Transcriptomics Analysis of the Influence of Nutrient-breaking on Nitrate Content in Brassica rapa L.ssp.chinensis L.

doi:10.7668/hbnxb.20193217

Abstract

Abstract:

In order to reduce the nitrate content of Brassica rapa L.ssp. chinensis L.,this study carried out nutrient-breaking treatment before the Brassica rapa L.ssp. chinensis L.was harvested.Physiological analysis of Brassica rapa L.ssp. chinensis L.nutrient-breaking treatment showed that Brassica rapa L.ssp.chinensis L.yield did not decrease significantly at 5 d,but the nitrate content of root,petiole and leaf decreased by 49.77%,23.90% and 33.39% respectively.Transcriptome comparison found that 301,2 270,and 2 271 differentially expressed genes(DEGs)were identified in Brassica rapa L.ssp.chinensis L.root,petiole,and leaf at 5 d,respectively.The Gene Ontology(GO)analysis revealed that these DEGs were mainly enriched in nitrogen(N),carbon(C)and reactive oxygen species(ROS)metabolism.In the process of N metabolism,nitrate uptake transporter NPF7.2 genes were down-regulated expression in root,petiole and leaf.The upstream regulator ERF104 of NPF7.2 were down-regulated expression in petiole and leaf.Nitrate retransporter NPF2.13 and NPF1.1 genes were up-regulated expression in leaf.Nitrate assimilation key enzyme glutamine synthetase(GS)genes were up-regulated expression in root and leaf.In the process of C metabolism,the sucrose phosphate synthase(SPS)genes,a key enzyme for sucrose synthesis in petiole and leaf,were up-regulated expression.In the process of ROS metabolism,superoxide dismutase(SOD)and ascorbate peroxidase(APX)genes were up-regulated expression in root.The expression of cytochrome P450,peroxisome and lipoxygenase genes were down-regulated expression in petiole and leaf.Catalase(CAT)and peroxidase(POD)genes were up-regulated expression in leaf.In summary,the nutrient-breaking treatment 5 d before harvesting can not only ensure that the yield of Brassica rapa L.ssp.chinensis L.was not affected,but also improve the quality of Brassica rapa L.ssp.chinensis L..

Key words: Brassica rapa L.ssp.chinensis L., Nitrate, Nutrient-breaking, N metabolism, C metabolism, ROS metabolism

SONG Danhua, JIAO Yonggang, SHI Linqi, YANG Yubo, GUO Jinghua, DONG Lingdi. Transcriptomics Analysis of the Influence of Nutrient-breaking on Nitrate Content in Brassica rapa L.ssp.chinensis L.[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 74-83. doi: 10.7668/hbnxb.20193217.

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Figures/Tables 12

Tab.1 The concentration of each ion in the nutrient solution

药品名称 Name of medicine	质量浓度/(mg/L) Concentration
$NO 3 -$	725.15
$SO 4 2 -$	721.54
K⁺	220.10
Mg²⁺	180.00
Ca²⁺	146.34
$PO 4 3 -$	125.74
EDTA^2-	21.67
Fe²⁺	3.33
$BO 3 3 -$	2.95
H⁺	2.80
Na⁺	1.72
Mn²⁺	0.87
Cl^-	0.49
Zn²⁺	0.27
$MoO 4 2 -$	0.19

Tab.1 The concentration of each ion in the nutrient solution

药品名称 Name of medicine	质量浓度/(mg/L) Concentration
$NO 3 -$	725.15
$SO 4 2 -$	721.54
K⁺	220.10
Mg²⁺	180.00
Ca²⁺	146.34
$PO 4 3 -$	125.74
EDTA^2-	21.67
Fe²⁺	3.33
$BO 3 3 -$	2.95
H⁺	2.80
Na⁺	1.72
Mn²⁺	0.87
Cl^-	0.49
Zn²⁺	0.27
$MoO 4 2 -$	0.19

