Gene Identification and Functional Analysis of Yellowish and Early Heading Mutant hz1 in Rice

doi:10.13560/j.cnki.biotech.bull.1985.2024-0132

Abstract

Abstract:

【Objective】 The heading date of rice（Oryza sativa）is an important agronomic trait and crucial for the regional adaptability and yields. Identification and functional analysis of heading date-associated genes may provide important gene resources for the breeding in rice. 【Method】 BSA-seq was performed to identify target gene of a yellowish and early heading mutant huangzao 1（hz1）. RT-qPCR was used to analyze the expression profile of target genes. The subcellular localization of HZ1 was determined by transient transformation between rice protoplasts and tobacco cells. Meanwhile, the heading dates, chlorophyll content and hydrogen peroxide content were measured to analyze the phenotype of hz1 mutant. 【Result】 Field phenotypic observations found that hz1 showed early heading phenotype. The heading date of hz1 mutant was the same under long-day（LD）and short-day（SD）conditions, which were 43 d and 26 d earlier than those of wild type（WT）respectively, indicating that hz1 was a photoperiod-insensitive mutant. Moreover, hz1 demonstrated a yellowish phenotype, with a decrease in chlorophyll content compared with WT. Genetic analysis revealed that hz1 was controlled by a recessive gene. BSA-seq（Bulk segregation analysis with whole-genome sequencing）of F₂ pools indicated that the causal gene was linked with molecular markers on the chromosome 6 with 17.8 Mb region. Further analysis demonstrated that LOC_Os06g40080 site with a T-DNA insertion was completely linkage with hz1 mutant. LOC_Os06g40080 site was an identified SE5 gene encoding heme oxygenase 1（HO1）. Expression pattern analysis revealed that HZ1/SE5 was highly expressed in the leaves and dhad diurnal rhythm expression. Subcellular localization assay showed that HZ1/SE5 protein was localized in chloroplasts. Gene expression analysis showed that HZ1/SE5 regulated the expression of florigen genes Hd3a and RFT1 to modulate the heading date in rice. In addition, HZ1/SE5 can also regulate the expression of genes associated with chlorophyll synthesis pathway to modulate the change of chlorophyll levels. 【Conclusion】 hz1 mutant is a photoperiodic insensitive mutant due to the mutation of heme oxygenase gene SE5. HZ1/SE5 may regulate the expression of the florigen genes and chlorophyll synthesis-associated genes to influence the heading date and leaf yellowish in rice.

Key words: rice, yellowish and early heading mutant, gene cloning, heme oxygenase 1

PANG Meng-zhen, XU Han-qin, LIU Hai-yan, SONG Juan, WANG Jia-han, SUN Li-na, JI Pei-mei, YIN Ze-zhi, HU You-chuan, ZHAO Xiao-meng, LIANG Shan-shan, ZHANG Si-ju, LUAN Wei-jiang. Gene Identification and Functional Analysis of Yellowish and Early Heading Mutant hz1 in Rice[J]. Biotechnology Bulletin, 2024, 40(7): 125-136.

Figures/Tables 9

Table 1 Primer sequence

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	内切酶 Endonuclease
SE5-GFP-F	GTTGGTACCCGCTATAAGAGGGAGAGAGG	Kpn Ⅰ
SE5-GFP-R	CCGTCTAGAGGTGAATATGTGACGGAGGA	Xba Ⅰ
LB-R	ACGATGGACTCCAGTCCGGCCcttgaccaactctatcagagcttgg
HZ1-F	CAAGGACCAGGCCAAGGAAG
HZ1-R	ACCAAACTCAAAACAGGGGG
InDel1-F	CAGGTATTCTGAGGTTGTATCC
InDel1-R	CACGGTCAAATTCAACATTCC
dCAP3-F	CACCAACTGAGCTCACTAGCCATAT	NdeⅠ
dCAP3-R	GGCCCAATGACTACCTTCTACTTTA	NdeⅠ
SE5-ex-F	GGCTGAAAAGGACTCCCAAG
SE5-ex-R	GTCCAGCTAGAGGCGACTTC
Actin1-ex-F	GACTCTGGTGATGGTGTCAGC
Actin1-ex-R	GGCTGGAAGAGGACCTCAGG
Hd3a-ex-F	TTGGTAGGGTTGTGGGTGATGTGC
Hd3a-ex-R	AGGTTAGGGTCACTTGGGCTTGGT
RFT1-ex-F	TCCGAGCCCAAGCAACCCTAAC
RFT1-ex-R	AGTTCCTGGTGCTGAAGTTCTG
HEMA-ex-F	GATGCAATCACTGCTGGAAAGCGT
HEMA-ex-R	CCATCTTGCCAGCACCAATCAACA
PORA-ex-F	TCGTCGGCCTCGTCTGAGTTTATT
PORA-ex-R	AGGCCTCTCTCACTGAAAGCTGAA
YGL1-ex-F	CCAGCCACTGATGAAAGCAGCAAT
YGL1-ex-R	AGAGCGCTAATACACTCGCGAACA
CAO1-ex-F	CTTGTCGTATTCTTGGCGAG
CAO1-ex-R	ATCCCGTGATGCTGTCGCTA

