黍稷落粒的生物学基础研究及落粒调控基因的鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2025-0150

摘要/Abstract

摘要：

目的黍稷对我国旱作农业、盐碱地开发利用和救灾补种有着重要的作用，但是落粒是黍稷重要的产量限制因素。研究黍稷落粒的机制，对选育抗落粒的黍稷新品种，提高黍稷产量具有重要的意义。方法以落粒性强的‘野糜子’和落粒性弱的‘红粘糜’为材料，利用石蜡切片，研究不同落粒性的黍稷品种间离层细胞的结构差异。其次运用反向遗传学策略，鉴定与水稻落粒基因同源的黍稷落粒基因。结果首先，通过对‘野糜子’和‘红粘糜’的离区组织的电镜观察发现，‘野糜子’在开花后，形成明显的离层，而在‘红粘糜’中，没有观察到完整的离层。其次，对黍稷落粒基因在‘野糜子’小穗开花后1、20和35 d的表达情况进行分析发现，大部分基因在开花后1 d的表达量最高。最后，通过比较‘野糜子’和‘红粘糜’PmSh1-1基因的cDNA序列发现，‘红粘糜’PmSh1-1的cDNA比‘野糜子’的cDNA短，缺少第3个外显子。结论黍稷离层发育不完整，能够导致黍稷落粒性降低。水稻OsSh1的同源基因PmSh1-1可能是调控黍稷落粒的候选基因。

关键词: 黍稷, 落粒性, 离层, 石蜡切片, 转录组测序, 趋同驯化, Sh1, 同源基因

Abstract:

Objective Proso millet (Panicum miliaceum L.) plays an important role in dryland agriculture, saline alkali land development and utilization, and disaster relief and replanting in China. Grain shattering is a crucial factor affecting yield of proso millet. Investigating the mechanism of grain shattering in proso millet is very important for breeding new varieties with low grain shattering and improving crop production. Method Having the proso millet gerplasm 'Yemizi' with strong grain shattering and 'Hongnianmi' with low grain shattering by paraffin sections, we studied the structural differences of abscission layer cells among millet varieties with different grain shattering characteristics. In addition, we identified proso millet grain shattering genes using reverse genetics strategies. Result Our results showed that 'Yemizi' had a distinct abscission layer, while no complete abscission layer was observed in 'Hongnianmi'. In addition, the homologous genes of rice (Oryza sativa) grain shattering genes were identified in proso millet and their expression in 'Yemeizi' at 1, 20, and 35 d after flowering was analyzed. The results showed that most genes had the highest gene expression at 1 d after flowering. Finally, PmSh1-1, was further analyzed, and the results showed that the expression of the PmSh1-1 in 'Yemizi' and 'Hongnianmi' was not significantly different, but the cDNA sequence of the PmSh1-1 in 'Hongnianmi' was shorter compared with it in 'Yemizi'. Conclusion We found that incomplete abscission layer may lead to a reduction of grain shattering and PmSh1-1, and a homologous gene of rice OsSh1 could be a potential grain shattering gene in proso millet.

Key words: proso millet, grain shattering, abscission zone, paraffin sectioning, transcriptome sequencing, parallel domestication, Sh1, homologous genes

王月琛, 韩鑫骐, 魏文敏, 崔兆兰, 罗阳美, 陈鹏如, 王海岗, 刘龙龙, 张莉, 王纶. 黍稷落粒的生物学基础研究及落粒调控基因的鉴定[J]. 生物技术通报, 2025, 41(7): 164-171.

WANG Yue-chen, HAN Xin-qi, WEI Wen-min, CUI Zhao-lan, LUO Yang-mei, CHEN Peng-ru, WANG Hai-gang, LIU long-long, ZHANG Li, WANG Lun. Biological Basis Study for Grain Shattering in Proso Millet and Identification of Genes Regulating Grain Shattering[J]. Biotechnology Bulletin, 2025, 41(7): 164-171.

