一个水稻粉质胚乳突变体的表型分析和基因定位

doi:10.13560/j.cnki.biotech.bull.1985.2025-0670

摘要/Abstract

摘要：

目的水稻粉质胚乳突变体因淀粉或蛋白合成、积累异常呈现粉质不透明表型，是研究胚乳发育及调控网络的理想材料。本研究旨在解析粉质胚乳突变体M97的籽粒表型特征并定位其突变基因。方法采用扫描电镜、荧光辅助毛细管电泳、X射线粉末衍射仪和快速黏度分析仪等技术，分析M97的淀粉颗粒形态、链长分布及理化特性；通过MutMap方法结合SNP/Indel过滤进行突变基因定位。结果野生型Kitaake籽粒断面透明，M97胚乳中心粉质不透明、四周透明。M97的粒长、粒宽、粒厚显著小于野生型，导致百粒重下降。扫描电镜显示，M97胚乳中心淀粉颗粒疏松、间隙大，四周区域则与野生型相似，颗粒排列紧密呈规则多角形。与野生型相比，M97总淀粉含量显著降低，可溶性糖显著升高，表观直链淀粉显著降低。两者淀粉均为A型晶体，但M97相对结晶度显著升高；其淀粉膨胀势升高，水溶性显著降低，峰值黏度和崩解值升高，热浆黏度和终止黏度降低。MutMap将突变基因定位于2号染色体，M97中BT1基因第3外显子存在T到C的替换，导致第322位异亮氨酸变为苏氨酸。KASP标记检测证实该突变与粉质表型共分离。M97被替换的异亮氨酸在各物种BT1同源蛋白中高度保守。结论鉴定出BT1基因新等位突变体，明确单氨基酸替换对水稻胚乳形态和淀粉特性的影响，为解析BT1调控淀粉合成的分子机制奠定基础，也为稻米品质改良提供理论参考。

关键词: 水稻, 粉质胚乳, BT1, 淀粉特性, 胚乳发育

Abstract:

Objective Rice floury endosperm mutants, which show an opaque endosperm phenotype due to abnormal starch or protein synthesis and accumulation, serve as ideal genetic materials for studying endosperm development and its regulatory networks. This study is aimed to conduct grain phenotypic analysis and mutant gene mapping of the floury endosperm mutant M97. Method The starch granule morphology, starch chain length distribution and physicochemical properties of mutant M97 were analyzed using scanning electron microscopy, fluorescence-assisted capillary electrophoresis, X-ray powder diffractometer, and rapid viscosity analyzer. Additionally, the mutated gene of M97 was identified via the MutMap method combined with SNP/Indel filtering. Result The whole cross-section of Kitaake grain was transparent, and the central area of M97 endosperm showed floury and opaque, and the periphery remained transparent. The grain length, width and thickness of M97 brown rice were significantly lower than those of the wild type, resulting in a decrease in its 100-brown rice weight. Observation via scanning electron microscopy showed that the starch granules in the central area of M97 endosperm were loose with large gaps, while those in the peripheral region were similar to those of the wild type, showing tightly arranged and regular polygonal starch granules. Compared with the wild type, the total starch content of M97 decreased significantly, the soluble sugar content increased significantly, and the apparent amylose content decreased significantly. Both M97 and wild-type starch belonged to A-type crystals, but the relative crystallinity of M97 significantly increased. The starch of M97 showed increased swelling power, decreased water solubility, higher peak viscosity and breakdown values, and lower hot paste viscosity and final viscosity. The mutant gene was mapped to chromosome 2 by the MutMap method. SNP filtering identified a T-to-C substitution in the third exon of the BT1 gene, leading to the substitution of isoleucine (Ile) at position 322 with threonine (Thr). Moreover, the KASP marker verified that this mutation site co-segregated with the floury endosperm phenotype. Homologous protein sequence analysis of BT1 showed that isoleucine was highly conserved across species. Conclusion This study identified a new allelic mutant of the BT1 gene and investigated the effects of a single amino acid substitution on rice endosperm morphology and starch properties, laying a foundation for further study on the mechanism of BT1 participating in regulating rice starch synthesis and providing insights for rice quality improvement.

Key words: rice, floury endosperm, BT1, starch characteristics, endosperm development

王程程, 黄天宇, 张晴, 史来权, 方慧敏, 张龙. 一个水稻粉质胚乳突变体的表型分析和基因定位[J]. 生物技术通报, 2026, 42(2): 169-177.

WANG Cheng-cheng, HUANG Tian-yu, ZHANG Qing, SHI Lai-quan, FANG Hui-min, ZHANG Long. Phenotypic Analysis and Genetic Mapping of a Rice Floury Endosperm Mutant[J]. Biotechnology Bulletin, 2026, 42(2): 169-177.

图/表 6

图1 糙米的外观表型和组分A、B：糙米籽粒；C、D：糙米横断面；E-H：糙米的粒长（E）、粒宽（F）、粒厚（G）、百粒重（I）；I：总淀粉含量；J：可溶性糖含量。比例尺为1 mm；数据进行t测验分析，*P<0.05， **P< 0.01，下同

Fig. 1 Appearance phenotype and components of brown riceA, B: Brown rice. C, D: Cross-section of brown rice. E-H: Quantification of brown rice length (E), width (F), thickness (G), 100-brown rice weight (H). I: Total starch content. J: Soluble sugar content. Scale bars, 1 mm. The data were analyzed by the t-test, *P<0.05, **P<0.01, the same below

图2 淀粉颗粒的形态A-F：淀粉颗粒的扫描电镜照片；比例尺=10 μm

Fig. 2 Morphologies of starch granulesA-F: Scanning electron microscope photograph of starch granules. Scale bars, 10 μm

图3 淀粉的链长分布和功能特性的测定A：碘吸收波谱；B：支链淀粉链长分布；C：淀粉X射线衍射图谱；D：淀粉的RVA图谱。RC：相对结晶度

Fig. 3 Determination of starch chain length distribution and functional propertiesA: Iodine absorbance spectra. B: Chain length distribution of amylopectin. C: X-ray diffraction patterns of starches. D: RVA patterns of the starch. RC: Relative crystallinity

表1 淀粉的膨胀势、水溶性和黏滞特性参数

Table 1 Swelling power, water solubility and pasting parameters of starches

Genotype	Watering solubility	Swelling power	RVA parameters of the starch （mPa/s）
Genotype	Watering solubility	Swelling power	PV	HV	BV	FV	SV
WT	6.2±0.1	17.2±1.0	2 480±9	2 393±9	87±18	2 863±2	470±7
M97	5.5±0.1**	24.5±1.3**	2 568±53	2 205±44*	363±9**	2 668±41*	463±3

图4 M97突变基因的定位A：MutMap定位突变基因流程图；B：候选位点的SNP/Indel-Index过滤原则；C：候选基因结构，第965位碱基由T替换为C；D：突变位点的测序峰图，第322位氨基酸Ile被替换为Thr，突变位点用红色方框标出；E：利用KASP标记进行共分离分析

Fig. 4 Mapping of the mutated gene in M97A: Flowchart of MutMap method for locating mutant gene. B: SNP/Indel-Index filtering principle of candidate sites. C. Candidate gene structure. The 965th base with T is replaced by C. D: Sequence chromatograms of the mutation site. The 322nd amino acid Ile is replased with Thr, and the mutation site is marked with a red box. E: Co-separation analysis was performed using KASP marker

图5 不同物种中BT1蛋白突变区域的氨基酸序列比对

Fig. 5 Amino acid sequence alignment of BT1 protein mutation region in different species

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