Fig. 1 Phenotype analysis of hz1 mutant A: The phenotype of hz1 mutant in the field, bar=10 cm. B-D: The heading dates of hz1 under natural long day（NLD）, long day（LD）, and short day（SD）conditions. Values are shown as mean ± SD（n ≥ 25）. E: The phenotype of hz1 during the seedling stage, bar=1 cm. F: The leaves of hz1 at heading stage. G: Measurement of chlorophyll content during seedling stage. Values are shown as mean ± SD（n=3）. H: The chlorophyll content of flag leaves during the heading stage. Values are shown as mean ± SD（n=3）. I: The chlorophyll content of the penultimate leaves. Values are shown as mean ± SD（n=3）（Chl a: chlorophyll a; Chl b: chlorophyll b; Chl T: total chlorophyll content）. J: The DAB staining results of WT and hz1. Red arrows indicate brown spots, bar=1 cm. K: Relative intensity of DAB staining for WT and hz1. Values are shown as mean ± SD（n=3）. L: The measurement of hydrogen peroxide（H2O2）content in leaves. Values are shown as mean ± SD（n=3）. M and N: Enzyme activity measurements of catalase（CAT）and peroxidase（POD）in leaves. Values are shown as mean ± SD（n=3）. *: P<0.05; **: P<0.01; ***: P<0.001, the same below

Fig. 2 Analysis of agronomic traits of hz1 mutant A-G: Statistical analysis of plant height, tiller number, panicle length, number of primary branches, number of secondary branches, seed setting rate, and 100-grain weight. Values are shown as mean ± SD（n=30）. H: The panicle of hz1, bar=10 cm. I: Grain number and seed setting rate of hz1, bar=10 cm. J and K: The pollen staining of hz1. The magnified times are 400 times, and the arrows indicate abortive pollens

Table 2 Genetic analysis of hz1 Mutant

组合 Cross	正常表型植株数 Number of wild plants	突变表型植株数 Number of mutant plants	总植株数 Number of total plants	ꭕ²（3∶1）	P value
ZH11/hz1	458	130	588	2.621 3	0.105 4

Fig. 3 BSA-seq analysis and gene mapping A: BSA-seq analysis. HZ1 is closely linked with the molecular marker on rice chromosome 6. B: T-DNA insertion site is co-segregated with HZ1. C: Sequencing analysis of T-DNA insertion site

Fig. 4 Detection and linkage analysis of T-DNA insertion sites A: Schematic diagram of LOC_Os06g40080 site and T-DNA insertion location. HZ1-F, HZ1-R, and LB-R indicate the positions of the detection primers. B: RT-PCR analysis of HZ1/SE5 gene. Actin1 is an internal control in rice. C: The linkage analysis of T-DNA insertion sites. LD is DL 2000 marker, WT is wild-type, F1 is F1 plant, F2 mutant and F2 WT are plants with mutant phenotype and WT phenotype in the F2 population, respectively. D: Schematic diagram of InDel1, dCAP3 markers in the chromosome 6. E and F: PCR amplification of InDel1 and dCAP3. LD is DL 2000 marker, P1 is ZH11 plant, P2 is hz1 mutant, F1 is the F1 plants, and F2 mutant are the plants with hz1 phenotype in the F2 population

Fig. 5 Expression patterns of HZ1/SE5 gene A: Tissue-specific expression analysis of HZ1/SE5（SAM: stem apical meristem）. B: Expression analysis of HZ1/SE5 at different growth stages in rice. C and D: Expression analysis of HZ1/SE5 under LD (C) and SD (D) conditions, respectively. ZT（zeitgeber time, h）indicates the time of light on. The white hollow box indicates the light period, and the black solid box indicates the dark period. The same below

Fig. 6 Subcellular localization of HZ1/SE5 protein A and B: Transient expression of empty vector and HZ1-GFP fusion protein in rice protoplasts, bar=10 μm. C and D: Transient expression of empty vector and HZ1-GFP fusion protein in tobacco epidermal cells, bar=20 μm

Fig. 7 Expression analysis of key genes involved in photoperiodic regulatory pathway and chlorophyll synthesis related genes in hz1 mutant A, B: Expression of key genes associated with photoperiodic regulatory pathway in WT and hz1 mutants under LD and SD conditions. C: Expression of chlorophyll synthesis related genes in WT and hz1 mutants

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