabolism in Arabidopsis thaliana[J]. Plant Sci, 2006, 171(3): 360-366. doi: 10.1016/j.plantsci.2006.04.008 URL
[7]	Hildebrandt T, Nunes Nesi A, Araújo W, et al. Amino acid catabolism in plants[J]. Mol Plant, 2015, 8(11): 1563-1579. doi: 10.1016/j.molp.2015.09.005 pmid: 26384576
[8]	Xu JJ, Fang X, Li CY, et al. General and specialized tyrosine metabolism pathways in plants[J]. aBIOTECH, 2020, 1(2): 97-105. doi: 10.1007/s42994-019-00006-w
[9]	Schenck CA, Maeda HA. Tyrosine biosynthesis, metabolism, and catabolism in plants[J]. Phytochemistry, 2018, 149: 82-102. doi: S0031-9422(18)30034-7 pmid: 29477627
[10]	Jin X, Chen X, et al. Imbalance of tyrosine by modulating TyrA arogenate dehydrogenases impacts growth and development of Arabidopsis thaliana[J]. Plant J, 2019, 97(5): 901-922. doi: 10.1111/tpj.2019.97.issue-5 URL
[11]	Han CY, Ren CM, Zhi TT, et al. Disruption of fumarylacetoacetate hydrolase causes spontaneous cell death under short-day conditions in Arabidopsis[J]. Plant Physiol, 2013, 162(4): 1956-1964. doi: 10.1104/pp.113.216804 URL
[12]	Zhou Z, Zhi T, Han C, et al. Cell death resulted from loss of fumarylacetoacetate hydrolase in Arabidopsis is related to phytohormone jasmonate but not salicylic acid[J]. Sci Rep, 2020, 10(1): 13714. doi: 10.1038/s41598-020-70567-0 pmid: 32792583
[13]	Maeda H, Dudareva N. The shikimate pathway and aromatic amino acid biosynthesis in plants[J]. Annu Rev Plant Biol, 2012, 63: 73-105. doi: 10.1146/annurev-arplant-042811-105439 pmid: 22554242
[14]	Singh S, Mishra VK, Kharwar RN, et al. Genetic characterization for lesion mimic and other traits in relation to spot blotch resistance in spring wheat[J]. PLoS One, 2020, 15(10): e0240029. doi: 10.1371/journal.pone.0240029 URL
[15]	Yu Y, Zhang Q, Sun S, et al. Upregulated expression of respiratory burst oxidase homolog d underlies lesion-mimic phenotype in dark-treated Arabidopsis pheide a oxygenase mutant leaves[J]. Planta, 2022, 255(6): 110. doi: 10.1007/s00425-022-03895-2
[16]	Mu XH, Li JK, Dai ZZ, et al. Commonly and specifically activated defense responses in maize disease lesion mimic mutants revealed by integrated transcriptomics and metabolomics analysis[J]. Front Plant Sci, 2021, 12: 638792. doi: 10.3389/fpls.2021.638792 URL
[17]	Zhao Y, Xu W, Wang L, et al. A maize necrotic leaf mutant caused by defect of coproporphyrinogen III oxidase in the porphyrin pathway[J]. Genes: Basel, 2022, 13(2): 272.
