Acta Botanica Sinica Volume 44 Issue 3, Pages 307-313.
Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

KONG Fan Jing, CHEN Xiao, MA You Zhi, XIN Zhi Yong, LI Lian Cheng,ZHANG Zeng Yan, LIN Zhi Shan

Abstract:

The material T240-6 derived from SC2 young embryo of the 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 32 P as probe. The PCR products were purified and ligated into clone vector—pGEM-T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl2. 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 H. 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 specific 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. 

簇毛麦端体6VS的显微切割及其专化DNA序列的克隆和分析

孔凡晶  陈孝  马有志*  辛志勇  李连成  张增艳  林志姗

(中国农业科学院作物育种栽培研究所,农业部作物遗传育种重点实验室,北京,100081)

摘要:从簇毛麦(Haynaldia villosa (L.) Schur.)组合CA9211/RW15(6D/6V异代换系)幼胚培养SC2后代中,用原位杂交方法鉴定出T240-66VS端体异代换系. 以此为材料,采用微细玻璃针切割法及"单管反应"技术体系,6VS进行切割分离及LA (Linker adaptor)-PCR扩增.扩增带在1003 000 bp 之间,大部分集中在6001 500 bp.利用32P标记的簇毛麦基因组为探针进行Southern杂交,证实扩增产物来源于簇毛麦.扩增产物纯化后,连接到pGEM-T载体上,构建了6VS DNA质粒文库.对文库的分析表明,文库大约有17 000个白色克隆;插入片段分布在1001 500 bp,平均600 bp.点杂交结果表明,37%克隆有中度到强烈的杂交信号,证明含有中度或高度重复序列;63%克隆有较弱的信号或没有信号,证明为单/低拷贝序列克隆.从文库中获得8个簇毛麦特异克隆,对其中两个克隆pHVMK22 pHVMK134进行了RFLP分析和序列分析,并利用该探针对小麦抗白粉病基因Pm21进行了检测.RFLP 结果表明,两个克隆一个为低拷贝序列克隆(pHVMK22),另一个为高度重复序列克隆,均为簇毛麦专化DNA序列.pHVMK22为探针对抗、感病小麦(Triticum aestivum L.)品系的Southern杂交发现抗病品系有一条2 kb的特征带, 该探针可能作为检测抗病基因Pm21的探针.

关键词 染色体显微切割及微克隆 簇毛麦 原位杂交 端体异代换系 专化DNA序列

*通讯作者 Email: mayzh@mail.caas.net.cn

 


Keywords:
microdissection and microcloning of chromosome; Haynaldia villosa; genomic in situ hybridization; alien substitution of telosome; species specific DNA sequences; RFLP

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