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作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2362-2372.doi: 10.3724/SP.J.1006.2023.22062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻OsCDF1基因突变效应及其基因组变异分析

胡艳娟(), 薛丹, 耿嫡, 朱末, 王天穹, 王晓雪()   

  1. 沈阳农业大学水稻研究所, 辽宁沈阳 110866
  • 收稿日期:2022-10-28 接受日期:2023-02-21 出版日期:2023-09-12 网络出版日期:2023-03-16
  • 通讯作者: *王晓雪, E-mail: wangxx@syau.edu.cn
  • 作者简介:胡艳娟, E-mail: 2020200056@stu.syau.edu.cn
  • 基金资助:
    国家重点研发计划项目(2017YFD0300107);国家自然科学基金项目(32070642)

Mutation effects of OsCDF1 gene and its genomic variations in rice

HU Yan-Juan(), XUE Dan, GENG Di, ZHU Mo, WANG Tian-Qiong, WANG Xiao-Xue()   

  1. Rice Research Institute, Shenyang Agricultural University, Shenyang 110866, Liaoning, China
  • Received:2022-10-28 Accepted:2023-02-21 Published:2023-09-12 Published online:2023-03-16
  • Supported by:
    National Key Research and Development Program of China(2017YFD0300107);National Natural Science Foundation of China(32070642)

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摘要：

开花期(又叫抽穗期)影响水稻的产量、品质和地区适应性。在拟南芥中, Cycling DOF Factor 1 (CDF1)蛋白质是CONSTANS (CO)的转录抑制因子, 负向调节拟南芥的开花期。但是, 水稻中的同源蛋白OsCDF1的功能尚不完全清楚。为了揭示OsCDF1在水稻中的生物学功能及其对开花期的影响, 本研究利用CRISPR/Cas9基因编辑技术设计定向敲除水稻OsCDF1基因的2个靶位点, 构建OsCDF1基因的敲除载体; 通过农杆菌介导的方法分别转化北方粳稻品种沈农9816, 创制OsCDF1基因的突变体; 分析沈农9816和oscdf1突变体在田间种植下的开花期和产量性状的差异。主要研究结果为: 创制了在第1个外显子的第16 bp处缺失5个碱基和在第2个外显子的338 bp处单碱基A插入的oscdf1纯合突变。序列比对分析表明, 这2种类型的突变均造成移码和蛋白翻译提前终止。在自然长日照条件下, oscdf1突变体的开花期比野生型沈农9816晚4 d以上, 其产量高于野生型。对OsCDF1单倍型和单倍型网络分析发现, OsCDF1在不同品种中进化出高度多样性。本研究成功利用CRISPR/Cas9基因编辑技术敲除了水稻OsCDF1基因, 为进一步研究OsCDF1基因功能提供了理论参考, 也为水稻遗传改良提供了潜在的基因和种质资源。

关键词: 水稻, CRISPR/Cas9, OsCDF1, 开花期, 产量相关性状, 单倍型

Abstract:

Flowering time (heading date) affects yield, quality, and regional adaptability of rice. The Cycling DOF Factor 1 (CDF1) protein is a transcriptional repressor of CONSTANS (CO) and negatively regulates flowering time in Arabidopsis. However, the biological functions of OsCDF1 in rice is not quite clear. To explore the biological functions of OsCDF1 and its effects on flowering time control in rice, we constructed two binary vectors carrying guide RNAs targeting OsCDF1 gene via CRISPR/Cas9 system. The resultant plasmids were transferred into SN9816 which was the variety widely cultivated in northern China by using an Agrobacterium-mediated transformation, and the mutations of OsCDF1 was firstly generated in SN9816. The flowering time and yield related traits of SN9816 and oscdf1 mutants were investigated in the paddy field. The main results were as follows: Two homozygous oscdf1 lines were identified, including a five bp deletion at 16th bp of the first exon and a single base pair A insertion at 338th bp of the second exon. Sequence alignment analysis revealed that the two types of mutations resulted in frame-shift and premature translation termination. Mutations of OsCDF1 delayed flowering time, but increased yield under natural long day conditions in rice. Analysis of OsCDF1 genetic variations and haplotype networks revealed that the rice accessions had evolved high genomic diversity in OsCDF1 locus. The knockout mutants of OsCDF1 created by CRISPR/Cas9 provided the theoretical basis to further study the role of OsCDF1 gene in rice and the potential gene and germplasm resources for genetic improvement in rice.

