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作物杂志,2023, 第4期: 22–30 doi: 10.16035/j.issn.1001-7283.2023.04.004

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

水稻粉质胚乳突变体cse的表型分析及基因定位

花芹1(), 林泉祥1, 宋远辉1, 孙家猛1, 张祖普2,3, 陈庆全1, 李金才1, 张海涛1()   

  1. 1安徽农业大学农学院，230036，安徽合肥
    2江苏红旗种业科技有限公司，225311，江苏泰州
    3江苏省（红旗）稻麦良种繁育工程技术研究中心，225311，江苏泰州
  • 收稿日期:2022-03-09 修回日期:2022-05-28 出版日期:2023-08-15 发布日期:2023-08-15
  • 通讯作者: 张海涛，研究方向为作物遗传育种，E-mail：43647174@qq.com
  • 作者简介:花芹，研究方向为作物遗传育种，E-mail：2227028367@qq.com
  • 基金资助:
    国家自然科学基金(31871603);国家留学基金委项目(201708340008);安徽省科技厅项目(KJ2017A143);安徽省科技厅项目(gxfxZD2016022);江苏现代农业产业体系(JATS[2021]295)

Phenotypic Analysis and Map-Based Cloning of Chalkiness and FlouryEndospermMutantcsein Rice (Oryza sativa L.)

Hua Qin1(), Lin Quanxiang1, Song Yuanhui1, Sun Jiameng1, Zhang Zupu2,3, Chen Qingquan1, Li Jincai1, Zhang Haitao1()   

  1. 1College of Agronomy, Anhui Agricultural University, Hefei230036, Anhui, China
    2Jiangsu Hongqi Seed Co., Ltd., Taizhou225311, Jiangsu, China
    3Jiangsu Province (Hongqi) Engineering Research Center for Rice and Wheat Seed Breeding, Taizhou225311, Jiangsu, China
  • Received:2022-03-09 Revised:2022-05-28 Online:2023-08-15 Published:2023-08-15

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摘要：

利用突变体挖掘与稻米品质相关基因，有助于阐明与品质相关的淀粉和贮藏蛋白生物合成的调控机制，促进水稻优质育种。通过筛选中花11组织培养突变体库，获得具有粉质、垩白和皱缩外观胚乳的突变体cse（chalkinessandshrunkenendosperm），并进行籽粒表型鉴定和理化性质分析。通过F2群体分析cse遗传行为和目的基因的图位克隆，利用qRT-PCR分析基因的时空表达模式。与野生型相比，突变体的农艺性状和淀粉理化性质均有显著差异，突变体淀粉颗粒呈不规则球型，且相互间隙较大。遗传分析表明，突变体性状受单隐性核基因控制。借助图位克隆将候选基因定位在4号染色体长臂端Os4-14-8和Os4-15之间的40kb范围内。测序结果表明，定位区间内仅有一个编码TPR蛋白的候选基因LOC_Os04g55230发生了突变。第1个外显子处缺失3个碱基GGC，导致一个甘氨酸缺失，第14个外显子处存在1个碱基G替换成碱基A，导致精氨酸突变为赖氨酸。qRT-PCR结果表明，LOC_Os04g55230在授粉后14d的胚乳中表达量达到最大值。突变的LOC_Os04g55230为已报道基因OsFLO2/OsCNY8的新等位基因，但突变体与已知的OsFLO2突变体表型不完全相同，cse籽粒伴有皱缩外观，有效分蘖数、贮藏蛋白和脂肪酸含量等均显著增加。

关键词: 水稻（Oryza sativa L.）, 粉质胚乳, 淀粉颗粒, 理化性质, 图位克隆

Abstract:

It is helpful to clarify the genetic regulatory mechanism of starch and storage protein biosynthesis related to quality and promote rice quality breeding by using mutants related to rice quality. The chalkiness and shrunken endosperm mutantnamed cse was obtained by screening a mutant library of tissue culture of Zhonghua 11, and the phenotype identification and physicochemical properties analysis of grains were carried out. Analysis of genetic behavior of cse and map-based cloning of target gene by F2 population. qRT-PCR was used to analyze the spatiotemporal expression pattern of relatedgenes.Compared with the wild type, the mutant showed significantly different in agronomic traits and starch physicochemical characteristics. The mutant starch granules were irregularly spherical with large interspaces. Genetic analysis showed that the mutant trait was controlled by a pair of recessive nuclear genes. Map-based cloning revealed that cse is located on the long arm of chromosome 4 between Os4-14-8 and Os4-15 with a physical interval of 40kb. Sequencing results showed that only one candidate gene LOC_Os04g55230 encoding TPR protein had mutation in the mapping interval. The deletion of three bases GGC in the first exon led to the deletion of one glycine, and the substitution of one base G in the fourteenth exon for base A led to the mutation of arginine to lysine. The results of qRT-PCR showed that the expression level of LOC_Os04g55230 reached the maximum in endosperm 14 days after fertilization.The mutant LOC_Os04g55230was a new allele of the reported gene OsFLO2/OsCNY8, but the phenotype of the mutant was not completely same as the known OsFLO2 mutant. The cse grains had shrunken appearance, and the effective tiller number, storage protein and fatty acid content were significantly increased.

