本文選題：小麥 + TaCHP��； 參考：《山東大學(xué)》2016年碩士論文

【摘要】：小麥?zhǔn)翘鹜磷魑?土壤鹽堿化是限制小麥生長、發(fā)育和產(chǎn)量提高的主要因素。隨著分子生物學(xué)的發(fā)展,利用轉(zhuǎn)基因技術(shù)進(jìn)行小麥耐鹽堿遺傳改良是分子育種的新方向。實(shí)驗(yàn)室前期利用不同轉(zhuǎn)化法,經(jīng)抗生素篩選和分子鑒定獲得兩組轉(zhuǎn)TaCHP基因小麥株系：以普通小麥濟(jì)南17(JN 17)為親本,經(jīng)莖尖生長點(diǎn)轉(zhuǎn)化法獲得的T6代轉(zhuǎn)基因系No.25-2-5和No.33-2-3；以普通小麥周麥22(Zhou 22)為親本,經(jīng)愈傷組織轉(zhuǎn)化法獲得的T4代轉(zhuǎn)基因系No.21-1-3、No.26-12-5 和 No.27-6-8。本課題以這兩組轉(zhuǎn)基因系和各自的親本對照為研究對象,進(jìn)行如下實(shí)驗(yàn)：1)、實(shí)驗(yàn)室中,進(jìn)行了轉(zhuǎn)基因小麥在鹽(NaCl)、堿鹽(NaHCO3: Na2CO3=9:1)及混合鹽堿[NaCl+(NaHCO3:Na2CO3=9:1)]的水溶液脅迫下種子萌發(fā)和早期幼苗生長發(fā)育實(shí)驗(yàn)；營養(yǎng)土和鹽堿土中,種子萌發(fā)和早期幼苗的生長發(fā)育實(shí)驗(yàn)。2)、普通土和鹽堿土試驗(yàn)田中,對兩組轉(zhuǎn)基因系進(jìn)行了小麥部分生長發(fā)育期(苗期、返青期和灌漿期)形態(tài)特征觀察記錄、理化指標(biāo)測定和農(nóng)藝性狀統(tǒng)計,并進(jìn)行產(chǎn)量相關(guān)性分析。3)、在梯度鹽池和堿池中,對T4代轉(zhuǎn)基因系進(jìn)行農(nóng)藝性狀和產(chǎn)量相關(guān)性分析。主要研究成果：1、實(shí)驗(yàn)室中,在萌發(fā)期,NaCl水溶液處理的兩組轉(zhuǎn)基因系表現(xiàn)出比各自親本更強(qiáng)的耐鹽性；堿性鹽水溶液處理下差異不顯著；混合鹽堿[25 mM NaCl+50 mM (NaHCO3:Na2CO3=9:1)]水溶液處理下,兩組轉(zhuǎn)基因系的生長狀態(tài)與各自的親本對照差異不顯著；混合鹽堿[50 mM NaCl+25 mM (NaHCO3: Na2CO3=9:1)]水溶液處理組,兩組轉(zhuǎn)基因系的相對胚芽長度大于各自的親本對照。在苗期,NaCl水溶液處理的轉(zhuǎn)基因系表現(xiàn)出與萌發(fā)期一致的強(qiáng)耐鹽性；兩組轉(zhuǎn)基因系在苗期均表現(xiàn)出高于各自親本對照的耐混合鹽堿脅迫特性。2、實(shí)驗(yàn)室中,點(diǎn)種在營養(yǎng)土中的種子,24 h后即開始萌動和快速生長,萌發(fā)至第4天時,種子幾乎全部發(fā)芽；點(diǎn)種在鹽堿土中的種子,48 h后才陸續(xù)開始萌動,隨后幾天陸續(xù)出芽和生長,且發(fā)芽率低于前者；兩組轉(zhuǎn)基因系種子的萌動及發(fā)芽率都優(yōu)于各自的親本對照。生長在營養(yǎng)土中20天的轉(zhuǎn)基因植株生長量與親本差異不顯著,生長在鹽堿土中的轉(zhuǎn)基因株系生長量均高于各自的親本對照,且總體顯著低于營養(yǎng)土中。3、通過對生長在鹽堿土試驗(yàn)田中的兩組轉(zhuǎn)基因系的葉片和根系的理化指標(biāo)分析發(fā)現(xiàn),各發(fā)育期(除返青期根系)的K+/Na+值顯著高于各自的親本對照,且葉片高于根中。兩組轉(zhuǎn)基因系各時期葉片和根系的MDA含量都顯著低于親本對照(除返青期葉片)。與K+/Na+值不同的是,隨著發(fā)育進(jìn)程的推進(jìn),葉片及根中MDA含量都明顯升高。