CN1052985C - 从大赖草中提取与穗形相关成分的高活性dna的方法 - Google Patents

从大赖草中提取与穗形相关成分的高活性dna的方法 Download PDF

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CN1052985C
CN1052985C CN97123423A CN97123423A CN1052985C CN 1052985 C CN1052985 C CN 1052985C CN 97123423 A CN97123423 A CN 97123423A CN 97123423 A CN97123423 A CN 97123423A CN 1052985 C CN1052985 C CN 1052985C
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dna
dalaicao
total dna
wheat
extracting
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CN1221750A (zh
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赵民安
刘敏
戚家华
李维琪
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Xinjiang Technical Institute of Physics and Chemistry of CAS
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Xinjiang Technical Institute of Physics and Chemistry of CAS
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Abstract

本发明涉及一种从大赖草核酸中高活性与穗形相关成份的确定，首先将大赖草叶片先经液氮研磨，抽提，低速离心进行两相分离，沉淀提出总DNA，再经乙醇洗涤后抽干、灭菌，经RNasp保温后，过Sepherose－28柱，分离器ONA和RNA，再进行纯化处理，经超声波及限制性内切酶酶切成DNA片段，通过花粉管通道将其转化到栽培小麦中去，该方法经过多次反复试验证明，小麦穗形均都产生了大穗形变异转化株，经过分子杂交检测，证实穗形小麦是由于大赖草基因转化后产生的结果。

Description

从大赖草中提取与穗形相关成分的高活性DNA的方法
本发明涉及一种从大赖草中提取与穗形相关成分的高活性DNA的方法，尤其是从大赖草中提取总DNA，通过花粉管通道将其转化到小麦中去。
大赖草(Leymus raceaus)[lam]Jzvel是一种生长在半干旱荒漠边缘的多年生杂草，为禾本科赖草属(Leymus Bochst)植物，它具有抗干旱，耐碱，高度抗病、抗虫、穗大、粒多、籽粒蛋白质含量高等多种优良遗传性状，因而在分子育种工作中是个比较理想的优良基因供体。本发明经过多年田间试验证明，大赖草基因中含有高活性的与穗形相关的成分，将该成分通过花粉管通道转化外源基因至培育小麦中去，它能较早的影响小麦的穗形，并最终产生出大穗形小麦。
本发明目的在于，研究确定大赖草核酸中高活性与穗形相关的成分，首先将大赖草叶片经液氮研磨、抽提、低速离心进行两相分离，沉淀出总DNA，再经乙醇洗涤抽干，灭菌，经RNase保温处理后过Sepberose-2B柱分离总DNA和RNA，收集总DNA再进行纯化处理，将纯化后的总DNA经超声波破碎成DNA片段，通过花粉管通道将其转化到小麦中去即可。该方法经过反复试验证明，采用该方法确定大赖草核酸中高活性与穗形相关的成分，是行之有效的，其小麦的穗形均都产生了大穗形变异转化株，经过分子杂交检测，证实大穗小麦是由于大赖草基因转化后产生的结果。
本发明所述的从大赖草中提取与穗形相关成分的高活性DNA的方法，首先将大赖草叶片经液氮研磨，用三羟基氨基甲烷盐酸(Jris-HCl)缓冲液及十二烷基磺酸钠抽提，60℃热酚—氯仿处理，低速离心进行两相分离，水相经异丙醇沉淀，提出总DNA，再经70％乙醇洗涤后抽干，溶于灭菌水中，经RNase保温处理后，过Sepberose-2B柱分离总DNA和RNA，收集总DNA，将收集的总DNA峰，经乙醇沉淀后抽干，溶于JE缓冲液中，得纯化后的总DNA，然后再经超声波及限制性内切酶酶切成大小为0.5-10kb的DNA片段，通过花粉管通道将其转化到载培小麦中去即可。
实施例
取1克大赖草叶片，经液氮研磨，加入10ml三羟基氨基甲烷盐酸缓冲液及1/10体积10％的十二烷基磺酸钠抽提，等体积各50％热酚—氯仿处理，于60℃保温10分钟，低速离心进行两相分离，水相加入0.6倍体积的异丙醇沉淀，提出总DNA，再用70％乙醇洗涤后抽干，溶于灭菌水中，加入RNasp50mg/ml，37℃、0.5小时保温，过Sepherose-2B柱，收集第一峰，再经乙醇沉淀后抽干，溶于TE缓冲液中，得纯化后的总DNA，再经超声波及限制性内切酶酶切成大小为0.5-10Kb的DNA片段，再通过花粉管通道将其转化到栽培小麦中去即可，可获得大穗形变异转化株。

Claims (3)

1、一种从大赖草中提取与穗形相关成分的高活性DNA的方法，其特征在于，首先将大赖草叶片经液氮研磨，用三羟基氨基甲烷盐酸(Tris-HCl)缓冲液及十二烷基磺酸钠抽提，60℃热酚—氯仿处理，低速离心进行两相分离，水相经异丙醇沉淀，提出总DNA，再经70％乙醇洗涤后抽干，溶于灭菌水中，经RNase保温处理后，过Sepherose-2B柱分离总DNA和RNA，收集总DNA。
2、根据权利要求1所述的从大赖草中提取与穗形相关成分的高活性DNA的方法，其特征在于，将收集的总DNA峰，经乙醇沉淀后抽干，溶于JE缓冲液中，得纯化后的总DNA。
3、一种从大赖草中提取与穗形相关成分的高活性DNA的应用，其特征在于，将收集的总DNA再经超声波及限制性内切酶切成大小为0.5-10kb的DNA片段，通过花粉管通道将其转化到栽培小麦中去即可。
CN97123423A 1997-12-27 1997-12-27 从大赖草中提取与穗形相关成分的高活性dna的方法 Expired - Fee Related CN1052985C (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1104251A (zh) * 1993-07-29 1995-06-28 农林水产省农业生物资源研究所 稻瘟病抗性基因的核酸标记物以及通过使用这些标记物分离到的稻瘟病抗性基因

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1104251A (zh) * 1993-07-29 1995-06-28 农林水产省农业生物资源研究所 稻瘟病抗性基因的核酸标记物以及通过使用这些标记物分离到的稻瘟病抗性基因

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