[18]	Kang SG, Lee KE, Singh M, et al. Rice Lesion Mimic Mutants(LMM): The current understanding of genetic mutations in the failure of ROS scavenging during lesion formation[J]. Plants, 2021, 10(8): 1598. doi: 10.3390/plants10081598 URL
[19]	Sindhu A, Janick-Buckner D, Buckner B, et al. Propagation of cell death in dropdead1, a sorghum ortholog of the maize lls1 mutant[J]. PLoS One, 2018, 13(9): e0201359. doi: 10.1371/journal.pone.0201359 URL
[20]	Wang J, Ye BQ, Yin JJ, et al. Characterization and fine mapping of a light-dependent leaf lesion mimic mutant 1 in rice[J]. Plant Physiol Biochem, 2015, 97: 44-51. doi: 10.1016/j.plaphy.2015.09.001 URL
[21]	Yang S, Hua JA. haplotype-specific resistance gene regulated by BONZAI1 mediates temperature-dependent growth control in Arabidopsis[J]. Plant Cell, 2004, 16: 1060-1071. doi: 10.1105/tpc.020479 URL
[22]	Gou M, Su N, Zheng J, et al. An F-box gene, CPR30, functions as a negative regulator of the defense response in Arabidopsis[J]. Plant J, 2009, 60: 757-770. doi: 10.1111/tpj.2009.60.issue-5 URL
[23]	Liu Q, Ning Y, Zhang Y, et al. OsCUL3a negatively regulates cell death and immunity by degrading OsNPR1 in rice[J]. Plant Cell, 2017, 29: 345-359. doi: 10.1105/tpc.16.00650 URL
[24]	Wolter M, Hollricher K, Salamini F, et al. The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype[J]. Mol Genet Genomics, 1993, 239: 122-128. doi: 10.1007/BF00281610
[25]	Li T, Bai G. Lesion mimic associates with adult plant resistance to leaf rust infection in wheat[J]. Theor Appl Genet, 2009, 119: 13-21. doi: 10.1007/s00122-009-1012-7 pmid: 19330313
[26]	Wang F, Wu W, Wang D, et al. Characterization and genetic analysis of a novel Light-Dependent lesion mimic mutant, lm3, showing adult-plant resistance to powdery mildew in common wheat[J]. PLos One, 2016, 11: e155358.
[27]	Liang BB, Wang H, Yang C, et al. Salicylic acid is required for broad-spectrum disease resistance in rice[J]. Int J Mol Sci, 2022, 23(3): 1354. doi: 10.3390/ijms23031354 URL
[28]	Cox KL. Stronger together: Ethylene jasmonic acid, and MAPK signaling pathways synergistically induce camalexin synthesis for plant disease resistance[J]. Plant Cell, 2022, 34(8): 2829-2830. doi: 10.1093/plcell/koac155 URL
[29]	Bouchez O, Huard C, Lorrain S, et al. Ethylene is one of the key elements for cell death and defense response control in the Arabidopsis lesion mimic mutant vad1[J]. Plant Physiol, 2007, 145(2): 465-477.
[30]	Wang Q, Chen X, Chai X, et al. The involvement of jasmonic acid, ethylene, and salicylic acid in the signaling pathway of clonostachys rosea-induced resistance to gray mold disease in tomato[J]. Phytopathology, 2019, 109(7): 1102-1114. doi: 10.1094/PHYTO-01-19-0025-R URL
[31]	Ding LN, Li YT, Wu YZ, et al. Plant disease resistance-related signaling pathways: recent progress and future prospects[J]. Int J Mol Sci, 2022, 23(24): 16200. doi: 10.3390/ijms232416200 URL
[32]	Yan J, Fang Y, Xue D. Advances in the genetic basis and molecular mechanism of lesion mimic formation in rice[J]. Plants, 2022, 11(16): 2169. doi: 10.3390/plants11162169 URL
[33]	王汉中. 我国油菜产业发展的历史回顾与展望[J]. 中国油料作物学报, 2010, 32(2): 300-302.
	Wang HZ. Review and future development of rapeseed industry in China[J]. Chin J Oil Crop Sci, 2010, 32(2): 300-302.
[34]	Neik TX, Barbetti MJ, Batley J. Current status and challenges in identifying disease resistance genes in Brassica napus[J]. Front Plant Sci, 2017, 8: 1788. doi: 10.3389/fpls.2017.01788 URL
[35]	Nagaharu U. Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization[J]. Japanese J Bot, 1935, 7: 389-452.