图/表 8

表1 水稻和黍稷落粒基因

Table 1 Grain shattering genes in rice (Oryza sativa) and proso millet (Panicum miliaceum)

基因名称 Gene name	基因编号 Gene ID	编码蛋白 Protein name	黍稷同源基因1 Homologous gene 1	黍稷同源基因2 Homologous gene 2
qSH1	Os01g0848400	BEL1型同源异型蛋白	PM13G13360	PM07G28580
OsSh1	Os03g0650000	YABBY转录因子	PM01G33800	PM02G06310
sh4/SHA1	Os04g0670900	MYB转录因子	PM16G23590	PM15G25020
SHAT1	Os04g0649100	AP2转录因子	PM16G22000	PM15G23430
SH5	Os05g0455200	BELI型同源异型蛋白	PM05G36060	PM06G19790
OsCPL1	Os07g0207700	CTD磷酸化酶	PM15G01070	PM16G00940

图1 ‘野糜子’和‘红粘糜’的离区的组织学观察A：成熟‘野糜子’的小穗；B：‘野糜子’小穗石蜡切片；C：成熟‘红粘糜’的小穗；D：‘红粘糜’小穗石蜡切片

Fig. 1 Histological observation of the abscission zone of 'Yemizi' and 'Hongnianmi'A: Mature spikelet of 'Yemizi'. B: Paraffin section of 'Yemizi' spikelet. C: Mature spikelet of 'Hongnianmi'. D: Paraffin section of 'Hongnianmi' spikelet

表2 黍稷落粒同源基因的染色体位置

Table 2 Chromosome location of homologous grain shattering genes in proso millet

基因名称 Gene name	基因编号 Gene ID	‘0390’染色体位置 Chromosome location in ‘0390’	‘晋黍7号’染色体位置 Chromosome location in ‘Jinsu7’	‘隆糜4号’染色体位置 Chromosome location in ‘Longmei4’
PmSH5-1	PM05G36060	05G	3B	Chr03
PmSH5-2	PM06G19790	06G	3A	Chr10
PmqSH1-1	PM07G28580	07G	5B	Chr05
PmqSH1-2	PM13G13360	13G	5A	Chr08
PmSh1-1	PM01G33800	01G	9B	Chr01
PmSh1-2	PM02G06310	02G	9A	Chr04
PmCPL1-1	PM15G01070	15G	7B	Chr14
PmCPL1-2	PM16G00940	16G	7A	Chr15
PmSHAT1-1	PM15G23430	15G	7B	Chr14
PmSHAT1-2	PM16G22000	16G	7A	Chr15
Pmsh4/SHA1-1	PM15G25020	15G	7B	Chr14
Pmsh4/SHA1-2	PM16G23590	16G	7A	Chr15

图2 黍稷落粒基因的染色体分布A：0390；B：陇糜4号；C：晋黍7号

Fig. 2 Chromosome distribution of grain shattering genes in proso milletA: 0390; B: Longmi 4; C: Jinsu 7

图3 落粒同源蛋白的系统进化树

Fig. 3 Phylogenetic relationships of homologous shattering proteins of grain shattering

图4 黍稷落粒同源基因的组织特异性表达热图橘红色代表表达量上调，蓝色代表表达量下调，表达差异大于2倍视为显著；GFS1表示开花后1 d，GFS20表示开花后20 d，GFS35表示开花后35 d

Fig. 4 Heat-map showing the tissue-specific expressions of homologous grain shattering genes in proso milletOrange-red indicates up-regulated expression, blue indicates down-regulated, and expression difference greater than 2 times is considered significant. GFS1 refers to 1 d after flowering, GFS20 refers to 20 d after flowering, GFS35 refers to 35 d after flowering

图5 PmSh1-1 cDNA的琼脂糖凝胶电泳检测图M：分子量标准；1和3：野糜子；2和4：红粘糜

Fig. 5 Detection of PmSh1-1 cDNA by agarose gel electrophoresisM: Marker; 1 and 3: Yemizi; 2 and 4: Hongnianmi

