Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (6): 1081-1094.doi: 10.3864/j.issn.0578-1752.2020.06.001

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

Transcriptome Research of Erect and Short Panicle Mutant esp in Rice

KunNeng ZHOU,JiaFa XIA,Peng YUN,YuanLei WANG,TingChen MA,CaiJuan ZHANG,ZeFu LI()   

  1. Rice Research Institute, Anhui Academy of Agricultural Sciences/Key Laboratory for Rice Genetics and Breeding of Anhui Province, Hefei 230001
  • Received:2019-08-05 Accepted:2019-10-21 Online:2020-03-16 Published:2020-04-09
  • Contact: ZeFu LI E-mail:lizefu@aliyun.com

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Abstract:

【Objective】In this study, we aimed to identify the ESP gene, whose mutation caused a phenotype, namely erect and short panicle, and to determine its regulatory role in the gene network that controls the related agronomic traits (e.g., plant types and panicle length).【Method】In this study, agronomic traits, such as plant height, panicle length and grain length at mature stages, were used as phenotypic marks to trace the esp mutant. Individuals carrying mutant phenotypes were selected from the F2 population to cross with indica and japonica for further gene mapping and genome sequencing that were used to map the potential mutation region/sites. The bioinformatics software was used to analyze phylogenetic tree and gene expression. The total RNAs isolated from wild type and mutants were used for transcriptome RNA-seq analysis. The differential expressed genes and expression levels of genes related to plant hormone signal transduction and protein processing in endoplasmic reticulum were analyzed by GO software and KEGG database. The transcriptome data were verified by qRT-PCR.【Result】Phenotypic analysis showed that the esp mutant exhibited a erect panicle architecture. The plant height, panicle length, grain length and the number of spikelets per panicle were decreased in the esp mutants, when compared to wild type control, whereas grain width and the 1 000-grain weight were increased, although no obvious difference in the number of effective panicles between mutants and control. The ESP gene was mapped to a 7.58 Mb interval between markers C7-11 and C7-14 on the long arm of rice 7th chromosome by using the F2 population of esp mutant and PA64. Genome sequencing demonstrated that a single nucleotide change (G to A) at the junction of 6th intron and 7th exon of LOC_Os07g42410, which led to a splicing defect, causing premature protein translation. The ESP was allelic to OsDEP2/OsEP2. Phylogenetic analysis revealed that the ESPs are widely present in monocot and dicot plants. Expression analysis predicted that ESP gene was highly expressed in stem, inflorescence, pistil, glumelle, lemma and ovary, and the expression level was gradually decreased with the ovary inflation. Transcriptome RNA-seq analysis of young panicle identified 630 differential expressed genes in esp mutants versus wild type, including 235 up-regulated and 395 down-regulated. GO and qRT-PCR analysis revealed that genes involved in plant hormone signal transduction and protein processing in endoplasmic reticulum were misregulated in esp mutants.【Conclusion】The ESP gene was allelic to OsDEP2/OsEP2, the mutation of which leads to multiple phenotypes, such as decreased plant height and shorter panicle length. Transcriptome analysis suggested that the ESP gene might affect plant development by regulating genes expression associated with plant hormone signal transduction and protein processing in endoplasmic reticulum.

Key words: rice (Oryza sativa L.), erect and short panicle mutant, gene cloning, phylogenetic analysis, transcriptome analysis

Table 1

Agronomic traits of the esp mutant and WT"

农艺性状Agronomic traits 野生型WT 突变体esp
株高Plant height (cm) 79.1±1.7 67.2±1.5**
有效穗数Number of effective panicles 14.8±1.7 14.9±1.2
穗长Panicle length (cm) 21.06±0.85 17.03±0.70**
每穗粒数Grain number per panicle 190.5±15.2 136.7±8.8**
剑叶长Flag leaf length (cm) 26.60±1.53 36.07±2.64**
剑叶宽Flag leaf width (cm) 1.86±0.05 1.83±0.08
千粒重1000-grain weight (g) 24.33±0.34 25.34±0.42*

Fig. 1

Phenotypic characteristics of the esp mutant and wild type (WT) A: Plant phenotypes of mature stage; B: Panicle phenotypes of mature stage; C, D and E: Determination of seed width and length"

