Cloning and Functional Analysis of Rice Tillering Regulatory Gene HTD3

doi:10.16819/j.1001-7216.2021. 210205

Abstract

Abstract:

【Objective】Cloning of rice tiller-related genes lays a theoretical basis and provides beneficial genetic resources for constructing ideal plant-type rice and increasing grain yield.【Method】We compared phenotypes and main agronomic traits between the mutant htd3 (high-tillering dwarf 3) and its wild type under conventional field planting conditions, used map-based cloning method to clone candidate genes, analyzed the expression levels of HTD3, SL and ABA-related genes by fluorescence quantitative real-time PCR. Sequencing comparison was performed to analyze the natural variation of HTD3 in 147 germplasm resources.【Result】Compared with the wild type, the axillay bud of the mutant htd3 grow faster, the number of tillers and effective panicles were significantly increased, the plant height, the number of primary rachis branches and grains per panicle were significantly decreased, and the seed setting rate and 1000-grain weight were not significantly changed. Genetic analysis indicated that the high-tillering trait of htd3 was controlled by single recessive nuclear gene, which was mapped to a 63.5 kb region between the marker CM8 and CM10 on chromosome 12, and the complementary transgenic experiment proved that LOC_Os12g21710 was the gene controlling the high-tillering phenotype. HTD3 is constitutively expressed in wild-type and mutant, and the mutation of this gene could up-regulate the expression level of some stratolactones and ABA-related genes. The natural variation of G2674A in the HTD3 coding region in rice varieties significantly increase the number of tillers.【Conclusion】HTD3 is a new allele of the recently reported T20/MIT1 gene. The HTD3 mutation leads to the phenotype of moderate increase in tillers and slightly shorter plant height in rice, which has great application potential in cultivating ideal plant type rice and high-yield breeding.

Key words: rice, high-tillering, gene cloning, biological function

摘要：

【目的】克隆水稻分蘖相关基因,为构建理想株型水稻,提高粮食产量提供理论基础及有利基因资源。【方法】在常规大田种植条件下分别比较突变体htd3（high-tillering dwarf 3）与野生型在幼苗期、抽穗期和成熟期表型及主要农艺性状差异,利用图位克隆方法克隆候选基因,利用荧光定量PCR方法分析HTD3及独脚金内酯和脱落酸相关基因的表达水平,测序比对分析HTD3在147份种质资源中的自然变异情况。【结果】与野生型相比,突变体htd3的分蘖芽生长较快,分蘖数和有效穗数显著增多,株高、一次枝梗数和每穗粒数显著降低,结实率和千粒重没有显著变化。遗传分析表明,htd3多分蘖的性状受一对隐性核基因控制。图位克隆将HTD3基因定位在第12染色体CM8和CM10之间约63.5 kb的物理区间内,互补转基因实验证明该区间内LOC_Os12g21710为控制突变体多分蘖表型的基因。HTD3在野生型和突变体中呈组成型表达,该基因突变会引起部分独脚金内酯和脱落酸相关基因的表达水平上调。水稻品种中HTD3编码区G2674A自然变异使得分蘖数显著增多。【结论】HTD3是最近报道的T20/MIT1基因的一个新的等位基因,HTD3突变导致水稻出现分蘖适度增加,株高略矮的表型,在培育理想株型水稻和高产育种上具有较大的应用潜力。

关键词: 水稻, 多分蘖, 图位克隆, 生物学功能

Xianmei WU, Sanfeng LI, Ping HU, Rui HE, Ran JIAO, Yijian MAO, Caolin LU, Juan HU, Han LIN, Rongliang WU, Xudong ZHU, Yuchun RAO, Yuexing WANG. Cloning and Functional Analysis of Rice Tillering Regulatory Gene HTD3[J]. Chinese Journal OF Rice Science, 2021, 35(6): 535-542.

吴先美, 李三峰, 胡萍, 何瑞, 焦然, 毛一剑, 鲁草林, 胡娟, 林晗, 吴荣梁, 朱旭东, 饶玉春, 王跃星. 水稻分蘖调控基因HTD3的克隆与功能研究[J]. 中国水稻科学, 2021, 35(6): 535-542.

Figures/Tables 9

Table 1 Sequences of markers for HTD3 mapping.

标记 Marker	正向引物 Forward primer (5′→3′)	反向引物 Reverse primer (5′→3′)
B12-6	TGAAGCGGTTTGACTTTGACC	GGGGTGAAAACTGGTAGGGT
B12-9	TCCTTCTCGTTTATGAACTTATGG	TAGAGCAAAGCAGAGCCCAG
M9	TAATCCCCTGCACTCCATCC	GTGAACAACCAGCCGAGAAT
M17	GACTTTCTAGCATTGCCCACA	GCATTAACTGGGGCCATTGT
M25	GAGACGGCCAGCTTAGGTAG	CAGTGCTACAGAAACAGGGC
M29	CCGAACTCCAGTTTGTGAGG	CGGAAATCTGACGCTGGTAT
M35	AAAAGAACAACACAGCCCCT	ACAAGGAGCAATCTGGACCA
CM5	TTGTGAACAAGAGCCAACGG	CGCTGTTGGGCATTCTTTAAAG
CM8	GTGTGATCCATGGGTAGCCT	CAGAGCCCTATTAGTCTATTGCT
CM10	TCACATGATACCTCGCGAGT	CAGACGATTCTACACAACAGGA
CM13	AGCAGCCAAGATTAAGGAGGA	CGTCAGAGTGATTAGCAAAAGGA

Table 2 Primers for amplifying the complete expression unit of HTD3.

