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作物学报 ›› 2021, Vol. 47 ›› Issue (5): 827-836.doi: 10.3724/SP.J.1006.2021.02044

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻单倍体诱导基因OsMATL突变体的创制与分析

文钦(), 贾思思, 王加峰, 黄翠红, 王慧, 陈志强, 郭涛*()   

  1. 华南农业大学/国家植物航天育种工程技术研究中心, 广东广州 510642
  • 收稿日期:2020-06-29 接受日期:2020-11-13 出版日期:2021-05-12 网络出版日期:2020-12-23
  • 通讯作者: 郭涛
  • 作者简介:E-mail: wenqin111222@163.com
  • 基金资助:
    广东省现代种业重大专项(2018B020206002);国家现代农业产业技术体系建设专项(CARS-01-17)

Construction and identification of haploid induction gene OsMATL mutants in rice

WEN Qin(), JIA Si-Si, WANG Jia-Feng, HUANG Cui-Hong, WANG Hui, CHEN Zhi-Qiang, GUO Tao*()   

  1. National Engineering Research Center of Plant Space Breeding, South China Agricultural University, Guangzhou 510642, Guangdong, China
  • Received:2020-06-29 Accepted:2020-11-13 Published:2021-05-12 Published online:2020-12-23
  • Contact: GUO Tao
  • Supported by:
    Research and Development Plant for the Key Areas in Guangdong Province(2018B020206002);China Agriculture Research System(CARS-01-17)

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摘要：

MTL基因是玉米中控制单倍体诱导性状的关键基因, 该基因在作物中的功能高度保守。OsMATL是MTL在水稻中的同源基因, 已有研究证实该基因的突变可以诱导水稻单倍体产生, 但不同遗传背景下, OsMATL基因的突变效应有待明确。本研究以日本晴(粳稻)和华航48 (籼稻)为材料, 利用CRISPR/Cas9技术对OsMATL基因启动子区和编码区的不同位点进行编辑, 成功获得OsMATL基因不同遗传背景系列突变体。分析了不同突变位点对结实率的影响, 发现OsMATL基因编码区突变后结实率为2%~15%, 且存在不同类型的败育籽粒, 而启动子区域的大片段缺失对结实率没有显著影响; 此外, 籼稻背景突变体的结实率高于粳稻突变体。本研究创制的不同类型OsMATL突变体为进一步研究水稻孤雌生殖单倍体诱导的机制提供了基础材料。

关键词: 水稻, 单倍体诱导基因, 突变体

Abstract:

MTL is the key gene for control haploid induction traits in maize, which is highly function conserved in crops. OsMATL is a homologous gene of MTL in rice. It has been confirmed that OsMATL mutation can induce haploid in rice, but the effect of OsMATL mutation in different genetic backgrounds remains to be determined. In this study, Nipponbare (Oryza sativa japonica) and Huahang 48 (Oryza sativa indica) were used as research materials. CRISPR/Cas9 technology was used to edit different sites in the promoter region and coding region of OsMATL, and a series of OsMATL mutations with different genetic backgrounds were successfully obtained. The effect of different mutation sites on seed setting rate was analyzed, and it was found that the seed setting rate was 2%-15% in the coding region of OsMATL. There were different types of abortion seeds, while large fragment deletion in the promoter region had no significant differences on seed setting rate. In addition, the background of indica rice mutants of seed setting rate was higher than japonica rice mutants. OsMATL mutants of different types created in this study provided basic materials for further research on the mechanism of parthenogenetic haploid induction in rice.