[36]	Allender CJ, King GJ. Origins of the amphiploid species Brassica napus L. investigated by chloroplast and nuclear molecular markers[J]. BMC Plant Biol, 2010, 10: 54. doi: 10.1186/1471-2229-10-54 URL
[37]	Lysak MA, Koch MA, Pecinka A, et al. Chromosome triplication found across the tribe Brassiceae[J]. Genome Res, 2005, 15(4): 516-525. doi: 10.1101/gr.3531105 pmid: 15781573
[38]	Chalhoub B, Denoeud F, Liu SY, et al. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome[J]. Science, 2014, 345(6199): 950-953. doi: 10.1126/science.1253435 pmid: 25146293
[39]	Beszterda M, Nogala-Kałucka M. Current research developments on the processing and improvement of the nutritional quality of rapeseed(Brassica napus L.)[J]. Eur J Lipid Sci Technol, 2019, 121(5): 1800045. doi: 10.1002/ejlt.v121.5 URL
[40]	沈金雄. 甘蓝型油菜杂种优势及其遗传分析[D]. 武汉: 华中农业大学, 2003.
	Shen JX. Heterosis and genetic analysis of Brassica napus L.[D]. Wuhan: Huazhong Agricultural University, 2003.
[41]	Wang Y, Hou YP, Chen CJ, et al. Detection of resistance in Sclerotinia sclerotiorum to carbendazim and dimethachlon in Jiangsu Province of China[J]. Australas Plant Pathol, 2014, 43(3): 307-312. doi: 10.1007/s13313-014-0271-1 URL
[42]	Clarkson JP, Fawcett L, Anthony SG, et al. A model for Sclerotinia sclerotiorum infection and disease development in lettuce, based on the effects of temperature, relative humidity and ascospore density[J]. PLoS One, 2014, 9(4): e94049. doi: 10.1371/journal.pone.0094049 URL
[43]	Rushton PJ, Reinstädler A, Lipka V, et al. Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling[J]. Plant Cell, 2002, 14(4): 749-762. doi: 10.1105/tpc.010412 pmid: 11971132
[44]	Xie Z, Zhang ZL, Zou XL, et al. Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells[J]. Plant Physiol, 2005, 137(1): 176-189. doi: 10.1104/pp.104.054312 pmid: 15618416
[45]	Qu D, Show PL, Miao X. Transcription factor ChbZIP1 from alkaliphilic microalgae Chlorella sp. BLD enhancing alkaline tolerance in transgenic Arabidopsis thaliana[J]. Int J Mol Sci, 2021, 22(5): 2387. doi: 10.3390/ijms22052387 URL
[46]	Buchel AS, Brederode FT, Bol JF, et al. Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo[J]. Plant Mol Biol, 1999, 40(3): 387-396. pmid: 10437823
[47]	Jeong YM, Chung WH, Mun JH, et al. De novo assembly and characterization of the complete chloroplast genome of radish(Raphanus sativus L.)[J]. Gene, 2014, 551(1): 39-48. doi: 10.1016/j.gene.2014.08.038 URL
[48]	Liu SY, Liu YM, Yang XH, et al. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes[J]. Nat Commun, 2014, 5: 3930. doi: 10.1038/ncomms4930

