含草甘膦抗性标记的水稻核不育繁殖系载体构建与验证

doi:10.16819/j.1001-7216.2022.211226

摘要/Abstract

摘要：

【目的】使用草甘膦抗性基因替代潮霉素抗性基因，构建核不育繁殖系的转化载体，赋予繁殖系对除草剂草甘膦的抗性，规避现行政策对转基因作物中使用抗生素标记基因的限制。【方法】利用同源重组的方法，构建含草甘膦抗性基因Epsps^#、花粉致死基因ZmAA1、核不育恢复基因OsEat1和红色荧光基因DsRed2的水稻eat1核不育基因繁殖系载体pC3300-Epsps-AA-Eat-Red;采用农杆菌介导法转化水稻。【结果】通过转化籼稻品系9K19-5获得普通核不育繁殖系Eat9K的转化植株318个。表型鉴定表明，单拷贝转化体Eat9K-3的花粉有可育和不育2种类型，种子有具有和不具有荧光信号2种类型，具有荧光信号的种子在发芽期能耐受浓度至少为1 g/L的草甘膦。建立的四引物检测法能够区分野生型Eat1基因和核不育eat1基因。【结论】证明载体pC3300-Epsps-AA-Eat-Red有效，创制了具有除草剂抗性的普通核不育繁殖系新种质，建立了区分野生型Eat1基因和核不育eat1基因的四引物检测法。

关键词: 草甘膦抗性, 花粉致死, 育性恢复, 普通核不育繁殖系

Abstract:

【Objective】 The hygromycin resistance gene is often used as a selection marker in expression vectors for multiplication of common genic male sterile (CGMS) lines in rice. The glyphosate resistance gene replaces the hygromycin resistance gene as it not only gives the multiplication line glyphosate resistance, but also bypasses the current policy restrictions on the use of antibiotic marker genes in genetically modified crops. 【Methods】 An expression vector pC3300-Epsps-AA-Eat-Red was constructed by using homologous recombination method in order to multiply the rice CGMS line with eat1 mutation gene, which harbors the glyphosate resistance gene Epsps^#, pollen lethal gene ZmAA1, CGMS restorer gene OsEat1 and red fluorescent gene DsRed2. Agrobacterium-mediated transformation method was adopted. 【Results】 Transformation of indica rice line 9K19-5 produced 318 regenerated plants of the CGMS multiplication line Eat9K that could restore the sterility of eat1 and resist glyphosate. The phenotype identification of single copy transformant Eat9K-3 showed that: there were two kinds of pollens in anthers, one was fertile and the other was sterile; the seeds included two types, one was with fluorescence and the other was without fluorescence; the seedlings from fluorescent seeds tolerated at least 1 g/L glyphosate. In addition, this study established a four-primer detection method to distinguish the wild-type gene Eat1 from the genic male sterile gene eat1. 【Conclusion】 This study not only verified the effectiveness of the expression vector, but also created the new germplasm of CGMS multiplication line with herbicide resistance, as well as established a four-primer detection method to distinguish the wild-type gene Eat1 from genic male sterile gene eat1.

Key words: glyphosate resistance, pollen lethality, sterility recovery, common genic male sterile multiplication line

李焱瑶, 邓力华, 李昕晏, 李华, 秦冠男, 翁绿水, 于江辉, 李锦江, 肖国樱. 含草甘膦抗性标记的水稻核不育繁殖系载体构建与验证[J]. 中国水稻科学, 2022, 36(5): 467-475.

LI Yanyao, DENG Lihua, LI Xinyan, LI Hua, QIN Guannan, WENG Lüshui, YU Jianghui, LI Jinjiang, XIAO Guoying. Construction and Verification of Vector Containing Glyphosate Resistance Selection Marker for Multiplication of Common Genic Male Sterile Lines in Rice[J]. Chinese Journal OF Rice Science, 2022, 36(5): 467-475.

图/表 8

表1 本研究用到的引物序列

Table 1. Primer sequences used in the study.

