本文選題：大白菜 + 核基因雄性不育　； 參考：《沈陽農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：大白菜核復(fù)等位基因遺傳的雄性不育材料的發(fā)現(xiàn),為核不育系的選育和利用開辟了新途徑。本實(shí)驗(yàn)室前期已將大白菜核不育復(fù)等位基因中的Ms基因,初步定位在A07染色體上。本研究以大白菜核雄性不育“兩用系”AB01中的不育植株為母本,與大白菜小孢子DH系FT雜交,雜交后代的不育株與FT回交,構(gòu)建大規(guī)模BC1精細(xì)定位群體,在A07染色體上繼續(xù)開發(fā)與不育基因Ms連鎖更緊密的SSR標(biāo)記。同時(shí),構(gòu)建大白菜基因組BAC文庫,篩選定位區(qū)間所在的染色體片段,為克隆Ms基因奠定基礎(chǔ)。主要研究結(jié)果如下：1.利用構(gòu)建的BC1分離群體,篩選到了與Ms基因緊密連鎖的SSR分子標(biāo)記LZY6和RF37,其與目的基因Ms的遺傳距離分別為0.45cM和0.62cM。RF37與LZY6之間的遺傳距離為0.17cM,確定Ms基因在A07染色體上的方向。2.構(gòu)建了含有51840個(gè)克隆的BAC文庫,其單個(gè)孔的原始克隆數(shù)為540個(gè),文庫的菌株為大腸桿菌DH10B,采用的載體是plndigoBAC-5。對BAC文庫質(zhì)量進(jìn)行鑒定,隨機(jī)選取文庫中的9個(gè)單克隆,經(jīng)過搖菌、酶切等檢測到單克隆的平均插入片段大小為160Kb。基因組覆蓋率為15x,能篩到陽性單克隆的幾率在99.9%以上。隨機(jī)選取的9個(gè)單克隆都含有插入片段,空載率為0%。3.設(shè)計(jì)引物SKRF1,利用PCR對BAC文庫進(jìn)行陽性混合池、陽性孔、陽性單克隆的逐級篩選,獲得了陽性單克隆SRF,其片段大小約為140Kb。對SRF進(jìn)行第二代測序,拼接成5個(gè)scaffold,預(yù)測編碼37個(gè)基因。對37個(gè)預(yù)測基因在大白菜數(shù)據(jù)庫中搜索,其中11個(gè)基因來自數(shù)據(jù)庫中的已知基因。對37個(gè)預(yù)測基因在擬南芥中進(jìn)行同源搜索(Blastx, E-10)其中18個(gè)基因可以找到同源基因,包括功能已知基因12個(gè),功能未知基因6個(gè)。其余的19個(gè)基因在Nr數(shù)據(jù)庫中搜索,能匹配到基因序列的有18個(gè)基因,包括功能已知基因1個(gè),功能未知基因17個(gè)。
[Abstract]:The discovery of male sterile materials inherited by nuclear multiple alleles in Chinese cabbage opens up a new way for breeding and utilization of GMS lines. In our laboratory, the Ms gene of gene-sterile complex allele in Chinese cabbage was preliminarily located on chromosome A07. In this study, the sterile plants in "dual-purpose line" AB01 of Chinese cabbage were used as female parent, and FT hybridization with microspore DH line of Chinese cabbage was carried out. The sterile plants of hybrid progenies were backcrossed with FT, and a large scale BC1 fine localizing population was constructed. SSR markers linked to sterility gene Ms continued to be developed on chromosome A07. At the same time, the genomic BAC library of Chinese cabbage was constructed, and the chromosomal fragments with the location interval were screened, which laid the foundation for the cloning of Ms gene. The main results are as follows: 1. The SSR markers LZY6 and RF37, which were closely linked to Ms gene, were screened by using the constructed BC1 segregated population. The genetic distance between LZY6 and target gene Ms was 0.45cM and 0.62cM.RF37, respectively, and the genetic distance between LZY6 and LZY6 was 0.17cM. The BAC library containing 51840 clones was constructed. The original clone number of 51840 clones was 540, the strain of the library was E. coli DH10B, and the vector was plndigo BAC-5. The quality of the BAC library was identified. Nine monoclonal clones were randomly selected. The average size of the inserted fragment was 160 kb by shaking bacteria and enzyme digestion. The genome coverage was 15 x, and the probability of screening positive monoclonal was over 99.9%. The randomly selected nine monoclonal clones all contained inserted fragments with a no-load rate of 0. 3. The primers SKRF1 were designed, and the positive mixed pool, positive pore and positive monoclonal were screened step by PCR, and the positive monoclonal SRFs were obtained, the size of which was about 140 kb. The SRF was sequenced in the second generation and spliced into 5 scaffolds and encoded 37 genes. Thirty-seven predictive genes were searched in Chinese cabbage database, 11 of which were from known genes in the database. In Arabidopsis thaliana, 18 of the 37 predicted genes were homologous to Blastx (E-10), including 12 genes with known function and 6 genes with unknown function. The remaining 19 genes were searched in Nr database and 18 genes were found to match the gene sequence, including 1 gene with known function and 17 genes with unknown function.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S634.1

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本文編號：2012814