Tab.2 DEGs and their primer sequences in qRT-PCR

基因名称 Gene name	基因ID Gene ID	功能注释 Gene annotation	引物序列(5'—3') Primer sequence (5'—3')
PDE	BnaC05G0211900ZS	丙酮酸脱氢酶E1组分亚基α-2	F: ATGGTATGGATGTACTGGCTGTG
			R: CAATTTTCGCACCCTTTCAAT
NRT1	BnaA01G0117900ZS	蛋白质NRT1/ PTR家族7.2	F: GTGATTGCGGTAATGGCGAT
			R: GACGATGGAAACCAGCAGACTA
NADH	BnaC06G0258800ZS	硝酸还原酶	F: CCGTTGGGCATATCGACTT
			R: TGGCAAACTTAGGTTTACCGTT
GD3	BnaC01G0508900ZS	谷氨酸脱氢酶3	F: TCAACAGGGAGAATGCAAATG
			R: GAAGCCGCGAGTCATGTAGTT
PLTC	BnaA07G0357800ZS	质体蓝素次要异构体	F: ATCGTGTTCAAGAACAACGCAG
			R: ACGGTGAGCTTTCCAACCAT
PL18	BnaA03G0481700ZS	果胶裂解酶18	F: TGTTGCTTGGGCATAGTGATAC
			R: GGAGCCACAAATCTATTCCCTT
CCM	BnaA02G0349200ZS	细胞色素P450	F: CCCTCACATGGCGTCAGAA
			R: ACGAACCCAGAGCCAAAGTC
LPG3	BnaA08G0261700ZS	脂肪氧化酶3	F: ACCGTCCACTTGGACCGG
			R: CTCGTAAGGCAAAACTCCAGC
IP1	BnaA07G0336100ZS	无机焦磷酸酶1	F: TATTTAGGAGATGGAGCTGGTGAT
			R: GCTTTCACCATCGGTCCATT
β-Actin			F: GGAATTGCTGACCGTATGAGC
			R: ACCACGAACCAGAAGGCAGA

Fig.1 Growth status of Brassica rapa L.ssp.chinensis L. after 5 d of broken nutrition treatment

Tab.3 Weight per plant in different periodskg

处理 Treatment	断营养 Broken nutrition	对照 CK
T₀	0.068±0.006a	0.068±0.006a
T₁	0.070±0.009a	0.072±0.008a
T₂	0.075±0.007a	0.080±0.008a
T₃	0.079±0.007a	0.086±0.007a
T₄	0.084±0.006a	0.094±0.008a
T₅	0.087±0.007a	0.102±0.007a
T₆	0.093±0.009a	0.112±0.008b

Fig.2 Nitrate content in different parts of Brassica rapa L.ssp.chinensis L.in different periods The dotted line indicates the nitrate content 3 100 mg/kg.

Tab.4 Quality statistics of filtered transcript group sequencing data

样品名称 Sample name	clean reads 数量 Number of clean reads	比对到参考基因组唯一位置的reads占 clean reads 的百分比 Percentage of unique mapped reads to clean reads	比对到参考基因组多处位置的reads 占clean reads 的百分比 Percentage of multiple mapped reads to clean reads	GC 含量 /% GC content	Q30 /%
根(对照-1)Root(CK-1)	26 835 645	62.73	2.80	47.56	92.31
根(对照-2)Root(CK-2)	23 523 067	81.04	4.17	47.71	92.51
根(对照-3)Root(CK-3)	22 008 544	81.30	3.98	47.49	93.10
根(断营养-1)Root(Broken nutrition-1)	24 949 367	81.56	4.07	47.57	91.95
根(断营养-2)Root(Broken nutrition-2)	24 268 227	81.41	3.99	47.61	92.73
根(断营养-3)Root(Broken nutrition-3)	22 717 607	81.62	4.18	47.62	92.16
叶柄(对照-1)Petiole(CK-1)	24 677 764	84.27	3.91	47.84	93.03
叶柄(对照-2)Petiole(CK-2)	25 146 080	84.17	4.17	47.73	93.36
叶柄(对照-3)Petiole(CK-3)	23 717 251	83.88	4.02	47.75	92.41
叶柄(断营养-1)Petiole(Broken nutrition-1)	23 112 395	84.08	3.92	47.62	92.85
叶柄(断营养-2)Petiole(Broken nutrition-2)	22 899 957	84.09	3.91	47.65	92.13
叶柄(断营养-3)Petiole(Broken nutrition-3)	23 787 469	84.63	3.80	47.88	93.10
叶片(对照-1)Leaf(CK-1)	21 507 242	80.93	4.44	47.48	91.87
叶片(对照-2)Leaf(CK-2)	23 149 355	83.73	4.49	47.66	93.02
叶片(对照-3)Leaf(CK-3)	21 890 795	83.46	4.53	47.91	92.36
叶片(断营养-1)Leaf(Broken nutrition-1)	24 079 426	83.57	5.23	48.32	93.19
叶片(断营养-2)Leaf(Broken nutrition-2)	22 826 628	83.86	4.60	47.82	92.99
叶片(断营养-3)Leaf(Broken nutrition-3)	22 926 956	83.10	5.31	48.07	92.80
平均Average	23 556 876	81.86	4.20	47.74	92.66

