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

Abstract

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

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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Figures/Tables 9

Table 1

Components of different culture medium for genetic transformation of broccoli"

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

Development of Ogura CMS Restorers of Broccoli via Genetic Transformation of Rfo

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