应用CRISPR-Cas9技术对大麦棱型皮裸基因进行基因编辑的初步研究

发布时间：2018-04-24 04:20

本文选题：大麦 + 农杆菌介导法　；参考：《华中农业大学》2017年硕士论文

【摘要】：大麦是世界上第四大谷类作物,可作为饲料、食品生产或啤酒酿造的原料,是典型的“三元作物”。控制大麦棱型的六棱基因vrs1,为位于2H染色体长臂上的隐性基因,是独立遗传的,它的等位基因Vrs1能够抑制侧小穗的发育,从而导致二棱的形成。很多研究表明,大麦棱型性状和大麦的产量有着密切联系。单隐性基因nud位于大麦7H染色体长臂上,控制裸颖果的性状,像棱型基因一样,Nud基因对大麦的品质性状也有着显著的影响。为了获得高产量和高品质的大麦品种或种质,本研究使用农杆菌介导法,利用CRISPR-Cas9技术对棱型Vrs1基因和皮裸基因Nud进行基因组的定点编辑,以期获得二棱性状向六棱性状转变以及皮颖果性状向裸颖果性状转变的大麦基因编辑植株。文中分别选取了华大麦5号,华大麦6号和浙农大3号的幼胚愈伤组织转化以棱型基因Vrs1为靶基因的载体,华大麦4号和华大麦7号的幼胚愈伤组织转化以皮裸基因Nud为靶基因的载体,同时对华大麦5号和华大麦6号的成熟胚愈伤组织进行了棱型基因Vrs1为靶基因的转化,最终以潮霉素为筛选标记基因,成功获得了部分不同大麦品种的转基因阳性植株,同时对幼胚愈伤分化率与培养时间的关系进行分析,主要结果如下:(1)在农杆菌介导棱型基因Vrs1对大麦幼胚愈伤组织的转化试验中,共得到14株抗性植株,其中华大麦5号未得到再生植株。经PCR检测,共有潮霉素基因阳性植株有4株,分别为浙农大3号和华大麦6号各2株。(2)在农杆菌介导棱型基因Vrs1对大麦成熟胚愈伤组织的转化试验中,使用的大麦品种为华大麦5号和华大麦6号,最终没有得到抗性苗。(3)在农杆菌介导皮裸基因Nud对大麦幼胚愈伤组织的转化试验中,华大麦7号得到5株抗性苗,而华大麦4号未得到抗性苗。经PCR检测得到1株潮霉素基因阳性植株。(4)幼胚愈伤组织侵染后在分化培养基上分化时,华大麦6号和浙农大3号在培养时间8周时愈伤组织分化率最高,而华大麦7号在培养12周时分化率最高。
[Abstract]:Barley is the fourth largest cereal crop in the world. It can be used as raw material for feed, food production or beer brewing. It is a typical "ternary crop". The six-rowed gene vrs1, a recessive gene located on the long arm of chromosome 2H, is independently inherited, and its allele Vrs1 can inhibit the development of lateral spikelet and lead to the formation of dirowths. Many studies have shown that barley prism traits are closely related to barley yield. The single recessive gene nud is located on the long arm of barley chromosome 7H and controls the characters of naked caryopsis. In order to obtain barley varieties or germplasm with high yield and high quality, this study used Agrobacterium tumefaciens (Agrobacterium tumefaciens) to modify the genomes of Vrs1 gene and naked gene Nud by CRISPR-Cas9. The purpose of this study was to obtain barley gene editing plants with the transformation from two-rowed traits to six-rowed traits and from caryopsis traits to naked caryopsis traits. In this paper, the callus of immature embryos of Huaheng No. 5, Huaheng No. 6 and Zhejiang Nongda No. 3 were selected respectively. The Vrs1 gene was used as the target gene vector. The immature embryogenic calli of Huamai 4 and Huahai 7 were transformed into calli with the naked gene Nud as the target gene, and the mature embryogenic callus of Huaheng 5 and Huahai 6 were transformed with the row-type gene Vrs1 as the target gene. Finally, using hygromycin as the marker gene, transgenic positive plants of some different barley varieties were successfully obtained, and the relationship between callus differentiation rate of immature embryos and culture time was analyzed. The main results were as follows: 1) in the transformation of callus of barley immature embryos mediated by Agrobacterium tumefaciens (Agrobacterium tumefaciens), 14 resistant plants were obtained, and no regenerated plants were obtained from Zhonghua Barley No. 5. According to PCR analysis, there were 4 hygromycin positive plants, 2 plants from Zhejiang province and 2 plants from Huaheng 6, respectively. The transformation of mature embryo callus mediated by Agrobacterium tumefaciens (Vrs1) gene was studied. In the transformation of callus of barley immature embryos mediated by Agrobacterium tumefaciens (Nud), five resistant seedlings were obtained. However, no resistant seedling was obtained from Huaheng No. 4. PCR analysis showed that one hygromycin gene-positive plant. 4) the callus differentiation rate was the highest at 8 weeks after the callus was infected and differentiated on the differentiation medium of Huamai 6 and Zhe Nongda 3. However, Huamai 7 had the highest rate of culture at 12 weeks.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S512.3

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