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作物学报 ›› 2022, Vol. 48 ›› Issue (8): 1948-1956.doi: 10.3724/SP.J.1006.2022.12049

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻落粒基因SH8的精细定位与克隆

王穆穆1(), 何艳芳2, 郑永胜1, 王晖1, 王丽媛1, 王东建1, 张晗1, 李汝玉1,*()   

  1. 1山东省农业科学院作物研究所, 山东济南 250100
    2山东省种子管理总站, 山东济南 250100
  • 收稿日期:2021-07-22 接受日期:2021-11-29 出版日期:2022-08-12 网络出版日期:2021-12-21
  • 通讯作者: 李汝玉
  • 作者简介:E-mail: opmcy@163.com
  • 基金资助:
    国家自然科学基金项目(31801340)

Fine mapping and cloning of a seed shattering gene SH8 in rice (Oryza sativa L.)

WANG Mu-Mu1(), HE Yan-Fan2, ZHENG Yong-Sheng1, WANG Hui1, WANG Li-Yuan1, WANG Dong-Jian1, ZHANG Han1, LI Ru-Yu1,*()   

  1. 1Crop research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
    2Shandong Seed Administration Station, Jinan 250100, Shandong, China
  • Received:2021-07-22 Accepted:2021-11-29 Published:2022-08-12 Published online:2021-12-21
  • Contact: LI Ru-Yu
  • Supported by:
    National Natural Science Foundation of China(31801340)

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摘要：

落粒性是水稻的重要性状之一, 是野生稻种子传播和后代繁衍的重要保障, 但是容易落粒在栽培稻的生产中会造成产量损失。目前, 在水稻中已经克隆了一些落粒相关基因, 但是这些基因并不能解释水稻落粒的全部表型变异。本研究从白谷(Pei-kuh)和普通野生稻(W1944)构建的重组自交系中选取落粒性强且不含有SH4基因的材料E6-5, 与Pei-kuh进行杂交构建分离群体, 将以前检测到位于8号染色体的落粒QTL精细定位于26.4 kb的区间内, 并把该位点的落粒基因命名为SHATTERING8 (SH8)。该区段共有4个编码基因, Pei-kuh和E6-5的测序比对发现LOC_Os08g41950基因在编码区有3个碱基的差异, 同时实时定量PCR分析表明该基因在离层部位表达, 因此我们把LOC_Os08g41950作为SH8的目标候选基因。为了验证LOC_Os08g41950是否参与水稻落粒的调控, 我们利用来自E6-5的LOC_Os08g41950编码序列构建了过表达载体并遗传转化Pei-huh。落粒表型鉴定结果表明, 与Pei-huh相比, 转基因植株的落粒性明显增强, 不仅种子脱落所需的拉力值显著降低, 而且还具有更光滑的离层断裂面。本研究有助于促进水稻驯化和水稻落粒调控机制的研究。

关键词: 普通野生稻, 落粒性, 离层, 基因克隆, SH8基因

Abstract:

Seed shattering is an important trait for seed dispersal and reproduction of offspring in wild rice. It is a vital guarantee for seed dispersal and offspring reproduction of wild rice, but easy grain dropping will cause yield loss in domesticated rice production. Although some genes related to seed shattering have been cloned in rice, these genes were not responsible for all phenotype variation of rice seed shattering. In this study, a recombinant inbred line (E6-5) was selected from a cross between an indica variety Pei-kuh and an Asian common wild rice W1944 and hybridized with Pei-kuh to construct the isolated population. E6-5 harbored W1944 introgression segment on chromosome 8, but it had not carried SH4 gene and exhibited a very easy seed shattering phenotype. By analyzing an F2 segregation population constructed using E6-5 and Pei-kuh, we narrowed down the SHATTERING8 (SH8, qSHT-8) in the 26.4 kb interval. There were four protein-coding genes in this region. The comparison of PEI-Kuh and E6-5 sequencing revealed that LOC_Os08g41950 contained three single nucleotide polymorphisms (SNPs) in coding region. Meanwhile, qRT-PCR revealed that LOC_Os08g41950 was expressed in the junction of flower and pedicel, where the abscission layer was formed and shed off. We considered LOC_Os08g41950 as the candidate gene for SH8. To verify whether LOC_Os08g41950 was responsible for rice seed shattering, we constructed an overexpression vector using LOC_Os08g41950 encoding sequence from E6-5 and transformed it into Pei-huh. Compared with Pei-kuh, the shattering ability of transgenic plants was significantly enhanced. In addition, consistent with the BTS testing results, transgenic plants had smoother fracture surface of pedicel compared with that of Pei-kuh in scanning electron microscopy photos. These results would facilitate the study of rice domestication and seed shattering regulation mechanism.

