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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (2): 197-209.doi: 10.3724/SP.J.1006.2021.02034

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Effect of OsPAL2;3 in regulation of rice allopathic inhibition on barnyardgrass (Echinochloa crusgalli L.)

LI Lan-Lan(), MU Dan, YAN Xue, YANG Lu-Ke, LIN Wen-Xiong*(), FANG Chang-Xun*()   

  1. Institute of Agroecology, School of Life Sciences, Fujian Agriculture and Forestry University / Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fuzhou 350002, Fujian, China
  • Received:2020-05-14 Accepted:2020-08-19 Online:2021-02-12 Published:2020-11-19
  • Contact: LIN Wen-Xiong,FANG Chang-Xun E-mail:1244311185@qq.com;lwx@fafu.edu.cn;changfangxingyx@163.com
  • Supported by:
    National Natural Science Foundation of China(31871556);Outstanding Youth Scientific Fund of Fujian Agriculture and Forestry University(xjq201805);Science and Technology Innovation of Fujian Agriculture and Forestry University(CXZX2018042)

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Abstract:

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the key enzyme in regulation of the synthesis of phenolic acid allelochemicals. PAL gene in rice belongs to a multigene family. In allelopathic accession rice PI312777 and non-allelopathic rice accession Lemont, the promoter sequences of the same PAL gene member were different, and there was the largest difference in OsPAL2;3 and OsPAL2;4 gene promoter sequence. Gene promoter of OsPAL2;3 from PI3127777 showed higher activity than the corresponding promoter from Lemont. Overexpression of OsPAL2;3 in PI312777 and Lemont resulted in increasing allelopathic inhibition on barnyardgrass (Echinochloa crusgalli L.), and the inhibitory ratios was increased by 11.11% in PI312777 and 5.56% in Lemont. Gene expression level of OsPAL2;3, OsC4H, OsCCA, OsCOL, and OsOMT was up-regulated in the OsPAL2;3-overexpressed transgenic rice compared with that of wild-type rice, and the contents of protocatechuic acid and vanillic acid were also increased. The results from Co-IP combined with mass spectrometry showed that transketolase, carbonic anhydrase, fructose-bisphospate aldolase isozyme, ATP synthase subunit alpha and ATP synthase subunit beta were interacted with OsPAL2;3 protein, resulting in regulating the phenylalanine pathway in rice. Our study indicated that the transcriptional activity of OsPAL2;3 contributed to the alleloapthic activity between PI312777 and Lemont, OsPAL2;3 was interacted with a couple of proteins to jointly regulate the synthesis of phenolic acids, and OsPAL2;3 could be considered as a candidate gene to improve the allelopathy of rice in breeding.

Key words: rice, allelopathy, PAL gene, promoter, phenolic acid, protein interaction

Table 1

Primers used in this study"

