【摘要】：miRNAs是植物體內(nèi)一類長(zhǎng)度在21個(gè)核苷酸左右、非編碼具有調(diào)控功能的小RNA分子,它們通過(guò)參與基因的轉(zhuǎn)錄后調(diào)控,在植物生長(zhǎng)發(fā)育和抗病抗逆方面發(fā)揮著至關(guān)重要的作用。前人研究表明,miR396廣泛參與到植物的細(xì)胞分裂、葉片性狀形成、花器官變異和耐旱性等過(guò)程。為有效利用該優(yōu)良基因資源,本研究使用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化技術(shù),將來(lái)源于水稻的miR396基因?qū)氪篼溨羞^(guò)量表達(dá),以期獲得性狀變異的優(yōu)良轉(zhuǎn)基因植株,尤其是在耐旱性方面表現(xiàn)突出的材料。文中選取了6個(gè)大麥品種的幼胚愈傷組織和3個(gè)品種的成熟胚愈傷組織進(jìn)行遺傳轉(zhuǎn)化,以抗潮霉素hptⅡ基因?yàn)楹Y選標(biāo)記,成功獲得了部分含有目的基因Os-miR396的不同大麥品種的轉(zhuǎn)基因陽(yáng)性株,同時(shí)對(duì)大麥轉(zhuǎn)基因過(guò)程的影響因素進(jìn)行了分析和優(yōu)化,主要結(jié)果如下:(1)基因型的差異依然是影響幼胚和成熟胚愈傷誘導(dǎo)率的主要因素;(2)當(dāng)成熟胚采用只接種盾片誘導(dǎo)時(shí),不需要添加ABA便可有效減少發(fā)芽率,添加ABA反而會(huì)抑制愈傷生長(zhǎng)發(fā)育;(3)相同潮霉素濃度對(duì)不同品種幼胚愈傷的分化篩選效果存在差異,25mg/L的潮霉素劑量對(duì)美里黃金、華大麥4號(hào)和華大麥7號(hào)而言篩選壓可能過(guò)高,對(duì)愈傷的生長(zhǎng)及增殖可能有著不良影響;(4)不同濃度的Timentin抑菌效果不同,當(dāng)侵染濃度高時(shí),需要提高Timentin的濃度才能很好的抑制農(nóng)桿菌的污染;(5)農(nóng)桿菌介導(dǎo)法對(duì)幼胚愈傷轉(zhuǎn)化實(shí)驗(yàn)中,得到17株抗性大麥植株,經(jīng)PCR檢測(cè):miR396基因陽(yáng)性株為5株,分別為干木二條1株、蘇啤1號(hào)2株和浙農(nóng)大三號(hào)2株。(6)農(nóng)桿菌介導(dǎo)法對(duì)成熟胚愈傷轉(zhuǎn)化實(shí)驗(yàn)中,得到13株再生大麥植株,PCR檢測(cè)表明:miR396基因陽(yáng)性株為4株,均來(lái)自于大麥品種干木二條。
[Abstract]:MiRNAs is a kind of small RNA molecules which are about 21 nucleotides in length and are not encoded in plants. They play an important role in plant growth and development and resistance to disease and stress by participating in the post-transcriptional regulation of genes. Previous studies have shown that miR396 is widely involved in plant cell division, leaf character formation, floral organ variation and drought tolerance. In order to effectively utilize this excellent gene resource, the miR396 gene derived from rice was introduced into barley by Agrobacterium tumefaciens-mediated genetic transformation in order to obtain excellent transgenic plants with variant characters. Especially in the field of drought resistance to outstanding materials. In this paper, the immature embryogenic callus of 6 barley varieties and the mature embryo callus of 3 varieties were selected for genetic transformation. The hygromycin resistant hpt 鈪，

本文編號(hào)：2364178