CN105219799A - 一种基于CRISPR/Cas系统的多年生黑麦草的育种方法 - Google Patents
一种基于CRISPR/Cas系统的多年生黑麦草的育种方法 Download PDFInfo
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Abstract
本发明提供了一种基于CRISPR/Cas系统的多年生黑麦草的育种方法，将CRISPR/Cas基因组编辑技术应用在多年生黑麦草品种改良上，对木质素合成关键酶基因LpCCR和LpCOMT进行基因定点敲除，筛选出纯合以及转化载体缺失的突变体，培育出木质素含量降低的多年生黑麦草的育种材料，对于牧草的育种研究和品种改良具有重要的意义。
Description
所属技术领域
本发明涉及植物基因工程技术领域，尤其涉及一种基于CRISPR/Cas系统的多年生黑麦草的育种方法。
背景技术
黑麦草是世界上最重要的温带牧草之一，优良的黑麦草品种对于改善动物的营养均衡、提高牛奶的产量和品质、增大乳品行业的利益，乃至改良人工草坪、防风固沙、保持水土、改善人类生存环境等都具有重要作用。黑麦草生长快、分蘖多、能耐牧，是优质的放牧用牧草，也是禾本科牧草中可消化物质产量最高的牧草之一。黑麦草营养价值高，富含蛋白质、矿物质和维生素，其中干草粗蛋白含量高达25％以上，且叶多质嫩，适口性好，可直接喂养牛、羊、马、兔、鹿、猪、鹅、鸵鸟、鱼等。传统杂交育种存在种质资源丰富度的硬性要求和远缘不亲和的技术瓶颈，如何通过新兴的生物技术获得黑麦草重大、突破性品种，成为黑麦草育种研究的突破口和关键技术。
发明内容
本发明的目的是针对现有技术中存在的上述问题，提供一种基于CRISPR/Cas系统的多年生黑麦草的育种方法，得到木质素含量降低的多年生黑麦草的育种材料。
本发明的技术方案为：
一种基于CRISPR/Cas系统的多年生黑麦草的育种方法，其特征在于：包括下列步骤：
(1)、筛选功能基因，所述功能基因为木质素合成的关键酶基因LpCCR和LpCOMT；
(2)、优化酿脓链球菌Cas9基因，并在基因编码序列的两端分别添加NLS核定位信号和限制性内切酶位点；
(3)、用限制性内切酶酶切优化的Cas9基因，得到基因片段，连入载体，得到重组质粒pA；
(4)、扩增多年生黑麦草的U3或U6RNA启动子，连接在gRNA骨架序列前，将U3或U6启动子和gRNA构建在pB载体上；
(5)、设计并合成带有粘性末端的靶位点引物，连接于pB载体上；
(6)、以黑麦草幼苗为起始材料，采用纤维素酶R-10和离析酶R-10，对黑麦草叶肉组织进行消化并利用密度梯度沉降的方法分离原生质体，获得高纯度的原生质体；
(7)、通过PEG介导法将目的基因导入黑麦草原生质体基因组中，通过后续原生质体基因组DNA提取，进行酶切和测序，检测构建载体pA、pB的活性。
(8)、选择高表达的靶位点引物进行遗传转化，在T0代转基因植株中筛选功能基因敲除的突变体；
(9)、在后代中筛选纯合突变体以及pA、pB质粒整合位点和靶基因位点不在同一染色体上的品系；
(10)、将步骤(9)筛选得到的纯合突变体和所述品系进行回交转育，得到木质素含量降低的多年生黑麦草品系；
本发明将CRISPR/Cas基因组编辑技术应用在多年生黑麦草品种改良上，对木质素合成关键酶基因LpCCR和LpCOMT进行基因定点敲除，筛选出纯合以及转化载体缺失的突变体，培育出木质素含量降低的多年生黑麦草的育种材料，对于牧草的育种研究和品种改良具有重要的意义。
具体实施方式
下面结合实施例对本发明做进一步说明：
本发明提供的基于CRISPR/Cas系统的多年生黑麦草的育种方法，包括以下步骤：
(1)、筛选功能基因，所述功能基因为木质素合成的关键酶基因LpCCR和LpCOMT；
(2)、优化酿脓链球菌Cas9基因，并在基因编码序列的两端分别添加NLS核定位信号和限制性内切酶位点；
(3)、用限制性内切酶酶切优化的Cas9基因，得到基因片段，连入载体，得到重组质粒pA；
(4)、扩增多年生黑麦草的U3或U6RNA启动子，连接在gRNA骨架序列前，将U3或U6启动子和gRNA构建在pB载体上；
(5)、设计并合成带有粘性末端的靶位点引物，连接于pB载体上；
(6)、以黑麦草幼苗为起始材料，采用纤维素酶R-10和离析酶R-10，对黑麦草叶肉组织进行消化并利用密度梯度沉降的方法分离原生质体，获得高纯度的原生质体；
(7)、通过PEG介导法将目的基因导入黑麦草原生质体基因组中，通过后续原生质体基因组DNA提取，进行酶切和测序，检测构建载体pA、pB的活性。
(8)、选择高表达的靶位点引物进行遗传转化，在T0代转基因植株中筛选功能基因敲除的突变体；
(9)、在后代中筛选纯合突变体以及pA、pB质粒整合位点和靶基因位点不在同一染色体上的品系；
(10)、将步骤(9)筛选得到的纯合突变体和所述品系进行回交转育，得到木质素含量降低的多年生黑麦草品系；
采用本发明的育种方法获得的木质素含量降低的多年生黑麦草种子产品与丹麦DLF-TrifoliumA/S公司产品“凯力”相比，具有如下优势：
以上通过实施例对本发明的进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。
Claims (1)
1.一种基于CRISPR/Cas系统的多年生黑麦草的育种方法，其特征在于：包括下列步骤：
(1)、筛选功能基因，所述功能基因为木质素合成的关键酶基因LpCCR和LpCOMT；
(2)、优化酿脓链球菌Cas9基因，并在基因编码序列的两端分别添加NLS核定位信号和限制性内切酶位点；
(3)、用限制性内切酶酶切优化的Cas9基因，得到基因片段，连入载体，得到重组质粒pA；
(4)、扩增多年生黑麦草的U3或U6RNA启动子，连接在gRNA骨架序列前，将U3或U6启动子和gRNA构建在pB载体上；
(5)、设计并合成带有粘性末端的靶位点引物，连接于pB载体上；
(6)、以黑麦草幼苗为起始材料，采用纤维素酶R-10和离析酶R-10，对黑麦草叶肉组织进行消化并利用密度梯度沉降的方法分离原生质体，获得高纯度的原生质体；
(7)、通过PEG介导法将目的基因导入黑麦草原生质体基因组中，通过后续原生质体基因组DNA提取，进行酶切和测序，检测构建载体pA、pB的活性。
(8)、选择高表达的靶位点引物进行遗传转化，在T0代转基因植株中筛选功能基因敲除的突变体；
(9)、在后代中筛选纯合突变体以及pA、pB质粒整合位点和靶基因位点不在同一染色体上的品系；
(10)、将步骤(9)筛选得到的纯合突变体和所述品系进行回交转育，得到木质素含量降低的多年生黑麦草品系。
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