引物名称 Primer name	引物序列 Primer sequences (5′→3′)
HTD3-CPT-EcoRⅠ-F	ACGAATTCGAGCTCGGTACCTGGTTCTGTGACTAAAGCGC
HTD3-CPT-HindⅢ-R	GGCCAGTGCCAAGCTTTCTCCGGGGCCCTGAATATTCCTCT

Fig. 1. Morphological characterization of WT and htd3. A~C, Plant phenotypes of wild-type (WT) and htd3 in the seedling stage (A), tillering stage (B) , and mature stage (C) , bar= 6 cm; D~E, Tiller number (D) and plant height (E) of WT and htd3 after sowing. Values are means ± SD (n=15), t-test, *, ** indicate significant difference at P<0.05 and P<0.01, respectively.

Table 3 Comparison of agronomic traits between WT and htd3.

农艺性状Agronomic trait	野生型WT	突变体htd3
株高Plant height /cm	97.08±2.78	83.76±1.54^*
有效穗数Effective panicles	11.0±1.0	28.0±2.0^**
穗长Panicle length/cm	21.52±0.98	20.82±0.62
一次枝梗数 Primary branches	12.0±2.0	9.0±1.0^*
二次枝梗数Secondary branches	26.0±3.0	23.0±4.0
每穗粒数Grains per panicle	121.0±11.0	104.0±16.0^*
每株总粒数Total grains per plant	1331±28	2018±36^**
千粒重1000-grain weight/g	23.7±2.1	23.9±1.3
结实率Seed setting rate/%	87.2±5.8	90.0±2.4

Fig. 2. Phenotypic observation of tillering buds of WT and htd3. A~F, Phenotype of tillering buds of WT and htd3 at five-, six- and seven-leaf stage; A-C represents WT; D-F represents htd3; bars=1 cm; G, Tillering bud length of WT and htd3 at each leaf stage, in n/0, n represents the leaf position, 0 represents the main stem, 1/0 represents the tiller bud corresponding to the first leaf of the main stem, 2/0 represents the tiller bud corresponding to the second leaf of the main stem, and so on.

Table 4 Genetic analysis of rice high-tillering mutant htd3.

组合Cross	F₁表型 Phenotype of F₁ plants	F₂群体 F₂ population			χ² _(3:1)	χ²_{0. 05}
组合Cross	F₁表型 Phenotype of F₁ plants	总株数 No. of plants	正常分蘖株数 No. of normal plants	多分蘖株数 No. of high-tillering plants	χ² _(3:1)	χ²_{0. 05}
中花11/ TN1 Zhonghua 11/TN1	正常分蘖Normal tiller	864	666	198	2	3. 84

Fig. 3. Map-based cloning of HTD3 and verification of complementary transgene. A, Initial location of HTD3; B, Fine mapping of HTD3; C, Predicted ORFs in the location interval; D, Gene structure of the candidate gene LOC_Os12g21710 and the sequence difference between WT and htd3, in which black boxes, white boxes, black lines represent exons, UTR and introns, respectively; E, Phenotype of WT, htd3 and complementary transgenic T2 generation plants in the tilling stage, bar=6 cm; F~G, Number of tillers (F) and plant height (G) of WT, htd3 and complementary transgenic T2 generation plants in the tillering stage, the data are means ± standard deviation (n=5). Significant difference by Duncan’s multiple range test. The same letters indicate no significant difference at P<0.05.

Fig. 4. Expression analysis of HTD3 and hormone related genes. A, HTD3 relative expression level in various tissues of WT and htd3; B-C, Relative expression of SLs (B) and ABA (C) related genes in WT and htd3. RNA was isolated from WT and htd3 leaves in B and C. Expression levels are represented as relative to the corresponding genes in WT (set as reference value of 1), and data are shown as means ± SD from three biological replicates. Asterisks indicate statistical significance between the WT and the mutant, as determined by Student’s t-test (*P < 0.05; **P < 0.01).

Fig. 5. Analysis of the HTD3 gene haplotypes. A, Gene structure of the HTD3, and nucleotide polymorphisms in the HTD3 5'-UTR and coding region, exons are indicated by black boxes, UTRs are indicated by white boxes, and the black lines between black boxes represent introns, hap means haplotypes, the number represents the base position relative to the start codon ATG. B, Tiller number and plant height of each haplotype. Significant difference by Duncan’s multiple range test. The same letters indicate no significant difference at P<0.05.

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