Key words: Rice(Oryza sativa L.), Floury endosperm, Starch granules, Physicochemical characteristic, Map-based cloning

表1

引物信息

名称Name 类型Type 正向引物序列（5′-3′）Forward primer sequence (5′-3′) 反向引物序列（5′-3′）Reverse primer sequence (5′-3′)
Os4-10 定位 ACCTTTTCTTGGCTTGAGGG GCTTTTGCTACTTTTGGGGG
Os4-11 定位 ACGTCCATGTCGGTGTACG CCACCACCTCTACTTCTTCAGTG
Os4-12 定位 ACGGAAACGTAGGTGGTCTG ACCCGCTAGCTCTTCGATCT
Os4-13 定位 TTGGTTGCTTCCTCCCATAC GGCATTGTACGACGGATCTC
Os4-14 定位 ACTGTGGAGTACAGGTCGGC GAAACGGAAACGAAACCCTC
Os4-15 定位 CCGCTACTAATAGCAGAGAG GGAGCTTTGTTCTTGCGAAC
Os4-10-13 定位 TCGAAGGACCTTGTGTGATTCC TAGCTATGTTTGCCGAATATTG
Os4-12-11 定位 CAGGTCAAAACTCCTCACGC TGCAGAGTGGCTTTCAATGG
Os4-13-16 定位 CTCGAAAGTATAGACAGACAGCT ACTACTCCTCCCTCTCGTGA
Os4-14-8 定位 GTATTATGCTAACGTCAGCATG CTAGGTGTCAGCATGGTACTG
Os4-15-5 定位 CACCGAATCACCTAAGAATAG CTGAATTCACTAATTCTGTCTC
Primer 1 实时荧光定量PCR CATTCTGAAGGATGAAGTGGTTC ACTCAGACATCGCTAGTACTCAG
Ubiquitin 实时荧光定量PCR GCCCAAGAAGAAGATCAAGAAC GACTTTTATGCTTCCGTTGTTATCT

表2

野生型和突变体农艺性状比较

农艺性状
Agronomic trait		 野生型
Wild type		 突变体
cse
株高Plant height(cm) 110.20±1.88 101.00±1.06**
主茎穗长Main panicle length (cm) 23.84±0.34 21.94±0.56*
第1节间长
The first internode length (cm)		 43.31±0.85 41.53±0.40
第2节间长
The second internode length (cm)		 22.40±0.40 19.62±0.56
第3节间长
The third internode length(cm)		 13.64±0.75 9.61±1.05**
第4节间长
The fourth internode length (cm)		 7.50±0.95 4.51±0.46**
有效分蘖数Effective tiller number 10.02±0.63 14.83±0.43**
一次枝梗数Primary branch number 13.33±1.05 13.40±0.91
二次枝梗数Secondary branch number 43.57±3.33 31.25±2.32**
穗粒数Grain number per panicle 243.88±22.03 172.12±14.21**
粒长Grain length(mm) 7.83±0.04 7.30±0.07*
粒宽Grain width (mm) 3.70±0.10 3.37±0.04**
粒厚Grain thickness (mm) 2.33±0.09 2.03±0.05**
千粒重1000-grain weight (g) 28.95±0.05 18.28±0.10**

图1

野生型和突变体cse的表型鉴定 (a) 抽穗期的植株形态；(b) ZH11（左）与cse（右）成熟节间；(c) 蜡熟期穗部形态；(d) 籽粒大小；(e) ZH11（外）与cse（内）成熟颖果外观；(f) 成熟颖果横截面。ZH11：中花11

图2

野生型和cse的不同时期干物质积累比较 “**”表示在P< 0.01水平上差异极显著，下同

图3

野生型和cse的成熟胚乳碘染和扫描电镜观察 a和b：野生型与cse胚乳碘染比较；c和d：野生型与cse扫描电镜观察

表3

野生型和突变体理化性质比较

指标Index 野生型Wild type 突变体cse
蛋白质Protein (%) 9.31±0.18 11.29±0.21**
总淀粉Total starch (%) 83.70±0.33 73.90±0.28**
直链淀粉Amylose (%) 17.65±0.48 8.81±0.25**
可溶性糖Soluble sugar (%) 4.64±2.38 6.21±0.57**
脂肪酸Fatty acid (%) 14.13±0.77 25.96±0.38**
垩白度Chalkiness (%) 17.23±0.24 76.84±2.30**
碱消值Alkali spreading value 8.06±0.27 7.64±0.08**
胶稠度Gel consistency (mm) 58.03±0.35 64.52±0.30**

表4

野生型和突变体糙米粉的热特性参数

材料
Material		 起始温度
T0 (℃)		 峰值
TP (℃)		 结束温度
TC (℃)		 焓变
?H (J/g)
野生型
Wild type		 66.60±0.65** 71.96±0.45** 76.09±0.29** 7.18±0.83**
cse 61.98±0.54 68.31±0.43 73.76±0.50 5.15±0.30

图4

野生型和cse的尿素膨胀分析 (a) 野生型与cse尿素膨胀试验；(b) 不同尿素浓度下野生型和cse米粉的膨胀体积比较

表5

突变基因cse的遗传分析

年份
Year		 调查总株数
Total number of
plants investigated		 野生型表型个数
Number of wild
type phenotypes		 突变体表型个数
Number of mutant
phenotypes		 χ2
2020 212 168 44 1.81
2021 743 567 176 0.68

图5

cse的精细定位 (a)~(d)横线的上方为定位所用的分子标记，横线下方为交换单株数，红色箭头框为目的基因；(e) 候选基因cse结构图，方框代表外显子内碱基的缺失和替换

表6

精细定位区间内注释基因

开放阅读框ORF 基因名称Gene name 注释功能Putative function
1 LOC_Os04g55180 含有α/β折叠家族结构域的蛋白质
2 LOC_Os04g55190 含SPOC结构域的蛋白质
3 LOC_Os04g55200 涂层亚基
4 LOC_Os04g55210 氯通道蛋白
5 LOC_Os04g55220 含有C2结构域的蛋白质
6 LOC_Os04g55230 含有四肽重复结构域的蛋白质

图6

CSE和CSE类蛋白的系统发育分析

图7

CSE在野生型不同组织及不同发育时期胚乳的表达模式分析

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