Key words: rice, haploid induction gene, mutant

图1

OsMATL基因靶点序列及位置 黑色加粗的字母为PAM序列; 红色三角形指向玉米MTL天然突变位点。"

表1

本研究所用引物"

引物名称 Primer name 引物序列Primer sequence (5'-3')
OsMATL-P1-F GCATCCATGTGTTCCTGATCC
OsMATL-P1-R GGTCCCATTAAGTTTGGCGG
OsMATL-P2-F GGGAGAGTTGGGAACGTTGA
OsMATL-P2-R TGTCGAGGTAGAAGCGGTTG
Recom-F GTGCAGATGATCCGT GGCAACAAAGCACCAGTGGT
Recom-R CTATTTCTAGCTCTAAAAC AAAAAAAAAAGCACCGACTCGGTG
OsMATL-Cas9-1-F taGGTCTCCCTACCTCGACAAgttttagagctagaa
OsMATL-Cas9-1-R cgGGTCTCAGTAGAAGCGGTTtgcaccagccgggaa
OsMATL-Cas9-3-F taGGTCTCCAGGTCGAACTCGgttttagagctagaa
OsMATL-Cas9-3-R cgGGTCTCAACCTCATCGACGtgcaccagccgggaa
OsMATL-Cas9-4-F taGGTCTCCCAACGTCGAGACgttttagagctagaa
OsMATL-Cas9-4-R cgGGTCTCAGTTGACCCTCGAtgcaccagccgggaa
OsMATL-Cas9-P1-F taGGTCTCCTTGCTGGAAGGAgttttagagctagaa
OsMATL-Cas9-P1-R cgGGTCTCAGCAAGGAAACTAtgcaccagccgggaa
OsMATL-Cas9-P2-F taGGTCTCCTAATCCCATATTgttttagagctagaa
OsMATL-Cas9-P2-R cgGGTCTCAATTAAACCCAGCtgcaccagccgggaa
Cas9-J-F AAGCATTTCGTAGTGGGCCA
Cas9-J-R TTTGGTGGTCGCCGTTAGGA
OsMATL-1-F GCATCAGGGGCCTCATCC
OsMATL-1-R GCCCAGCATCTTCCTGATCT
OsMATL-3-F GCGTTGATTCCATCCGCATT
OsMATL-3-R GCTCCTCCTTGTCCTTCACC
OsMATL-4-F GCGCCCATCATCGACATCTT
OsMATL-4-R TGCGTGTACAGTTACTAACGCT
OsMATL-P-F GATCGATCGCCCATAAGCCA
OsMATL-P-R TGACCAACTTGGCGTCATGA
q-ACTIN-F GAATGCTAAGCCAAGAGGAG
q-ACTIN-R AATCACAAGTGAGAACCACAG
q-OsMATL-F CCGAGGTACAACGGCAAGTA
q-OsMATL-R AGAAGATGGTTGGCTGGAGC

图2

CRISPR/Cas9载体构建示意图 A: 单靶点载体; B: 双靶点载体。"

图3

谷物和拟南芥中MTL同源基因氨基酸序列多重比对 红色方框处为玉米MTL天然突变位点。"

图4

OsMATL启动子序列及主要顺式作用元件 蓝色字母为TATA-box; 灰色阴影为CAAT-box; 其余元件均用下画线标出, 元件名字在序列下方。"

表2

T0代编码区纯合突变及启动子区大片段缺失突变类型"

