本文選題：大麥 + 農(nóng)桿菌介導(dǎo)法��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：大麥?zhǔn)鞘澜缟系谒拇蠊阮愖魑?可作為飼料、食品生產(chǎn)或啤酒釀造的原料,是典型的“三元作物”�？刂拼篼溊庑偷牧饣騰rs1,為位于2H染色體長臂上的隱性基因,是獨立遺傳的,它的等位基因Vrs1能夠抑制側(cè)小穗的發(fā)育,從而導(dǎo)致二棱的形成。很多研究表明,大麥棱型性狀和大麥的產(chǎn)量有著密切聯(lián)系。單隱性基因nud位于大麥7H染色體長臂上,控制裸穎果的性狀,像棱型基因一樣,Nud基因?qū)Υ篼湹钠焚|(zhì)性狀也有著顯著的影響。為了獲得高產(chǎn)量和高品質(zhì)的大麥品種或種質(zhì),本研究使用農(nóng)桿菌介導(dǎo)法,利用CRISPR-Cas9技術(shù)對棱型Vrs1基因和皮裸基因Nud進行基因組的定點編輯,以期獲得二棱性狀向六棱性狀轉(zhuǎn)變以及皮穎果性狀向裸穎果性狀轉(zhuǎn)變的大麥基因編輯植株。文中分別選取了華大麥5號,華大麥6號和浙農(nóng)大3號的幼胚愈傷組織轉(zhuǎn)化以棱型基因Vrs1為靶基因的載體,華大麥4號和華大麥7號的幼胚愈傷組織轉(zhuǎn)化以皮裸基因Nud為靶基因的載體,同時對華大麥5號和華大麥6號的成熟胚愈傷組織進行了棱型基因Vrs1為靶基因的轉(zhuǎn)化,最終以潮霉素為篩選標(biāo)記基因,成功獲得了部分不同大麥品種的轉(zhuǎn)基因陽性植株,同時對幼胚愈傷分化率與培養(yǎng)時間的關(guān)系進行分析,主要結(jié)果如下:(1)在農(nóng)桿菌介導(dǎo)棱型基因Vrs1對大麥幼胚愈傷組織的轉(zhuǎn)化試驗中,共得到14株抗性植株,其中華大麥5號未得到再生植株。經(jīng)PCR檢測,共有潮霉素基因陽性植株有4株,分別為浙農(nóng)大3號和華大麥6號各2株。(2)在農(nóng)桿菌介導(dǎo)棱型基因Vrs1對大麥成熟胚愈傷組織的轉(zhuǎn)化試驗中,使用的大麥品種為華大麥5號和華大麥6號,最終沒有得到抗性苗。(3)在農(nóng)桿菌介導(dǎo)皮裸基因Nud對大麥幼胚愈傷組織的轉(zhuǎn)化試驗中,華大麥7號得到5株抗性苗,而華大麥4號未得到抗性苗。經(jīng)PCR檢測得到1株潮霉素基因陽性植株。(4)幼胚愈傷組織侵染后在分化培養(yǎng)基上分化時,華大麥6號和浙農(nóng)大3號在培養(yǎng)時間8周時愈傷組織分化率最高,而華大麥7號在培養(yǎng)12周時分化率最高。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S512.3

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本文編號：1795123