引物名称 Primer name	引物序列(5′-3′) Primer sequence (5′-3′)	用途 Application
Epsps-F	CTGGCGACAAGTCTATCTCAC	Epsps^#基因检测 Epsps# gene detection
Epsps-R	CCTGGAGCATCTTCTCGGTAT	Epsps^#基因检测 Epsps# gene detection
ZmAA1-F	TTCCACGGTGGTTAGTGGTTACTTCT	ZmAA1基因检测 ZmAA1 gene detection
ZmAA1-R	CTGTAGCTCAGCGAGTTCCATATCTC	ZmAA1基因检测 ZmAA1 gene detection
Ltp-Eat1-F	TCCATGTAGTGGGTGGGATT	转化体中Eat1基因检测 Detection of eat1 gene in transformants
Ltp-Eat1-R	TAGGCCACGGTTATTCTCACG	转化体中Eat1基因检测 Detection of eat1 gene in transformants
Eat1-F	GGGTGTGAGCTTTCCGTCTT	载体中Eat1基因检测 Detection of Eat1 gene in vector
Eat1-R	GAAGATGGAGCCGTGGAGTT	载体中Eat1基因检测 Detection of Eat1 gene in vector
DsRed2-F	CCTCCGAGAACGTCATCACC	DsRed2基因检测 DsRed2 gene detection
DsRed2-R	TAGTCCTCGTTGTGGGAGGT	DsRed2基因检测 DsRed2 gene detection
Eat1af	GCAACAGATCCAGCAGTATGAT	区分Eat1与eat1基因 Distinguishing Eat1 from eat1 gene
Eat1ar	GTAGCAGCGGTTCCAGACAGG
Eat1bf	GCAAACATCAACCCACTCCCA
Eat1br	TTTCCCTTGCCCTTTCCGAAC

图1 载体pC3300-Epsps-AA-Eat-Red的T-DNA区域和分子验证 A−载体pC3300-Epsps-AA-Eat-Red的T-DNA区域。Epsps#―草甘膦抗性基因;TNOS―来源于胭脂碱合成酶基因的终止子;PG47―来源于玉米多聚半乳糖醛酸酶基因的花粉特异性启动子;ZmAA1―玉米的α-淀粉酶基因，赋予花粉致死性的花粉致死基因;Tin2-1―来源于In2-1基因的终止子;PEat1―Eat1基因的启动子，Eat1在绒毡层表达，具有育性恢复作用;TEat1―Eat1基因的终止子;Ltp2―来源于大麦糊粉层的特异性启动子;DsRed2―来源于珊瑚的DsRed2基因，编码红色荧光蛋白;CaMV 35S polyA―花椰菜花叶病毒多聚腺苷酸化信号。B−质粒pC3300-Epsps-AA-Eat-Red酶切结果。M−DL 15000 DNA分子量标记;泳道1−完整质粒;泳道2−质粒经PacⅠ酶切。C−PCR检测载体中Epsps#、ZmAA1、Eat1和DsRed2基因。M−DL2000 DNA分子量标记;泳道1−检测pC3300-UbiEpsps-35sCry1Ca;泳道2−检测pC1300-Eat1;泳道3−检测pC3300-Epsps-AA-Eat-Red。

Fig. 1. T-DNA region of vector pC3300-Epsps-AA-Eat-Red and molecular identification. A, T-DNA region of vector pC3300-Epsps-AA-Eat-Red. Epsps# stands for the glyphosate resistance gene Epsps#;TNOS, The terminator of nopaline synthase gene; PG47, The pollen-specific promoter from maize polygalacturonase gene; ZmAA1, The alpha-amylase gene of maize conferring pollen lethality; Tin2-1, The terminator from the In2-1 gene; PEat1, The promoter of Eat1 gene; Eat1, The Eternal Tapetum 1 gene that recovers the stertility of eat1 mutation; TEat1, The terminator of Eat1 gene; Ltp2, The promoter of barley aleurone-specific gene Lipid transfer protein 2, DsRed2, The DsRed2 gene from a marine coral-like anemone Discosoma coding red fluorescent protein; CaMV 35S PolyA, The cauliflower mosaic virus polyadenylation signal. B, Digestion of plasmid pC3300-Epsps-AA-Eat-Red. M, DL 15000 DNA marker; Lane 1, Intact plasmid; Lane 2, The plasmid digested by PacI. C, PCR detection of Epsps#, ZmAA1, Eat1 and DsRed2 genes. M, DL2000 DNA marker; Lane 1, Detecting vector pC3300-UbiEpsps-35sCry1Ca; Lane 2, Detecting vector pC1300-Eat1; Lane 3, Detecting vector pC3300-Epsps-AA-Eat-Red.