图6 PmSh1-1在‘野糜子’和‘红粘糜’中cDNA序列比对缺失的序列和点突变用红框标注

Fig. 6 cDNA sequence alignment of PmSh1-1 in 'Yemizi' and 'Hongnianmi'The indel sequence and SNPs are marked with red squares

参考文献 32

[1]	王星玉, 王纶, 温琪汾. 黍稷的名实考证及规范 [J]. 植物遗传资源学报, 2010, 11(2): 132-138.
	Wang XY, Wang L, Wen QF. Textual research and standardization of Chinese Name of broomcorn millet [J]. J Plant Genet Resour, 2010, 11(2): 132-138.
[2]	Lu HY, Zhang JP, Liu KB, et al. Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10, 000 years ago [J]. Proc Natl Acad Sci USA, 2009, 106(18): 7367-7372.
[3]	王纶, 王星玉. 中国黍稷种质资源研究 [M]. 北京: 中国农业科学技术出版社, 2018.
	Wang L, Wang XY. Study on millet germplasm resources in China [M]. Beijing: China Agricultural Science and Technology Press, 2018.
[4]	刘敏轩, 许月, 陆平. 中国野生黍稷资源收集保存与遗传多样性研究进展 [J]. 植物遗传资源学报, 2020, 21(6): 1435-1445.
	Liu MX, Xu Y, Lu P. Advances in germplasm collection and genetic diversity research of wild broomcorn millet in China [J]. J Plant Genet Resour, 2020, 21(6): 1435-1445.
[5]	苏占明, 李海, 皇甫红芳. 黍稷的营养成分与保健功效及种植建议 [J]. 种业导刊, 2020(4): 31-34.
	Su ZM, Li H, Huangfu HF. Nutritional components, health care effects and planting suggestions of millet [J]. J Seed Ind Guide, 2020(4): 31-34.
[6]	李新月, 董燕, 李颖, 等. 五谷为养之黍米历史溯源与功能考证 [J]. 中国食品药品监管, 2022(8): 118-125.
	Li XY, Dong Y, Li Y, et al. Historical traceability and functional research of broomcorn millet as a grain for raising [J]. China Food Drug Adm Mag, 2022(8): 118-125.
[7]	王纶, 王星玉, 温琪汾. 黍稷种质资源繁殖更新技术 [J]. 山西农业科学, 2012, 40(3): 227-232, 240.
	Wang L, Wang XY, Wen QF. Regeneration and renew technical specification of broomcorn millet germplasm resources [J]. J Shanxi Agric Sci, 2012, 40(3): 227-232, 240.
[8]	谢文刚, 万依阳, 张宗瑜, 等. 禾本科植物落粒机理研究进展 [J]. 草业学报, 2021, 30(8): 186-198.
	Xie WG, Wan YY, Zhang ZY, et al. Research progress on the seed-shattering mechanism of Poaceae plants [J]. Acta Prataculturae Sin, 2021, 30(8): 186-198.
[9]	Maity A, Lamichaney A, Joshi DC, et al. Seed shattering: a trait of evolutionary importance in plants [J]. Front Plant Sci, 2021, 12: 657773.
[10]	Yu YQ, Hu H, Doust AN, et al. Divergent gene expression networks underlie morphological diversity of abscission zones in grasses [J]. New Phytol, 2020, 225(4): 1799-1815.
[11]	Yu YQ, Kellogg EA. Multifaceted mechanisms controlling grain disarticulation in the Poaceae [J]. Curr Opin Plant Biol, 2024, 81: 102564.
[12]	Parker TA, Lo S, Gepts P. Pod shattering in grain legumes: emerging genetic and environment-related patterns [J]. Plant Cell, 2021, 33(2): 179-199.
[13]	Yu YQ, Beyene G, Villmer J, et al. Grain shattering by cell death and fracture in Eragrostis tef [J]. Plant Physiol, 2023, 192(1): 222-239.
[14]	Ning J, He W, Wu LH, et al. The MYB transcription factor Seed Shattering 11 controls seed shattering by repressing lignin synthesis in African rice [J]. Plant Biotechnol J, 2023, 21(5): 931-942.