引物名称Primer name	引物序列Primer sequence（5'-3'）
BnaC05FAH-ty-F	AACCTACCACTGCCCTTT
BnaC05FAH-ty-R	TTTCCACCAACTTCCTGC
BnaA06FAH-ty-F	ACCATACATCATCCGTTT
BnaA06FAH-ty-R	TCAAGGCAGTGAAGGTAAAA
BnaC05FAH-Xba I -F	GCTCTAGACCATGGCGTTGCTCAAGTCTTT
BnaC05FAH-Sac I -R	CGAGCTCCATCAAGGCAGTGAAGGTAAAA
BnaA06FAH-Xba I -F	GCTCTAGAATGGCGTTGCTCAAGTCTTTCG
BnaA06FAH-Sac I -R	CGAGCTCTCAAGGCAGTGAAGGTAAAAT
BnaC05FAHpro-Xba I -F	GCTCTAGATTAAGGTAATGCTTTGATTCG
BnaC05FAHpro-Bgl II -R	GGAAGATCTGGACGATAAACAAACAGAT
BnaA06FAHpro-Xba I -F	GCTCTAGAAGTTCCCTAACGTTGTCCTCACTTT
BnaA06FAHpro-Bgl II -R	GGAAGATCTTGTATGACGAAGTTTCCAAAGC
qBnaC05FAH-F	AACCTACCACTGCCCTTT
qBnaC05FAH-R	CGTATGACGATGTTTCCG
qBnaA06FAH-F	TTTGGAAACTTCACCGAG
qBnaA06FAH-R	GAGTGTGGAGCAACATCG
ACTIN2-F	AGCACTTGCACCAAGCAGCATG
ACTIN2-R	ACGATTCCTGGACCTGCCTCATC

引物名称Primer name	引物序列Primer sequence（5'-3'）
BnaC05FAH-ty-F	AACCTACCACTGCCCTTT
BnaC05FAH-ty-R	TTTCCACCAACTTCCTGC
BnaA06FAH-ty-F	ACCATACATCATCCGTTT
BnaA06FAH-ty-R	TCAAGGCAGTGAAGGTAAAA
BnaC05FAH-Xba I -F	GCTCTAGACCATGGCGTTGCTCAAGTCTTT
BnaC05FAH-Sac I -R	CGAGCTCCATCAAGGCAGTGAAGGTAAAA
BnaA06FAH-Xba I -F	GCTCTAGAATGGCGTTGCTCAAGTCTTTCG
BnaA06FAH-Sac I -R	CGAGCTCTCAAGGCAGTGAAGGTAAAAT
BnaC05FAHpro-Xba I -F	GCTCTAGATTAAGGTAATGCTTTGATTCG
BnaC05FAHpro-Bgl II -R	GGAAGATCTGGACGATAAACAAACAGAT
BnaA06FAHpro-Xba I -F	GCTCTAGAAGTTCCCTAACGTTGTCCTCACTTT
BnaA06FAHpro-Bgl II -R	GGAAGATCTTGTATGACGAAGTTTCCAAAGC
qBnaC05FAH-F	AACCTACCACTGCCCTTT
qBnaC05FAH-R	CGTATGACGATGTTTCCG
qBnaA06FAH-F	TTTGGAAACTTCACCGAG
qBnaA06FAH-R	GAGTGTGGAGCAACATCG
ACTIN2-F	AGCACTTGCACCAAGCAGCATG
ACTIN2-R	ACGATTCCTGGACCTGCCTCATC

基因 Gene	顺式作用元件 Cis-acting element	核心序列 Core sequence	生物学功能 Biological function
BnaC05FAH BnaA06FAH AtFAH	CAAT-box	CCAAT/CAAT/CAAAT	启动子和增强子区的一般顺式作用元件 Common cis-acting element in promoter and enhancer regions
	TATA-box	ATTATA/TATACA/TATAAGAA	转录起始-30核心启动子元件 Core promoter element around -30 of transcription start
	as-1	TGACG	胁迫响应元件Stress responsive element
	CGTCA-motif	CGTCA	参与茉莉酸甲酯响应的顺式作用元件 Cis-acting regulatory element involved in the MeJA-responsiveness
	GT1-motif	GGTTAA	光响应元件Light responsive element
	O2-site	GTTGACGTGA	参与玉米醇溶蛋白代谢调控的顺式作用元件 Cis-acting regulatory element involved in zein metabolism regulation
	STRE	AGGGG	胁迫响应元件Stress responsive element
	TCT-motif	TCTTAC	部分光响应元件Part of a light responsive element
	TGACG-motif	TGACG	参与茉莉酸甲酯响应的顺式作用元件 Cis-acting regulatory element involved in the MeJA-responsiveness
BnaA06FAH AtFAH	ABRE	ACGTG/CACGTG	脱落酸响应元件Abscisic acid responsive element
	ABRE3a	TACGTG	脱落酸响应元件Abscisic acid responsive element
	ABRE4	CACGTA	脱落酸响应元件Abscisic acid responsive element
	DRE core	GCCGAC	脱水响应元件Dehydration responsive element
