【摘要】：植物芽再生能力是遺傳轉(zhuǎn)化的基礎(chǔ)。幼胚是玉米(Zea mays L.)常用的遺傳轉(zhuǎn)化外植體,不同基因型玉米的芽再生能力差別很大。目前關(guān)于調(diào)控玉米芽再生能力的基因鮮有報(bào)道,因此挖掘調(diào)控玉米芽再生能力的重要基因不僅有利于提高玉米遺傳轉(zhuǎn)化頻率而且還有助于加深人們對(duì)芽再生能力遺傳調(diào)控分子基礎(chǔ)的理解。本研究選取367份來(lái)自全球不同地區(qū)的玉米自交系,先后在2013年和2014年夏天種植,取授粉后11d的幼胚進(jìn)行組織培養(yǎng)實(shí)驗(yàn)。在培養(yǎng)過(guò)程中,對(duì)誘導(dǎo)培養(yǎng)0d、誘導(dǎo)培養(yǎng)15d、誘導(dǎo)培養(yǎng)30d、分化培養(yǎng)0d、分化培養(yǎng)30d 5個(gè)時(shí)期的愈傷組織狀態(tài)及愈傷組織芽分化情況進(jìn)行觀察,并對(duì)初級(jí)愈傷組織誘導(dǎo)率、成熟愈傷組織誘導(dǎo)率、愈傷組織狀態(tài)、愈傷組織增殖能力、愈傷組織芽再生率及單個(gè)愈傷組織芽再生數(shù)6個(gè)性狀進(jìn)行量化分析。繼而通過(guò)全基因組關(guān)聯(lián)分析方法找到組織培養(yǎng)能力相關(guān)的基因。主要研究結(jié)果如下:(1)通過(guò)對(duì)授粉后11d的幼胚進(jìn)行組織培養(yǎng),得到了367份玉米自交系幼胚的組織培養(yǎng)表型數(shù)據(jù)。結(jié)果表明,不同基因型的玉米自交系之間表型差異很大,如CIMBL3、CML426、CML323等玉米自交系愈傷組織狀態(tài)較好,芽再生頻率分別能達(dá)到75%、65.5%、64.5%,而有些自交系形成的愈傷組織不能分化形成芽。(2)結(jié)合RNA測(cè)序獲得的高質(zhì)量的SNP數(shù)據(jù),通過(guò)全基因組關(guān)聯(lián)分析,共得到了132個(gè)標(biāo)記-性狀關(guān)聯(lián),并通過(guò)功能注釋得到了58個(gè)候選基因,這些候選基因分別與6個(gè)性狀相關(guān)。(3)對(duì)玉米芽再生過(guò)程6個(gè)性狀間的相關(guān)性分析發(fā)現(xiàn),初級(jí)愈傷組織誘導(dǎo)率與成熟愈傷組織誘導(dǎo)率存在相關(guān)性,而愈傷組織誘導(dǎo)率與愈傷組織芽再生率之間并不存在相關(guān)性,愈傷組織增殖能力與愈傷組織芽再生率之間也不存在相關(guān)性,愈傷組織誘導(dǎo)、愈傷組織的生長(zhǎng)速度和愈傷組織芽分化過(guò)程是相互獨(dú)立的,高愈傷組織誘導(dǎo)率并不一定伴隨高的愈傷組織芽分化率,因此,愈傷組織的誘導(dǎo)和愈傷組織芽分化過(guò)程是受不同遺傳因子調(diào)控的。(4)選擇4個(gè)玉米愈傷組織芽再生相關(guān)的基因進(jìn)行了初步功能分析。GRMZM2G074850和GRMZM2G045404基因的過(guò)表達(dá)能夠提高芽的再生頻率,而過(guò)表達(dá)GRMZM2G168393基因后則降低了愈傷組織芽再生率,過(guò)表達(dá)GRMZM2G138689基因后對(duì)愈傷組織芽再生率幾乎沒(méi)有影響。說(shuō)明GRMZM2G168393、GRMZM2G074850和GRMZM2G045404可能在調(diào)控玉米愈傷組織芽再生率中發(fā)揮重要作用。
[Abstract]:The ability of plant bud regeneration is the basis of genetic transformation. Immature embryos are corn (Zea mays L.) The shoot regeneration ability of different maize genotypes is very different from that of common genetic transformation explants. At present, there are few reports on genes regulating the regeneration ability of maize buds. Therefore, digging the important genes to regulate the regeneration ability of maize bud is not only helpful to improve the transformation frequency of maize, but also to deepen the understanding of the molecular basis of genetic regulation of shoot regeneration ability. In this study 367 maize inbred lines from different regions of the world were planted in the summer of 2013 and 2014 respectively. The immature embryos were pollinated 11 days after pollination for tissue culture. In the course of culture, the callus status and callus bud differentiation were observed at 0 d, 15 d, 30 d, 0 d, 30 d and 30 d, respectively, and the rate of callus induction was also studied. The induction rate of mature callus, the status of callus, the ability of callus proliferation, the rate of callus bud regeneration and the number of callus regeneration in single callus were analyzed quantitatively. Then the gene related to tissue culture ability was found by whole genome association analysis. The main results were as follows: (1) the phenotypic data of 367 maize inbred lines were obtained by tissue culture of immature embryos at 11 days after pollination. The results showed that the phenotypes of maize inbred lines with different genotypes varied greatly, such as CIMBL3, CML426CML323 and other maize inbred lines, and the callus status of maize inbred lines such as CIMBL3 and CML426CML323 was better. The frequency of bud regeneration could reach 75% 65.5% and 64.5%, respectively, while some inbred lines could not differentiate and form buds. (2) combined with the high quality SNP data obtained by RNA sequencing, 132 marker-trait associations were obtained by whole genome association analysis. Through functional annotation, 58 candidate genes were obtained, which were related to 6 characters respectively. (3) the correlation analysis of 6 characters in the regeneration process of maize bud was found. The primary callus induction rate was correlated with mature callus induction rate, but there was no correlation between callus induction rate and callus bud regeneration rate. There was no correlation between callus proliferation ability and callus bud regeneration rate. Callus induction, callus growth rate and callus bud differentiation were independent of each other. High callus induction rate is not always accompanied by high callus bud differentiation rate. Callus induction and callus bud differentiation were regulated by different genetic factors. (4) four genes related to callus regeneration in maize were selected for preliminary functional analysis. The overexpression of GRMZM2G074850 and GRMZM2G045404 genes could increase the regeneration frequency of maize callus. Overexpression of GRMZM2G168393 gene decreased callus bud regeneration rate, and overexpression of GRMZM2G138689 gene had little effect on callus bud regeneration rate. It was suggested that GRMZM2G168393GRMZM2G074850 and GRMZM2G045404 might play an important role in regulating the shoot regeneration rate of maize callus.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S513

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