Key words: O. rufipogon, seed shattering, abscission layer, gene cloning, SH8 gene

附表1

本研究所用的引物(酶切位点用小写字母表示)"

引物名称
Name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 用途
Application
FNP-SH4 ACTACCGCAAGGGGAACTG TGGGGAGGTTGCAGTCCTT 功能位点检测FNP detection
RM23469 CAACAGAAGCCATTAGGCATTAGC ACCGAAGTGATCAACAACCTACC 基因定位Gene mapping
RM23489 AGCAGGAGGAGGAGAAGGGAAGG CGAGCCACTGCTTCTTCTGTGC 基因定位Gene mapping
RM447 ACGGGCTTCTTCTCCTTCTCTCC TCCCTTGTGCTGTCTCCTCTCC 基因定位Gene mapping
RM23532 TATTGCCATGAATGCTGGGAAGG GGCATCGCAATGACAAACATCC 基因定位Gene mapping
RM23586 TTCTGTGGATAGCTGGAACATGC CCAGATACATCATCAGGAAGGAAGG 基因定位Gene mapping
M1(CAPS)a TGAATTGACAAAACGGACCA GGACCTGTTCTTGGGTTGAA 基因定位Gene mapping
M2(IN/DEL) AACATGATGGTGCCAAGTGA AGGATGGAGATGGTGAGTGG 基因定位Gene mapping
ACTIN TACAGTGTCTGGATTGGAGGAT TCTTGGCTTAGCATTCTTGGGT 实时定量PCR RT-qPCR
qPCR-950 ATTCATCCCTGAAGCACGTC GGCTGACGTTCATAGCCAAT 实时定量PCR RT-qPCR
qPCR-960 CTTCCACCTCCAGCATTGAT ATCGCTTCCTCCAAACCTCT 实时定量PCR RT-qPCR
950QC-1 AACATGATGGTGCCAAGTGA ACAGGACAGATCCAATCGAA 测序 Sequencing
950QC-2 GCTGAAGAGGATCGAGAACA CAGCGCTATGACATCGAAAA 测序 Sequencing
950QC-3 CTGAAGGCAAGGGTTGAAAA GTTGCTTTCCTCCAGCTACA 测序 Sequencing
950QC-4 TGCAGATATCATGGAATGGA TGCGACAGTGAATCTGTCAT 测序 Sequencing
960QC-1 TATCCGCGTACGGCTTAATG AATGTGGTGGTGAGGGAATC 测序 Sequencing
960QC-2 TCCGAGTGCTCCTAGTGGAT AGAAAGGTGGGAAGGGAAAA 测序 Sequencing
960QC-3 GAGGCGAAACACCAAATCTC TACCCCTAGCCTCTGCTTGA 测序 Sequencing
引物名称
Name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 用途
Application
960QC-4 TATCTCCACCATCCCCACTC GGCGTGCCAGTTAGTTTGAT 测序 Sequencing
960QC-5 GTTCGGTCCGATCTATCAAGA CTCACACAACCACCATCCTG 测序 Sequencing
960QC-6 ATGGCGACAACCTACTGACC GAACCAGGCAAAGGGTTTCT 测序 Sequencing
pSH8-0E AAAggatcc- ATGGGGAGGGGTCGGGTGGA AAAactagt- TCATGGTAGCCATGGGGGCAT 载体构建Vector construction
HYG AAGTTCGACAGCGTCTCCGAC TCTACACAGCCATCGGTCCAG 转基因检测Transgenic detection
CDS-1 ATGGGGAGGGGTCGGGTGGA CGGCTAGCTTTCAATTGCTC 转基因检测Transgenic detection

图1

E6-5和Pei-kuh的表型鉴定 A: 种子脱落所需的拉力值(BTS); B和C: 离层组织纵切后吖啶橙染色的荧光图片, AL表示离层; D和E: 离层断裂面的扫描电镜图片。B~E的标尺为50 μm。**表示在0.01水平上差异显著(t-test)。"

图2

SH8的图位克隆 N表示定位群体的植株数量。"

表1

定位区段内的日本晴基因注释"

基因名称
Locus name		 基因注释
Gene annotation
LOC_Os08g41950 OsMADS7-MADS-box family gene with MIKCc type-box, expressed
LOC_Os08g41960 OsMADS37-MADS-box family gene with MIKCc* type-box, expressed
LOC_Os08g41970 Conserved hypothetical protein
LOC_Os08g41980 Aminotransferase, putative, expressed

图3

候选区段内的MADS-box基因分析 A: E6-5抽穗期MADS-box基因的表达模式分析; B: Os08g41950基因在花柄和花中的表达; C: E6-5和Peikuh的Os08g41950基因编码序列比对, 差异碱基用红色加亮显示, 引起氨基酸变化的碱基用下划线标识。FP表示小花和柄的交界处, F表示小花。"

图4

转基因植株的检测 A: 潮霉素引物检测; B: SH8基因特异引物检测。"

图5

转基因植株的表型鉴定 A: SH8基因在离层部位的表达; B: 种子脱落所需的拉力值(BTS); C和D: 离层断裂面的扫描电镜图片。C和D的标尺为50 μm。**表示在0.01的水平上差异显著(t-test)。"

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