功能
Function		 基因名称
Gene name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
启动子克隆
Promoter cloning		 OsPAL2;1 GGTAGTGCTAGATATGAAGGGCTGC CACGAGGAGAAGAGAGGATTCGAT
OsPAL2;2 GTTTTCCGAACGGGTAACCGATGTG CTCACAGATGCAGTAGTAGCACACG
OsPAL2;3 GGTACTGGTACCGCCAGTACGCATA AGTTAGCTAGACGGCCGGAGAGAAC
OsPAL2;4 GGGTCATATGTGACACGTCATCACA CAGAAGATGGATCGGTTAACCTAGC
OsPAL4;1 ATGAGCTGTGAGGCCCAGCAAGCAG AACAACGACGAGGAGGAGGAGGAGG
OsPAL4;2 CGTCCCGTAATACAGGGGATTTTGG GAAGCAGAGGGGAGATCGATGTGTA
OsPAL11 TAGAAGATCAGTTCGCTCCCAGGCT TACAGGTCGCCGAGTACGTACGTAT
荧光定量PCR
qRT-PCR		 OsPAL2;3 CCGTGCTCTTTGAGGCTAAC GCTTGTGAGTCAGGTGGTCG
OsC4H ACCGCAGCGTCTCCTTC ACCACCCGAGCATCCAG
OsCOL TGGTGGAGTGCGTGCTG AGGCGTTGGCGTAGATG
OsOMT TGTCCTGTGAAATGGGTG CCTCGGAACAAGAACTG
OsCAD TCGGCGTCGCTAATTTCATCC TCGATGGAAGAACGGGCAGAG
β-actin CTGCGGGTATCCATGAGACT GCAATGCCAGGGAACATAGT
基因过表达载体
Gene overexpression vector		 OsPAL2;3 cgactctagaggatccATGGCGTGCGAGA ACGGTCAG ccatactagtggatccGCAGATGGGCAGGG GCGCGC
转基因水稻PCR鉴定
PCR identification of transgenic rice line		 OsPAL2;3-OX CTGAAGCTCATGTCCTCCACGTTC GATGTTGCCGTCCTCCTTGAAGTC
双分子荧光互补载体
Bimolecular fluorescent complimentary vector		 OsPAL2;3 atggcgcgccactagtATGGCGTGCGAGA ACGGTCAG cacctcctccactagtGCAGATGGGCAGGGG CGCGC
ATP synthase subunit alpha atggcgcgccactagtATGGCAACCCTTCG AGTCGACGA cacctcctccactagtAAGGGAAAACCGTTC GAGTTGTTCC
ATP synthase subunit beta atggcgcgccactagtATGGCGACTCGCCG GGCCCTCT cacctcctccactagtTGAAGCCGACTCCTTG GCGATC
Transketolase atggcgcgccactatATGGCCGCGCACTCC GTCG cacctcctccactagtCAGGCTCTTTGCTGTT GCGAT
Carbonic anhydrase atggcgcgccactagtATGTCGACCGCCGC CGCCGC cacctcctccactagtGGGCTCCCATAAGTCC AAGTTG
Frutose-bisphosphate
aldolase		 atggcgcgccactagtATGGCGTCTGCTAC TCTCCTCAA cacctcctccactagtGTAGACGTAGTTCTTG ACGAACA

Fig. 1

Agarose gel electrophoresis determination and partially different sequence on the gene promoters of PAL gene members from PI312777 and Lemont (a) Agarose gel electrophoresis determination on the fragment of PAL gene promoter; (b) Different sequence of OsPAL2;3 or OsPAL2;4 gene promoters from PI312777 and Lemont, respectively."

Fig. 2

Comparative determination on GFP fluorescence transcriptionally regulated by OsPAL2;3 and OsPAL2;4 gene promoters from PI312777 and Lemont"

Fig. 3

Detection of the protein expression level of eYFP and eYFP fluorescent intensity on OsPAL2;3 overexpressed transgenic rice (a) Western-blotting detection of the protein expression level of eYFP; (b) Laser scanning confocal microscope detection of the eYFP fluorescent intensity. WT: wild type."

Fig. 4

Expression level of OsPAL, OsOMT, OsC4H, OsCOL, and OsCAD from OsPAL2;3-OX transgenic rice line and its wild type"

Table 2

Contents of phenolic acids in the OsPAL2;3-OX transgenic rice lines and its wild type"

水稻株系
Rice lines		 酚酸含量Contents of phenolic acid (μg g-1)
原儿茶酸Protocatechuic acid 对羟基苯甲酸
p-hydroxybenzoic acid		 香草酸
Vanillic acid		 丁香酸
Syringic acid		 4-香豆酸
4-coumaric acid		 阿魏酸
Ferulic acid		 水杨酸
Salicylic acid		 肉桂酸
Cinnamic acid
PI32777野生型
Wild type of PI312777		 161.78±32.74 2.18±0.14 6.98±1.68 3.19±0.71 25.26±1.65 19.65±0.57 15.34±2.43 8.19±0.62
过表达OsPAL2;3转基因PI312777
OsPAL2;3-OX
transgenic PI312777		 165.11±2.41 1.74±0.41 10.05±0.97 4.77±2.40 31.42±2.30 15.56±0.60 10.64±0.92 8.07±0.20
Lemont野生型
Wild type of Lemont		 18.19±1.38 2.04±0.47 5.47±0.40 6.64±0.35 11.31±0.15 21.07±1.89 15.03±0.13 1.60±0.13
过表达OsPAL2;3转基因Lemont
OsPAL2;3-OX
transgenic Lemont		 26.29±1.81 1.83±0.14 6.45±0.65 3.61±0.37 9.01±4.28 17.64±1.74 17.06±5.61 0.85±0.56

