Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

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	Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences
	Kong Fanjing; Chen Xiao; Ma Youzhi; Xin Zhiyong; Li Liancheng; Zhang Zengyan; Lin Zhishan
刊名	Acta Botanica Sinica
	2002
卷号	44 期号:3 页码:307-313
关键词	microdissection and microcloning of chromosome Haynaldia villosa genomic in situ hy-bridization alien substitution of telosome species-specific DNA sequences RFLP
ISSN号	1672-6650
其他题名	簇毛麦端体6VS的显微切割及其专化DNA序列的克隆和分析
英文摘要	The material T240-6 derived from SC_2 young embryo of Lhe combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the "single tube", all the subsequence steps were conducted. After two round of LA (Linker adaptor)-PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600 - 1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32P as probe. The PCR products were purified and ligated into clone veetot-pGEM-T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl_2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100 - 1 500 bp, with average of 600 bp. Using II. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H, villosa -spec! fie clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.
学科主题	植物学
语种	英语
内容类型	期刊论文
源URL	[http://ir.nais.net.cn/handle/2HMLN22E/131315]
专题	作物科学研究所
作者单位	Institute of Crop Breeding and Cultivation, Glinese Academy of Agricultural Science, Key Lahorahiry of Crop Caustics and Breeding of Ministry of Agriculture, 北京, 100081
推荐引用方式 GB/T 7714	Kong Fanjing,Chen Xiao,Ma Youzhi,et al. Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences[J]. Acta Botanica Sinica,2002,44(3):307-313.
APA	Kong Fanjing.,Chen Xiao.,Ma Youzhi.,Xin Zhiyong.,Li Liancheng.,...&Lin Zhishan.(2002).Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences.Acta Botanica Sinica,44(3),307-313.
MLA	Kong Fanjing,et al."Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences".Acta Botanica Sinica 44.3(2002):307-313.