Key words: rice, CRISPR/Cas9, OsCDF1, flowering time, yield related traits, haplotype

表1

引物及序列信息"

引物名称
Primer name		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
OsCDF1gR1 ggcaGGGGAGTGCAAGGTGGGAGG aaacCCTCCCACCTTGCACTCCCC
OsCDF1gR2 ggcaGTGCCCCCGGTGTAGCAGCA aaacTGCTGCTACACCGGGGGCAC
gR ATTTCGTAGTGGGCCATGAA TAGTCCGTTTTTAGCGCGTG
Hyg GTGCTTGACATTGGGGAGTT GATGTTGGCGACCTCGTATT
JCDF1 gR1 TCGTCTCCGGGAGGAGTAGT GATGTGGCGATCGGAATTAG
JCDF1 gR2 GACACCGAGGACTCTTCAGC CTCCTCTCTATGCCCCAGTG
OsACT1 CTATGTTCCCTGGCATTGCT GGCGATAACAGCTCCTCTTG
OsCDF1re AACTACAACATCAACCAGCCG TGAGAACGGTGCCATTAGTCT

图1

OsCDF1 gRNA靶位点示意图及含有OsCDF1 gRNA的阳性克隆筛选 A: OsCDF1靶位点示意图。灰色矩形为5'和3' untranslated region (UTR); 黑色矩形为外显子; 黑线为内含子。比例尺为200 bp。PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列。B: PCR扩增连接到pRGEB32载体的gRNA。M: DNA marker III; 1~2: PCR产物。C: 含有OsCDF1的gRNA的阳性克隆测序结果。"

图2

纯合不含T-DNA的oscdf1突变体筛选及分析 A: PCR扩增Hyg基因片段结果。M: DNA marker III; 1~48: PCR产物, 1: 模板是ddH2O。B: T1代突变类型分析。C: T2代突变类型分析。红色箭头表示突变位点。D: 靶位点核苷酸变化。灰色矩形为5'和3' UTR; 黑色矩形为外显子; 黑线为内含子。PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列。标尺200 bp。"

图3

OsCDF1突变蛋白的氨基酸变化及三级结构分析 A: 靶位点氨基酸变化。标尺为50 aa。B: 突变蛋白比对分析。C: 三级结构分析。"

图4

oscdf1 突变体开花期表型调查 A: oscdf1突变体表型。白线为标尺, 10 cm。B: oscdf1突变体开花期表型分析(n ≥ 15)。*表示P < 0.05水平差异显著。"

图5

水稻oscdf1突变体和野生型的农艺性状比较 A: 野生型(WT)和oscdf1突变体穗部表型; B: oscdf1突变体抽穗期表型分析; C: 单株穗数; D: 穗长; E: 一次枝梗数; F: 二次枝梗数; G: 粒长; H: 粒宽; I: 粒长表型; J: 粒宽表型; K: 粒厚; L: 每穗粒数; M: 结实率; N: 千粒重; O: 单株产量。C~F和I~N的数据为平均值±标准差(n ≥ 15)。*表示P < 0.05水平差异显著; **表示P < 0.01水平差异显著; n.s.表示P > 0.05水平差异显著。"

图6

OsCDF1基因表达模式分析 A: OsCDF1组织特异性表达分析。B: OsCDF1在不同发育时期的表达量; A和B的数据为平均值±标准差。"

图7

OsCDF1基因变异与单倍型网络分析 A: OsCDF1基因结构及6种变异位置。黑色矩形代表外显子; 灰色矩形代表5'非编码区和3'非编码区; 黑线代表内含子。B: OsCDF1蛋白结构及6种变异位置。不同染色三角形的位置代表6种变异的位置。C: 6种基因变异的单倍型网络, 红线代表2种单倍型之间突变数量。"

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