Fig. 5

Phenotype of barnyardgrass growth in the solution with root exudates from OsPAL2;3-OX transgenic rice and the wild type"

Table 3

Root length, plant height and dry weigh of barnyardgrass growth in the solution with root exudates from OsPAL2;3-OX transgenic rice and the wild type"

处理方式
Treatment		 根长
Root length (cm)		 根长抑制率
IR of root length (%)		 株高
Plant height (cm)		 株高抑制率
IR of plant height (%)		 干重
Dry weight
(g)		 干重抑制率
IR of dry weight (g)
PI312777根系分泌物培养的稗草
Barnyardgrass cultured in the solution with PI31277 root exudates		 9.20±1.43 b 18.73 27.84±3.77 b 14.69 0.12±0.02 b 33.33
过表达OsPAL2;3转基因PI312777根系分泌物培养的稗草
Barnyardgrass cultured in the solution with OsPAL2;3-OX PI31277 root exudates		 6.87±2.00 c 39.31 27.57±2.87 b 15.61 0.10±0.02 b 44.44
对照组稗草
Control group of barnyardgrass		 11.32±2.11 a — 32.67±3.18 a — 0.18±0.01 a —
Lemont根系分泌物培养的稗草
Barnyardgrass cultured in the solution with Lemont root exudates		 9.94±1.20 b 12.19 28.89±3.19 b 11.57 0.14±0.02 b 22.22
过表达OsPAL2;3转基因Lemont根系分泌物培养的稗草
Barnyardgrass cultured in the solution with OsPAL2;3-OX Lemont root exudates		 10.36±1.28 b 8.48 27.32±3.33 b 16.38 0.13±0.01 b 27.78

Fig. 6

OsPAL2;3 interacted proteins from the OsPAL2;3-OX transgenic rice"

Table 4

Identification of the proteins interaction with OsPAL2;3"

蛋白登录号
Protein accession number		 蛋白名称
Protein name		 全部肽段
Peptide count		 匹配的肽段
Unique peptide count		 覆盖率
Coverage
(%)		 分子量
Molecular weight (kD)		 等电点
Isoelectric point
LOC_Os02g41670.1 Phenylalanine ammonia-lyase 31 26 36.19 76.58 5.84
LOC_Os10g21268.1 Ribulose bisphosphate carboxylase large chain precursor 29 16 30.99 53.71 6.58
LOC_Os04g16740.1 ATP synthase subunit alpha 15 15 27.22 55.66 5.95
LOC_Os01g45274.3 Carbonic anhydrase, chloroplast precursor 25 14 42.37 28.04 6.99
LOC_Os02g41650.3 Phenylalanine ammonia-lyase 15 13 17.69 77.75 6.04
LOC_Os10g21266.1 ATP synthase subunit beta 14 13 29.92 53.98 5.38
LOC_Os11g07020.1 Fructose-bisphospate aldolase isozyme 11 11 27.06 42.00 6.39
LOC_Os12g10580.1 Ribulose bisphosphate carboxylase large chain precursor 18 10 18.94 56.06 9.04
LOC_Os04g38600.2 Glyceraldehyde-3-phosphate
dehydrogenase		 12 10 24.94 43.01 7.61
LOC_Os04g43800.1 Phenylalanine ammonia-lyase 11 10 13.59 76.93 5.93
LOC_Os06g04270.1 Transketolase, chloroplast precursor 11 10 14.67 80.02 6.12
LOC_Os05g41640.2 Phosphoglycerate kinase protein 10 10 21.28 50.13 6.29
LOC_Os12g37260.1 Lipoxygenase 2.1, chloroplast precursor 10 10 9.00 10.46 5.87

Fig. 7

BiFC determination of the interaction between OsPAL2;3 and the interacted proteins"

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