[15]	Wu H, He Q, He B, et al. Gibberellin signaling regulates lignin biosynthesis to modulate rice seed shattering [J]. Plant Cell, 2023, 35(12): 4383-4404.
[16]	Jiang LY, Ma X, Zhao SS, et al. The APETALA2-like transcription factor SUPERNUMERARY BRACT controls rice seed shattering and seed size [J]. Plant Cell, 2019, 31(1): 17-36.
[17]	Hofmann NR. SHAT1, A new player in seed shattering of rice [J]. Plant Cell, 2012, 24(3): 839.
[18]	Zhang YL, Xia QY, Jiang XQ, et al. Reducing seed shattering in weedy rice by editing SH4 and qSH1 genes: implications in environmental biosafety and weed control through transgene mitigation [J]. Biology, 2022, 11(12): 1823.
[19]	Wu H, He Q, Wang Q. Advances in rice seed shattering [J]. Int J Mol Sci, 2023, 24(10): 8889.
[20]	吕树伟, 唐璇, 李晨. 水稻落粒性研究进展 [J]. 中国农业科学, 2025, 58(1): 1-9.
	Lü SW, Tang X, Li C. Research progress on seed shattering of rice [J]. Sci Agric Sin, 2025, 58(1): 1-9.
[21]	王星玉, 王纶. 黍稷种质资源描述规范和数据标准 [M]. 北京: 中国农业出版社, 2006.
	Wang XY, Wang L. Descriptors and data standard for broomcorn millet (Panicum miliaceum L.) [M]. Beijing: China Agriculture Press, 2006.
[22]	Hodge JG, Kellogg EA. Abscission zone development in Setaria viridis and its domesticated relative, Setaria italica [J]. Am J Bot, 2016, 103(6): 998-1005.
[23]	Ishikawa R, Castillo CC, Htun TM, et al. A stepwise route to domesticate rice by controlling seed shattering and panicle shape [J]. Proc Natl Acad Sci USA, 2022, 119(26): e2121692119.
[24]	张兰. 小麦落粒性与产量性状相关基因功能鉴定 [D]. 北京: 中国农业科学院, 2013.
	Zhang L. Functional identification of genes related to grain dropping and yield traits in wheat [D]. Beijing: Chinese Academy of Agricultural Sciences, 2013.
[25]	Liu HQ, Fang XJ, Zhou LN, et al. Transposon insertion drove the loss of natural seed shattering during foxtail millet domestication [J]. Mol Biol Evol, 2022, 39(6): msac078.
[26]	Lin ZW, Li XR, Shannon LM, et al. Parallel domestication of the Shattering1 genes in cereals [J]. Nat Genet, 2012, 44(6): 720-724.
[27]	Yu YQ, Hu H, Voytas DF, et al. The YABBY gene SHATTERING1 controls activation rather than patterning of the abscission zone in Setaria viridis [J]. New Phytol, 2023, 240(2): 846-862.
[28]	Ishikawa R. Genetic dissection of a reduced seed-shattering trait acquired in rice domestication [J]. Breed Sci, 2024, 74(4): 285-294.
[29]	Shi JP, Ma XX, Zhang JH, et al. Chromosome conformation capture resolved near complete genome assembly of broomcorn millet [J]. Nat Commun, 2019, 10(1): 464.
[30]	Zou CS, Li LT, Miki D, et al. The genome of broomcorn millet [J]. Nat Commun, 2019, 10: 436.
[31]	Sun YL, Liu Y, Shi JF, et al. Biased mutations and gene losses underlying diploidization of the tetraploid broomcorn millet genome [J]. Plant J, 2023, 113(4): 787-801.
[32]	Lu Q, Zhao HN, Zhang ZQ, et al. Genomic variation in weedy and cultivated broomcorn millet accessions uncovers the genetic architecture of agronomic traits [J]. Nat Genet, 2024, 56(5): 1006-1017.