Fig. 2

Map-based cloning of the ESP gene A: Initial mapping of the ESP gene; B: Structure of the ESP gene and mutant site of esp; ATG and TGA indicate start and stop codons, respectively"

Fig. 3

Phylogenetic analysis of ESP and its related proteins"

Fig. 4

Expression analysis of ESP gene A: Expression profile of ESP gene in different tissues at various stages; B: Expression analysis of ESP gene during the progress of ovary development; Data were collected from the rice expression profile database, RiceXPro"

Fig. 5

Transcriptome analysis of young panicles in esp mutant and WT A: Volcano Plot of transcriptome analysis of young panicles in esp mutant and wild type; Red and green dots separately represent up- and down-regulated genes, blue dots indicate genes with no significant difference; B: Numbers of up-regulated and down-regulated genes expressed differentially more than 1.5 time; C: Functional classification of differentially expressed genes"

Table 2

Metabolic pathways and the number of differentially expressed genes"

序号 No． 通路名称 Pathway name 差异基因数 DEGs P值 P value
1 次生代谢产物生物合成 Biosynthesis of secondary metabolites 86 5.47×10-3
2 乙醛酸和二羧酸代谢 Glyoxylate and dicarboxylate metabolism 12 6.19×10-3
3 淀粉和蔗糖代谢 Starch and sucrose metabolism 22 8.09×10-3
4 内质网蛋白加工 Protein processing in endoplasmic reticulum 28 9.59×10-3
5 苯丙素生物合成 Phenylpropanoid biosynthesis 19 1.30×10-2
6 脂肪酸伸长 Fatty acid elongation 6 2.36×10-2
7 植物激素信号转导 Plant hormone signal transduction 24 2.69×10-2
8 氮代谢 Nitrogen metabolism 6 3.63×10-2
9 氰基氨基酸代谢 Cyanoamino acid metabolism 7 4.23×10-2
10 酪氨酸代谢 Tyrosine metabolism 7 4.71×10-2
11 丙氨酸、天冬氨酸和谷氨酸代谢 Alanine, aspartate and glutamate metabolism 7 6.99×10-2

Fig. 6

Expression analysis of genes related to plant hormone signal transduction A: Pathway of plant hormone signal transduction; B: Expression of genes related to plant hormone signal transduction in transcriptome analysis"

Fig. 7

Expression analysis of genes related to protein processing in endoplasmic reticulum A: Pathway of protein processing in endoplasmic reticulum; B: Expression of genes related to protein processing of endoplasmic reticulum in transcriptome analysis"

Fig. 8

Quantitative PCR analysis of genes involved in plant hormone signal transduction and protein processing of endoplasmic reticulum between mutant and WT A: Expression analysis of genes related to plant hormone signal transduction; B: Expression analysis of genes related to protein processing of endoplasmic reticulum; * and ** separately indicate significance at P=0.05 and P=0.01 by Student's t test"

Table 3

Phenotypic characteristics of ESP allelic mutant"

突变体
Mutant		 野生型
Wild type		 株高
Plant height		 穗长
Panicle length		 每穗粒数
Grain number per panicle		 粒长
Grain length		 粒宽
Grain width		 千粒重
1000-grain weight
esp 宁粳36（粳）
Ningjing 36 (Japonica)		 降低
Decrease		 变短
Shorten		 减少
Reduce		 降低
Decrease		 增加
Increase		 轻微增加
Slightly increase
ep2-1 中籼3037（籼）
Zhongxian 3037 (Indica)		 降低
Decrease		 变短
Shorten		 减少
Reduce		 降低
Decrease		 增加
Increase		 降低
Decrease
ep2-2 9311（籼）
9311 (Indica)		 降低
Decrease		 变短
Shorten		 增加
Increase		 降低
Decrease		 增加
Increase		 轻微降低
Slightly decrease
dep2-1/dep2-2 中花11/日本晴（粳）
Zhonghua 11/Nipponbare (Japonica)		 降低
Decrease		 变短
Shorten		 无差别
No difference		 降低
Decrease		 增加
Increase		 降低
Decrease
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