	G-box	TACGTG	光响应元件Light responsive element
	Sp1	GGGCGG	光响应元件Light responsive element
	WRE3	CCACCT	创伤响应元件Wounding responsive element

基因 Gene	顺式作用元件 Cis-acting element	核心序列 Core sequence	生物学功能 Biological function
BnaC05FAH BnaA06FAH AtFAH	CAAT-box	CCAAT/CAAT/CAAAT	启动子和增强子区的一般顺式作用元件 Common cis-acting element in promoter and enhancer regions
	TATA-box	ATTATA/TATACA/TATAAGAA	转录起始-30核心启动子元件 Core promoter element around -30 of transcription start
	as-1	TGACG	胁迫响应元件Stress responsive element
	CGTCA-motif	CGTCA	参与茉莉酸甲酯响应的顺式作用元件 Cis-acting regulatory element involved in the MeJA-responsiveness
	GT1-motif	GGTTAA	光响应元件Light responsive element
	O2-site	GTTGACGTGA	参与玉米醇溶蛋白代谢调控的顺式作用元件 Cis-acting regulatory element involved in zein metabolism regulation
	STRE	AGGGG	胁迫响应元件Stress responsive element
	TCT-motif	TCTTAC	部分光响应元件Part of a light responsive element
	TGACG-motif	TGACG	参与茉莉酸甲酯响应的顺式作用元件 Cis-acting regulatory element involved in the MeJA-responsiveness
BnaA06FAH AtFAH	ABRE	ACGTG/CACGTG	脱落酸响应元件Abscisic acid responsive element
	ABRE3a	TACGTG	脱落酸响应元件Abscisic acid responsive element
	ABRE4	CACGTA	脱落酸响应元件Abscisic acid responsive element
	DRE core	GCCGAC	脱水响应元件Dehydration responsive element
	G-box	TACGTG	光响应元件Light responsive element
	Sp1	GGGCGG	光响应元件Light responsive element
	WRE3	CCACCT	创伤响应元件Wounding responsive element

基因 Gene	顺式元件 Cis-acting element	序列 Sequence（5'-3'）	生物学功能 Biological function
BnaC05FAH	AC-I	（T/C）C（T/C）（C/T）ACC（T/C）ACC	木质部特异性表达顺式元件 Cis-acting regulatory element of xylem specific expression
	AE-box	AGAAACAA	光响应元件的一部分Part of a light responsive element
	AT1-motif	AATTATTTTTTATT	光响应元件的一部分Part of a light responsive element
	Box 4	ATTAAT	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
	ERE	ATTTTAAA	乙烯响应元件Ethylene responsive element
	GA-motif	ATAGATAA	光响应元件的一部分Part of a light responsive element
	WUN-motif	CAATTACAT	创伤响应元件Wounding responsive element
	MRE	AACCTAA	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
BnaA06FAH	A-box	CCGTCC	瞬时顺式调节元件Instantaneous cis-regulating element
	AAGAA-motif	gGTAAAGAAA	胚乳发育相关顺式作用元件 Cis-acting regulatory element involved in endosperm development
	GARE-motif	TCTGTTG	赤霉素响应元件Gibberellin responsive element
	TGA-element	TGACGTAA	生长素响应元件Auxin responsive element
	W-box	（T）TGAC（C/T）	病原诱导相关基因的顺式作用元件 Cis-acting regulatory element of pathogen-induction-related genes
AtFAH	ATCT-motif	AATCTAATCC	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
	circadian	CAAAGATATC	参与昼夜节律控制的顺式作用调节元件 Cis-acting regulatory element involved in circadian rhythm control
	G-Box	CACGTG	参与光反应的顺式作用调节元件 Cis-acting regulatory element involved in light response