谷子叶绿体基因RNA编辑位点的鉴定与分析

doi:10.3724/SP.J.1006.2022.14043

Abstract

Abstract:

RNA editing is one of the post-transcriptional regulation mechanisms of gene expression in the chloroplast genomes of higher plants, which affects the chloroplast development and leads to albino phenotype or yellow phenotype of plant leaves. In this study, chlorophyll content was measured with a UV spectrophotometer at seeding stage among Changnong 35, E752, and E1005, and chloroplast structure of leaves was observed with a transmission electron microscopy; the online tool Prep-CP was used to predict the RNA editing sites of chloroplast genes; RNA editing site was verified by PCR, RT-PCR, and sequencing method, and the editing sites were compared and analyzed between foxtail millet and other monocotyledon. Furthermore, the relative expression patterns of rpoB and PEP-transcription-dependent photosynthetic pathway related genes (ndhG, psaA, psbA, and rbcL) were analyzed by qRT-PCR at different developmental stages, and the secondary structure of rpoB protein before and after editing was analyzed by bioinformatics. The results showed that chlorophyll content of E752 and E1005 were significantly lower and their chloroplasts were abnormal compared with Changnong 35. A total of 20 RNA editing sites of 10 chloroplast genes were identified, among which all were C to U conversion; the number of editing sites among chloroplast genes was varied, and ndhB had the most editing sites with the number of 6. Among the 20 editing sites, 19 editing sites were highly conserved in the evolution of species, however rpoC1-2753 was a unique editing site in foxtail millet. The editing efficiency of rpoB-467, rpoB-545, and rpoB-560 was distinctly different from those of the other editing sites among Changnong 35, E752, and E1005, leading to the expression level change of ropB, which might further affect the expression levels changes of ndhG, psaA, psbA, and rbcL. Bioinformatics analysis revealed that secondary structure of rpoB protein was changed due to the RNA editing of rpoB-467 and rpoB-560. Our results laid a foundation for the molecular mechanism analysis of chloroplast RNA editing in chloroplast development of foxtail millet.

Key words: foxtail millet, chloroplast, RNA editing, mutant

DU Xiao-Fen, WANG Zhi-Lan, HAN Kang-Ni, LIAN Shi-Chao, LI Yu-Xin, ZHANG Lin-Yi, WANG Jun. Identification and analysis of RNA editing sites of chloroplast genes in foxtail millet [Setaria italica (L.) P. Beauv.][J].Acta Agronomica Sinica, 2022, 48(4): 873-885.

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引物名称 Marker name	引物序列 Sequence (5'-3')	产物大小 Product size (bp)	引物名称 Marker name	引物序列 Sequence (5'-3')	产物大小 Product size (bp)
atpA-F1	TCGCTTAATTGAATCTCCTG	946	rpoB-F1	ATCAGGGCTTGGCAGAAGA	875
atpA-R1	CGTTCCGGTATAAATGGTAG		rpoB-R1	ATCCGCAACCGAACGAATA
ccsA-F1	TCTTAGTTTCTCGTTGGGTTTC	641	rpoC1-F1	AGGACGACTCAGAAGAAGAAT	1042
ccsA-R1	TGCAAATATGGTCCAGGTAAT		rpoC1-R1	GAACAGTTGGATGCCGAC
matK-F1	GAGGGGTATTCAGAAAAACA	918	rps8-F1	ATGGGCAAGGACACTATTG	404
matK-R1	TTGTGAGAAATTGACAAGGTAA		rps8-R1	ATATAACATAAGACTTCTCCCC
matK-F2	CACTTTTCTGGGAAGATGGA	780	rps14-F1	AAAAGTTTGATTCAGAGGGAG	302
matK-F2	CACCAGGTCATTGATACGG		rps14-R1	TACCAACTGGATCTTGTTGC
ndhA-F1	ATGGATTCTACCCATTTTGAC	982	ycf3-F1	CTAGATCCCGTATAAATGGAA	498
ndhA-R1	TGTTAATAAGAGATTGCCCAG		ycf3-R1	GCTTCGTAATCTTCAACCAGT
ndhB-F1	TTTATGTGGTGCTAACGATT	1073	ndhG-Real-Time-F	GCCTTTTCGCTGGGATTAG	155
ndhB-R1	GGTTCATTGATATTCCTGGT		ndhG-Real-Time-R	GACCACTCTGAGCCGTTTACA
ndhD-F1	ACTTGTTGTTTTGCCGATAT	759	psaA-Real-Time-F	TTTCTTAGGCGCTCATTTTGTC	151
ndhD-R1	ATCAATCCGTATGCTCCC		psaA-Real-Time-R	TATAATGCTCAAGGCTCTAGGC
ndhF-F1	TAATCCCTCTTCTCCCACTT	1132	psbA-Real-Time-F	TTATGATTGTATTCCAGGCGGA	154
ndhF-R1	AAACCACCCATAAGAACCAT		psbA-Real-Time-R	GCAGACTCATTTTCAGTGGTTT
ndhF-F2	TGATCACTCATGCTTATTCGA	1010	rbcL-Real-Time-F	CCGAAATCTTTGGAGACGATTC	105
ndhF-R2	ACCAGCAAGACCTACTCCAT		rbcL-Real-Time-R	CTTCTAAAGCCACACGATTAGC
petB-F1	ATGAGTATGAAATTTTCATATAC	689	rpoB-Real-Time-F	AAACGTATTCGTTCGGTTGC	135
petB-R1	ATACCTTGCTTACGTATCA		rpoB-Real-Time-R	TACCAAAGTTTGTGGAGTCGG
rpl20-F1	AGAGTTCCGCGAGGATAT	325	SiActin-7-F	TGATCTCACTGACAGTCTGATG	88
rpl20-R1	TCGTGTAAAGATTATTTGGATT		SiActin-7-R	GATGTCTCTTACAATTTCCCGC