图2 再生植株Eat9K的PCR检测结果 A―Epsps#基因检测;B―ZmAA1基因检测;C―Eat1基因检测;D―DsRed2基因检测。M―DL2000 DNA分子量标记;P―质粒pC3300-Epsps-AA-Eat-Red;CK―对照9K19-5;1~20―不同的Eat9K再生植株。

Fig. 2. PCR detection of regenerated Eat9K plant. A, Detection of Epsps# gene; B, Detection of ZmAA1 gene; C, Detection of Eat1 gene; D, Detection of DsRed2 gene. M, DL2000 DNA marker; P, Plasmid pC3300-Epsps-AA-Eat-Red; CK, Control 9K19-5; 1-20, Different regenerated plants of Eat9K.

图3 Southern杂交检测T2代Eat9K水稻中Epsps#基因 M―地高辛标记的DNA分子量标记;P―质粒pC3300-Epsps-AA- Eat-Red;CK―非转基因对照;3、6、12、15、17、23、24、28、35分别为Eat9K的不同转化株。

Fig. 3. Southern blotting of Epsps# gene in T2 generation of Eat9K. M, Dig labeled DNA marker II; P, Plasmid pC3300-Epsps-AA-Eat- Red; CK, Non-transgenic control; 3, 6, 12, 15, 17, 23, 24, 28 and 35 are different transformants of Eat9K, respectively.

图4 Eat9K-3的表型鉴定 A−花粉育性观察; B−种子荧光观察; C−除草剂耐受性观察。

Fig. 4. Phenotype identification of Eat9K-3. A, Observation of pollen fertility. B, Observation of fluorescent seeds; C, Assessment of herbicide tolerance.

表2 Eat9K-3的有荧光种子与无荧光种子卡平方检验

Table 2. Chi square test of seeds with and without red fluorescence in T2 generation of Eat9K-3.

株系编号 Number	重复 Repeat	种子有无荧光信号 Seed with or without red fluorescence	实际值 Real value	理论值 Theoretical value	χ²_(1:1)	χ²_{(0.05，1)(1:1）}
Eat9K-3	1	有 With	474	486.5	0.642	3.84
	1	无 Without	499	486.5	0.642
	2	有 With	549	561.0	0.513
	2	无 Without	573	561.0	0.513
	3	有 With	232	230.5	0.020
	3	无 Without	229	230.5	0.020

图5 eat1基因的分子标记选择 A−Eat1和eat1基因测序结果与四引物设计。B−PCR检测Eat1和eat1基因。M−DL2000 DNA分子量标记;WT−受体品种9K19-5;eat1−eat1基因纯合的不育株;H−eat1基因杂合的第三代杂交稻单株;1~3−Eat9K-223杂交转育后的不同单株;4~10−Eat9K-242杂交转育后的不同单株;11−Eat9K-62杂交转育后的不同单株;12~17−Eat9K-150杂交转育后的不同单株;18~20−Eat9K-152杂交转育后的不同单株。

Fig. 5. Marker-assisted selection of eat1 gene. A, Sequencing results of Eat1 and eat1 genes and the design of four primers to detect Eat1 and eat1 genes. B, Detection of Eat1 and eat1 genes by PCR. M, DL2000 DNA marker; WT, Receptor variety 9K19-5; eat1, Sterile plant with homozygous eat1 gene; H, Plant of the third-generation hybrid rice with heterozygous eat1 gene; 1-3, Different plants from cross with Eat9K-223; 4-10, Different plants from cross with Eat9K-242; 11, Different plants from cross with Eat9K-62; 12-17, Different plants from cross with Eat9K-150; 18-20, Different plants from cross with Eat9K-152.

图6 回交选育普通核不育繁殖系和不育系的育种程序

Fig. 6. Backcross breeding of common genic male sterile line and its multiplication line.

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