通过遗传转化Rfo获得青花菜Ogura CMS恢复系

doi:10.3864/j.issn.0578-1752.2023.15.011

摘要/Abstract

摘要：

【目的】将来自萝卜的胞质不育恢复基因Rfo导入到青花菜Ogura细胞质雄性不育（cytoplasmic male sterility，CMS）主栽商业品种‘耐寒优秀'（命名为SFB45）中，获得Ogura CMS恢复系，打破Ogura CMS材料在生产上无法再利用的现状，为优异种质的创制和改良提供新途径。【方法】利用基因合成的方法克隆萝卜Rfo 的CDS及其启动子序列，构建表达载体pRfo ::Rfo。通过农杆菌介导的遗传转化方法侵染SFB45的具柄子叶和下胚轴，进行再生、抗性苗筛选和PCR鉴定获得阳性植株，开花期观察转基因植株的育性恢复情况；用亚历山大染液分析对照组及阳性株的花粉活力；分别提取对照组及阳性株<3 mm和>3 mm花蕾的RNA并反转录成单链cDNA，设计特异引物，利用RT-PCR分析Rfo及绒毡层和花粉壁发育相关基因在SFB45及对应的育性恢复材料花蕾中的表达差异。【结果】通过遗传转化共获得10个阳性株系，其中8个株系育性得到不同程度的恢复，花粉活力为84.2%—90.4%。RT-PCR分析结果表明，育性恢复植株花蕾中Rfo表达，绒毡层发育关键调节因子DYT1和TDF1及花粉壁主要成分孢粉素合成所必需的基因ACOS5在育性恢复植株<3 mm花蕾中上调表达。绒毡层降解相关基因AMS、四分体胼胝质壁及花粉外壁发育所必需的基因CalS5和CYP703在育性恢复植株>3 mm花蕾中上调表达。分析阳性株系R-1、R-3和R-6自交及杂交后代发现转入的Rfo能稳定遗传，符合孟德尔遗传。【结论】通过遗传转化Rfo获得了优良青花菜Ogura CMS商品种SFB45的育性恢复系，且Rfo的转入使绒毡层及花粉壁发育相关基因恢复正常表达。

关键词: 青花菜, 遗传转化, Ogura CMS, Rfo, 绒毡层和花粉壁发育相关基因

Abstract:

【Objective】 The broccoli restorers were developed by transforming the matching fertility restorer gene of Rfo from radish into the leading cultivar Ogura CMS line of Nai Han You Xiu (named as SFB45), so as to efficaciously use Ogura cytoplasmic male sterility (CMS) hybrids in breeding and to provide resources for genetic improvement of germplasm.【Method】 The sequence of CDS with its preceding promoter of Rfo were synthesized and the pRfo::Rfo plant expression vector was constructed to infect the cotyledon with petiole, and hypocotyl explants of the SFB45 through Agrobacterium-mediated transformation method. After regeneration and screening of herbicide resistant seedlings, transgenic plants were identified by detection of the Bar resistant marker. The fertility of transgenic plants were observed at flowering stage. The pollen viability of SFB45 and the transgenic plants were analyzed using the alexander stain. Total RNA of <3 mm and >3 mm flower buds from SFB45 and the transgenic plants were isolated and transcribed into cDNA. The Rfo specific primers were designed, and RT-PCR was performed to analyze the expression levels of Rfo and also the genes related to tapetum and pollen wall development in flower buds of SFB45 and its restorer lines.【Result】 A total of 10 transgenic plants were obtained by genetic transformation. Among which, the fertilities of 8 were restored to varying degrees with the average pollen viability ranging from 84.2% to 90.4%. RT-PCR analysis showed that Rfo was expressed in flower buds of fertility restored plants. The key regulators of tapetum development (DYT1 and TDF1) and the essential gene (ACOS5) for the synthesis of sporopollenin, a major component of the pollen wall were up-regulated in <3 mm flower buds of Ogura CMS restore lines. The tapetum degradation related gene AMS and tetrad callose wall and pollen outer wall development related genes CalS5 and CYP703 were up-regulated in >3 mm flower buds of the restore lines. The analysis of the self- and hybrid-crossing progenies of the positive transgenic lines R-1, R-3 and R-6 showed that the introduced Rfo could be stably inherited, which was consistent with the Mendelian inheritance.【Conclusion】 The fertility restorer lines of Ogura CMS SFB45 were obtained by genetic transformation of Rfo, and the integration of Rfo into the genome of SFB45 recovered the expression of genes associated with tapetum and pollen wall development.

Key words: broccoli, genetic transformation, Ogura CMS, Rfo, tapetum and pollen wall development-related genes

邢苗苗, 许园园, 卢昱宇, 严继勇, 曾爱松. 通过遗传转化Rfo获得青花菜Ogura CMS恢复系[J]. 中国农业科学, 2023, 56(15): 2966-2976.

XING MiaoMiao, XU YuanYuan, LU YuYu, YAN JiYong, ZENG AiSong. Development of Ogura CMS Restorers of Broccoli via Genetic Transformation of Rfo[J]. Scientia Agricultura Sinica, 2023, 56(15): 2966-2976.