植株编号
Number of plants		 品种
Variety		 序列
Sequence (5′-3′)		 突变类型
Mutation type
Exon 1 WT AACCGCTTCTACCTCGACAACGGCCCACTCATC
1-1-1 Nipponbare AACCGCTTCTACCTCGAACAACGGCCCACTCATC +1 bp
1-1-2 Nipponbare AACCGCTTCTACCTCGATCAACGGCCCACTCATC +1 bp
1-1-3 Nipponbare AACCGCTTCTACCTCGAATTCG --------------------------- ATC -8 bp and substitute 3 bp
1-1-4 Nipponbare AACCGCTT --------------------------------- AACGGCCCACTCATC -10 bp
1-2-1 Huahang 48 AACCGCTTCTACCTCGATCAACGGCCCACTCATC +1 bp
Exon 3 WT CGTCGATGAGGTCGAACTCGCGGACCTTGCCGG
3-1-1 Nipponbare CGTCGATGAGGTCGAACTTCGCGGACCTTGCCGG +1 bp
3-1-2 Nipponbare CGTCGATGAGGTCGA-CTCGCGGACCTTGCCGG -1 bp
3-1-3 Nipponbare CGTCGATGAGGTCGAA-TCGCGGACCTTGCCGG -1 bp
3-1-4 Nipponbare CGTCGATGAGGTCGA ------ TCGCGGACCTTGCCGG -2 bp
3-1-5 Nipponbare CGTCGATGAGGTCG -------------------- CGGACCTTGCCGG -6 bp
3-1-6 Nipponbare CGTCGATGAGG ------------------------------ CGGACCTTGCCGG -9 bp
3-2-1 Huahang 48 CGTCGATGAGGTCGAAACTCGCGGACCTTGCCGG +1 bp
3-2-2 Huahang 48 CGTCGATGAGGTCGAA-TCGCGGACCTTGCCGG -1 bp
3-2-3 Huahang 48 CGTCGATGAGGTCGA ----- TCGCGGACCTTGCCGG -2 bp
3-2-4 Huahang 48 CGTCGATGAGGTC ------------ TCGCGGACCTTGCCGG -4 bp
3-2-5 Huahang 48 CGTCGATGAGGT --------------- TCGCGGACCTTGCCGG -5 bp
Exon 4 WT TCGAGGGTCAACGTCGAGACCGGCAGGTACGTC
4-1-1 Nipponbare TCGAGGGTCAACGTCGAAGACCGGCAGGTACGTC +1 bp
4-1-2 Nipponbare TCGAGGGTCAACGTCGATGACCGGCAGGTACGTC +1 bp
4-1-3 Nipponbare TCGAGGGTCAACGTC-AGACCGGCAGGTACGTC -1 bp
4-1-4 Nipponbare TCGAGGGTCAACGTC ------- GACCGGCAGGTACGTC -2 bp
4-2-1 Huahang 48 TCGAGGGTCAACGTCGAAGACCGGCAGGTACGTC +1 bp
4-2-2 Huahang 48 TCGAGGGTCAACGTCGATGACCGGCAGGTACGTC +1 bp
4-2-3 Huahang 48 TCGAGGGTCAACGT ------- AGACCGGCAGGTACGTC -2 bp
4-2-4 Huahang 48 TCGAGGGTCAACGTC ------ GAACCGGCAGGTACGTC -2 bp
4-2-5 Huahang 48 TCGAGGGT --------------------------------------------------------------------- ACGTC -20 bp
Promoter WT CCATCCTTCCAGCAAGGAAACTA(293 bp)CCGAATATGGGATTAAAC
CCAGC
P-1 Huahang 48 CCATCCT ------------------------------------- (-292 bp) -------------- ATGGGATTAAACCCAGC -292 bp
P-2 Huahang 48 CCATCC ---------------------------------------- (-293 bp) ------------- ATGGGATTAAACCCAGC -293 bp
P-3 Huahang 48 CCA -------------------------------------------------- (-304 bp) --------------------------------------- AAACCCAGC -304 bp
P-4 Huahang 48 TTAGTTA -------------------------------------- (-306 bp) ------------------- GGGATTAAACCCAGC -306 bp
P-5 Huahang 48 TTAGT --------------------------------------------- (-315 bp) ------------------------------------------ AACCCAGC -315 bp
P-6 Huahang 48 TTAGT --------------------------------------------- (-363 bp) -------------------- AGGAGTATATAATTA -363 bp
P-7 Huahang 48 CCAT ------------------------------------------------ (-722 bp) --------------------------------- TAGTTACGGAG -722 bp

图5

野生型和突变体OsMATL基因蛋白三级结构预测及相对表达量 A: 野生型OsMATL基因蛋白三级结构; B: 第1外显子上突变体OsMATL基因蛋白三级结构; C: 第3外显子上突变体OsMATL基因蛋白三级结构; D: 第4外显子上突变体OsMATL基因蛋白三级结构; E: 启动子区293 bp缺失突变体OsMATL基因相对表达量。WT为野生型, MT为突变体。"

图6

野生型和OsMATL突变体的结实率和败育率 **表示OsMATL突变体与野生型之间存在显著差异(P < 0.01)。"

图7

结实表型及不同类型败育籽粒表型 A, B: 结实表型; C, D: 败育表型; 红色三角形指向为实粒。标尺为0.5 cm。"

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