發(fā)布時(shí)間：2019-06-10 20:22

【摘要】：竹子傳統(tǒng)雜交與選擇育種困難,造成育種水平滯后,成為竹產(chǎn)業(yè)進(jìn)一步提升的限制因素。轉(zhuǎn)基因是有效育種手段,但是竹子再生比較困難,遺傳轉(zhuǎn)化成功的報(bào)道很少。本研究主要從兩方面入手,一方面通過非組培轉(zhuǎn)基因研究,避開竹子組培再生的難題;另一方面是對能夠促進(jìn)植物再生的基因如DhNiR(Dendrocalamus hamiltonii,Dh)進(jìn)行研究,通過將促進(jìn)再生基因整合入雙元表達(dá)載體,以提高竹子再生與轉(zhuǎn)化效率。本試驗(yàn)的目的是分別將這兩種方法應(yīng)用到竹子中,使竹子的轉(zhuǎn)化效率得到有效的提高。本項(xiàng)目首先以毛竹種子為實(shí)驗(yàn)材料,用農(nóng)桿菌介導(dǎo)植物萌動種子基因轉(zhuǎn)化方法將CP4-EPSPS基因成功轉(zhuǎn)化到毛竹種子中,成功獲得了轉(zhuǎn)基因植株。試驗(yàn)結(jié)果如下:在共培養(yǎng)時(shí),菌液濃度以O(shè)D600=0.07為最適濃度,太低或過高的濃度都不利于苗的轉(zhuǎn)化和萌發(fā);且共培養(yǎng)液中加入400μmol·L-1乙酰丁香酮,可使出苗率提高8%左右;經(jīng)PCR分子驗(yàn)證獲得1株毛竹轉(zhuǎn)基因植株。其次是對轉(zhuǎn)DhNiR基因水稻進(jìn)行后代性狀分析,試驗(yàn)結(jié)果如下:以轉(zhuǎn)DhNiR的T2代水稻成熟種子和野生型水稻成熟種子為試驗(yàn)材料,經(jīng)愈傷組織誘導(dǎo)及增殖后,選取生長良好的致密愈傷組織進(jìn)行分化培養(yǎng),愈傷誘導(dǎo)率和苗分化時(shí)間上均表現(xiàn)出差異,轉(zhuǎn)DhNiR的水稻愈傷組織在轉(zhuǎn)入分化培養(yǎng)基第25天時(shí)綠點(diǎn)愈傷率達(dá)到了100%,而野生型水稻綠點(diǎn)愈傷率僅為56.37%,且苗分化時(shí)間比野生型水稻早5天左右,表明DhNiR在水稻再生能力提高方面有促進(jìn)作用;以轉(zhuǎn)DhNiR基因的水稻和野生型水稻的葉片、莖、根、愈傷組織為樣本,測定各個(gè)樣本中的亞硝酸還原酶活性,轉(zhuǎn)DhNiR基因水稻各部分組織的亞硝酸還原酶活性均高于野生型水稻,且達(dá)到了極顯著差異;對轉(zhuǎn)DhNiR水稻和野生型水稻的種子、葉片及根分別進(jìn)行硝酸鹽和亞硝酸鹽含量測定,結(jié)果顯示轉(zhuǎn)DhNiR基因水稻各部分組織中的硝酸鹽和亞硝酸鹽含量也普遍高于野生型水稻。
[Abstract]:The difficulty of traditional hybrid and selection breeding of bamboo, which leads to the lag of breeding level, has become a limiting factor for the further promotion of bamboo industry. Transgenic is an effective breeding method, but bamboo regeneration is difficult, and there are few reports of successful genetic transformation. This study mainly starts from two aspects, on the one hand, through non-tissue culture transgenic research, to avoid the problem of bamboo tissue culture regeneration; On the other hand, genes that can promote plant regeneration, such as DhNiR (Dendrocalamus hamiltonii,Dh, are studied in order to improve the efficiency of bamboo regeneration and transformation by integrating the promoting genes into binary expression vectors. The purpose of this experiment is to apply these two methods to bamboo respectively, so that the transformation efficiency of bamboo can be effectively improved. In this project, using Phyllostachys pubescens seeds as experimental materials, CP4-EPSPS gene was successfully transformed into Phyllostachys pubescens seeds by Agrobacterium tumefaciens mediated seed gene transformation, and transgenic plants were successfully obtained. The results were as follows: in co-culture, OD600=0.07 was the optimum concentration, too low or too high concentration was not conducive to seedling transformation and germination; The emergence rate of Phyllostachys pubescens was increased by about 8% when 400 渭 mol 路L-1 acetyleugenone was added to the co-culture medium, and a transgenic plant of Phyllostachys pubescens was obtained by PCR molecular verification. Secondly, the characters of the progenies of transgenic rice with DhNiR gene were analyzed. The results were as follows: the mature seeds of T2 generation rice and wild type rice mature seeds transformed with DhNiR were used as experimental materials, and after Callus induction and proliferation, When the dense calli with good growth were selected for differentiation and culture, the calli induction rate and seedling differentiation time were different. The green spot healing rate of rice calli transferred to DhNiR reached 100% on the 25th day after being transferred to the differentiation medium. However, the green spot healing rate of wild type rice was only 56.37%, and the seedling differentiation time was about 5 days earlier than that of wild type rice, which indicated that DhNiR could promote the improvement of rice regeneration ability. Taking the leaves, stems, roots and calli of transgenic rice with DhNiR gene as samples, the activity of nitrate reductase in each sample was determined. The activity of nitrate reductase in each part of transgenic rice with DhNiR gene was higher than that in wild type rice. And reached a very significant difference; The contents of nitrate and nitrite in seeds, leaves and roots of transgenic DhNiR rice and wild type rice were determined respectively. The results showed that the contents of nitrate and nitrite in the tissues of transgenic rice with DhNiR gene were also generally higher than those in wild type rice.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S795.7;S511

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