Fig.3 Correlation heatmap of gene expression Y415,Y416,Y417 are three replicates of the root(CK);Y385,Y386,Y387 are three replicates of the root(broken nutrition);Y373,Y374,Y375 are three replicates of the leaf(broken nutrition);Y403,Y404,Y405 are three replicates of the leaf(CK);Y409,Y410,Y411 are three replicates of the petiole(CK);Y379,Y378,Y379 are three replicates of the petiole(broken nutrition).

Fig.4 DEGs in root,petiole and leaf of Brassica rapa L.ssp. chinensis L.under nutrient-breaking treatment

Fig.5 Venn diagram of DEGs in different parts of Brassica rapa L.ssp.chinensis L. G0.DEGs of leaf ;G1.DEGs of petiole;G2.DEGs of root.

Fig.6 The term of biological function GO was significantly enriched in Brassica rapa L.ssp. chinensis L.under nutrient-breaking treatment The percentage of DEGs number 1.Glutathione metabolic process;2.Reactive nitrogen species metabolic process;3.Glutamate metabolic process;4.Nitrogen cycle metabolic process;5.Regulation of protein metabolic process;6.Histidine metabolic process;7.Protein complex subunit organization;8.Cellulose biosynthetic process;9.β-glucan biosynthetic process;10.Cellular glucuronidation;11.Glycosphingolipid metabolic process;12.(+)-abscisic acid D-glucopyranosyl ester transmembrane transport;13.Xylan biosynthetic process;14.Glucosamine-containing compound metabolic process;15.Chitin catabolic process;16. Amino sugar metabolic process;17.Polysaccharide catabolic process.

Fig.7 The term of molecular function GO was significantly enriched in Brassica rapa L.ssp. chinensis L.under nutrient-breaking treatment The percentage of DEGs number 1.L-tryptophan aminotransferase activity;2.Glutathione binding;3.IAA-amino acid conjugate hydrolase activity;4.Receptor serine/threonine kinase binding;5.Transmembrane receptor protein tyrosine kinase activity;6.Glutathione transferase activity;7.High-affinity secondary active ammonium transmembrane transporter activity;8.Oxidoreductase activity,oxidizing metal ions,NAD or NADP as acceptor;9.Oxidoreductase activity,acting on diphenols and related substances as donors;10.Oxidoreductase activity,acting on the aldehyde or oxo group of donors,oxygen as acceptor;11.Oxidoreductase activity,acting on the CH-CH group of donors,NAD or NADP as acceptor;12.Oxidoreductase activity,oxidizing metal ions;13.α-D-xyloside xylohydrolase;14.Glucomannan 4-β-mannosyltransferase activity;15.Galactosidase activity;16.Mannose-6-phosphate isomerase activity;17.(+)-abscisic acid D-glucopyranosyl ester transmembrane transporter activity;18.Cellobiose glucosidase activity;19.Glucoside transmembrane transporter activity;20.UDP-glucosyltransferase activity;21.Cellulose synthase(UDP-forming)activity;22.β-glucanase activity;23.Xyloglucan-specific endo-β-1,4-glucanase activity;24.Chitin binding.

Fig.8 qRT-PCR verification of DEGs identified by transcriptome analysis PDE,NRT1,NADH and GD3 are derived from the root;PLTC and PL18 come from the petiole;CCM,LPG3 and IP1 come from the leaf.

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