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https://www.chinaagrisci.com/CN/Y2023/V56/I15/2966

图/表 9

表1

青花菜遗传转化的培养基配方"

培养基 Medium	成分 Component
MS培养基 MS medium	4.43 g∙L^-1 MS培养基，30 g∙L^-1蔗糖，8 g∙L^-1琼脂粉，pH 5.9 4.43 g∙L^-1 MS, 30 g∙L^-1 sugar, 8 g∙L^-1 agar, pH 5. 9
预培养基 Preculture medium	4.43 g∙L^-1 MS培养基，30 g∙L^-1蔗糖，8 g∙L^-1琼脂粉，1 mg∙L^-1 6-苄氨基嘌呤，0.1 mg∙L^-1 萘乙酸，pH 5.9 4.43 g∙L^-1 MS, 30 g∙L^-1 sugar, 8 g∙L^-1 agar, 1 mg∙L^-1 6-BA, 0.1 mg∙L^-1 NAA, pH 5. 9
共培养基 Cocultivation medium	4.43 g∙L^-1 MS培养基，30 g∙L^-1蔗糖，8 g∙L^-1琼脂粉，1 mg∙L^-1 6-苄氨基嘌呤，0.1 mg∙L^-1 萘乙酸，100 μmol∙L^-1乙酰丁香酮，pH 5.9 4.43 g∙L^-1 MS, 30 g∙L^-1 sugar, 8 g∙L^-1 agar, 1 mg∙L^-1 6-BA, 0.1 mg∙L^-1 NAA, 100 μmol∙L^-1Acetosyringone, pH 5. 9
延迟培养基 Delayed medium	4.43 g∙L^-1 MS培养基，30 g∙L^-1蔗糖，8 g∙L^-1琼脂粉，1 mg∙L^-1 6-苄氨基嘌呤，0.1 mg∙L^-1 萘乙酸，300 mg∙L^-1特美汀，pH 5.9 4.43 g∙L^-1 MS, 30 g∙L^-1 sugar, 8 g∙L^-1 agar, 1 mg∙L^-1 6-BA, 0.1 mg∙L^-1 NAA, 300 mg∙L^-1Timentin, pH 5. 9
选择培养基 Selective medium	4.43 g∙L^-1 MS培养基，30 g∙L^-1蔗糖，8 g∙L^-1琼脂粉，1 mg∙L^-1 6-苄氨基嘌呤，0.1 mg∙L^-1 萘乙酸，300 mg∙L^-1特美汀，pH 5.9 4.43 g∙L^-1 MS, 30 g∙L^-1 sugar, 8 g∙L^-1 agar, 1 mg∙L^-1 6-BA, 0.1 mg∙L^-1 NAA, 10 mg∙L^-1 basta, 300 mg∙L^-1 Timentin, pH 5. 9
长苗培养基 Seedling medium	4.43 g∙L^-1 MS培养基，30 g∙L^-1蔗糖，8 g∙L^-1琼脂粉，0.2 mg∙L^-1 6-苄氨基嘌呤，0.1 mg∙L^-1 萘乙酸，300 mg∙L^-1特美汀，pH 5.9 4.43 g∙L^-1 MS, 30 g∙L^-1 sugar, 8 g∙L^-1 agar, 0.2 mg∙L^-1 6-BA, 0.1 mg∙L^-1 NAA, 300 mg∙L^-1 Timentin, pH 5. 9
生根培养基 Rooting medium	4.43 g∙L^-1 MS培养基，30 g∙L^-1蔗糖，8 g∙L^-1琼脂粉，0.1 mg∙L^-1 6-苄氨基嘌呤，0.1 mg∙L^-1吲哚乙酸，pH 5. 9 4.43 g∙L^-1 MS, 30 g∙L^-1 sugar, 8 g∙L^-1 agar, 0.1 mg∙L^-1 NAA, 0.1 mg∙L^-1 IBA, pH 5. 9

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引物名称 Primer name	引物序列 Primer sequence
Bar-F	AAACCCACGTCATGCCAGTT
Bar-R	GTCTGCACCATCGTCAACCAC
Bo138₃₀₀-F	CCATGGACAATAATCTTAGT
Bo138₃₀₀-R	TAATGTGCATGAAGTTGAAG
PRfo-F	cagtGCTCTTCaagacatattcatagattttgttt
PRfo-R	cgatCGTCTCatttatttttgtttcgcctaa
Rfo-F	cagtCGTCTCataaaatgttggctagggtttgtgg
Rfo-R	cagtGCTCTTCaagttcatcccccaaatgatagat
Rfo-qF	TGCTCTAAGCTCCCCTTTGC
Rfo-qR	CAGAGCTACGGCTTCGACAA
Actin-F	CCAGAGGTCTTGTTCCAGCCATC
Actin-R	GTTCCACCACTGAGCACAATGTTAC
DYT1-F	TTATGAACAACAAACGGT
DYT1-R	ATCTCAAATCCCAACAGG
TDF1-F	GAGATGTGGGAAGAGTTG
TDF1-R	ATTAGGTGTGAGAAAGGC
BolC7t41091H-F	TTCCTTCTTCCATCCCTC
BolC7t41091H-R	GACCTCATCCGCTACCAT
BolC3t18036H-F	TGCCCATCTTCCTTCTTC
BolC3t18036H-R	CGTTACGGTTTCATCCAT
CYP703A2-F	CTGGTGAGAATGGGAAAG
CYP703A2-R	TCGTGAGGAATCAAGAAC
ACOS5-F	AAGAGCATACCAATCCTA
ACOS5-R	AGACACCTTCCTCAACAT
CalS5-F	TGGTGTCATTGGTGCCTT
CalS5-R	TTTTGCCGCTTCAGTTCT

自交/杂交Inbreds/Hybrids	阳性株数 Number of positive plants	阴性株数 Number of negative plants	检测株数 Number of total detective plants	比例 Ratio	χ²	χ²_0.05,1
R-1×R-1	23	8	31	3:1	0.01	3.84
R-3×R-3	17	9	26	3:1	1.28	3.84
R-6×R-6	15	10	25	3:1	3.00	3.84
MXS×R-3	25	25	50	1:1	0	3.84