材料 Material name	总叶绿素含量 Total chlorophyll	叶绿素a Chlorophyll a	叶绿素b Chlorophyll b	类胡萝卜素 Carotenoid
长农35号 Changnong 35	2.12±0.060	1.68±0.019	0.45±0.004	0.69±0.016
E752	1.55±0.050^**	1.28±0.072^*	0.27±0.031^**	0.36±0.010^**
E1005	0.99±0.017^**	0.76±0.018^**	0.24±0.002^**	0.34±0.008^**

基因 Gene ID	密码子位置 Codon position	密码子变化 Codon conversion	氨基酸转变 Amino acid conversion	长农35号 Changnong 35	E752	E1005
atpA	1148	uCa→uUa	S→L	+	+	+
ccsA	641	aCu→aUu	T→I	-	-	-
matK	331	Cuu→Tuu	L→F	-	-	-
	1258	Cau→Uau	H→Y	-	-	-
ndhA	47	uCg→uUg	S→L	+	+	+
	470	uCa→uUa	S→L	+	+	+
	560	uCa→uUa	S→L	+	+	+
ndhB	467	cCa→cUa	P→L	+	+	+
	586	Cau→Uau	H→Y	+	+	+
	611	uCa→uUa	S→L	+	+	+
	737	cCa→cUa	P→L	+	+	+
	830	uCa→uUa	S→L	+	+	+
	1481	cCa→cUa	P→L	+	+	+
ndhD	878	uCa→uUa	S→L	+	+	+
ndhF	62	uCa→uUa	S→L	-	-	-
	1834	Cuu→Tuu	L→F	-	-	-
	1909	Cuu→Tuu	L→F	-	-	-
petB	668	cCa→cUa	P→L	-	-	-
rpl20	308	uCa→uUa	S→L	+	+	+
rpoB^*	467	uCa→uUa	S→L	+/-	+/-	+/-
	545	uCa→uUa	S→L	+/-	+/-	+/-
	560	uCa→uUa	S→L	+/-	+/-	+/-
	617	cCa→cUa	P→L	+	+	+
rpoC1	2753	uCa→uUa	S→L	+	+	+
rps8	182	uCa→uUa	S→L	+	+	+
rps14	80	uCa→uUa	S→L	+	+	+
ycf3	44	uCc→uUc	S→F	-	-	-
	191	aCg→aUg	T→M	+	+	+

基因 Gene	密码子位置 Codon position	谷子 Setaria italica	水稻 Oryza sativa	黑麦 Lolium perenne	玉米 Zea mays	小麦 Triticum asetivum	大麦 Hordeum vulgare	野生二粒小麦 Triticum dicoccoides	乌拉尔图小麦 Triticum urartu
atpA	1148	+	+	+	+	+	+	+	+
ccsA	641	-
matK	1258	-	+	+/-			+	+	+
ndhA	47	+	-	+	+		+	-
	470	+	+	+	+	+	+	+	+
	560	+	+	+	+	+	+	+	+
ndhB	467	+	+	+	+	+	+	+	+
	586	+	+	+	+	+	+	+	+
基因 Gene	密码子位置 Codon position	谷子 Setaria italica	水稻 Oryza sativa	黑麦 Lolium perenne	玉米 Zea mays	小麦 Triticum asetivum	大麦 Hordeum vulgare	野生二粒小麦 Triticum dicoccoides	乌拉尔图小麦 Triticum urartu
	611	+	+/-	+	+	+	+	+	+
	737	+	+	+	+	+	+	+	-
	830	+	+	+	+	+	+	+	+
	1481	+	+/-	+	+	+	+	+	+
ndhD	878	+	+	+/-	+	+	+	+	+
ndhF	62	-	+	+	+	+	+	-	+
petB	668	-	-	+	+	-	+		+
rpl20	308	+	-	+/-	+	+	+	+	+
rpoB	467	+/-	+/-	+/-	+	+/-	+	+	+
	545	+/-	+/-	+/-	+	+/-	+	+	+
	560	+/-	+/-	+/-	+	+/-	+	+	+
	617	+	-	-	+		+	+	+
rpoC1	2753	+
rps8	182	+	+	+	+	+	-	+	+
rps14	80	+	+	-	+	-		-	+
ycf3	44	-	-	+	+	+		+	+
	191	+	+/-	+	+/-	+		+	+

Identification and analysis of RNA editing sites of chloroplast genes in foxtail millet [Setaria italica (L.) P. Beauv.]

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