4、普通土試驗(yàn)田中,莖尖轉(zhuǎn)化法獲得的兩個轉(zhuǎn)基因系產(chǎn)量較JN 17分別提高了3.37%和4.53%,但在經(jīng)愈傷組織法獲得的轉(zhuǎn)基因系與親本Zhou 22的對比發(fā)現(xiàn)無顯著差異；農(nóng)藝性狀中,小穗數(shù)和分蘗數(shù)與畝產(chǎn)相關(guān)性最大。鹽堿土試驗(yàn)田中,兩組轉(zhuǎn)基因系的產(chǎn)量較其各自的親本都有所提高；農(nóng)藝性狀中,株高、旗葉長度和小穗數(shù)與畝產(chǎn)相關(guān)性最大,其中,株高呈負(fù)相關(guān),后兩者呈正相關(guān)。5、對梯度鹽池及堿池的農(nóng)藝性狀分析顯示,在一定濃度的鹽(0-0.8%NaCl)和堿鹽(0-80 mM (NaHCO3:Na2CO3=9:1))脅迫的范圍內(nèi),小麥產(chǎn)量隨處理濃度的增加總體有降低的趨勢。但當(dāng)鹽、堿鹽處理濃度達(dá)到較大值[0.8%NaCl,80 mM堿池(NaHCO3:Na2CO3=9:1)]時,轉(zhuǎn)基因系產(chǎn)量高于親本對照Zhou 22,此時,在0.8% NaCl鹽池中,三個轉(zhuǎn)基因系的產(chǎn)量分別較親本對照提高了0.03、0.37和0.16倍,80 mM (NaHCO3:Na2CO3=91)堿池中,三個轉(zhuǎn)基因系的千粒重分別較親本對照提高了的0.18、0.37和0.57倍。綜上,實(shí)驗(yàn)室中,鹽堿脅迫下的兩組轉(zhuǎn)基因系,在種子萌發(fā)和苗期生長所測的各指標(biāo)均優(yōu)于各自的親本對照；鹽堿土試驗(yàn)田中,兩組轉(zhuǎn)基因系均通過離子調(diào)節(jié)和氧化酶活性調(diào)節(jié)保護(hù)質(zhì)膜免受鹽堿傷害,以維持植物體正常生長。研究結(jié)果可為小麥轉(zhuǎn)基因系耐鹽堿機(jī)制的探索提供依據(jù)和可參考的技術(shù)路線。
[Abstract]:Wheat is a sweet soil crop, and soil salinization is the main factor restricting the growth, development and yield of wheat. With the development of molecular biology, genetic improvement of salt tolerance in wheat is a new direction of molecular breeding with the development of molecular biology. In the early laboratory, two groups of TaCHP were obtained by different transformation methods, through the screening of antibiotics and molecular identification. Gene wheat lines: the T6 generation transgenic lines No.25-2-5 and No.33-2-3 obtained from Ji'nan 17 (JN 17) were obtained by the stem tip growth point transformation method. The T4 generation transgenic lines, No.21-1-3, No.26-12-5 and No.27-6-8., obtained by callus transformation method, were obtained by the transformation method of normal wheat 22 (Zhou 22). The two sets of transgenic lines were given by the two sets of transgenic lines. Compared with their parents, the experiment was carried out as follows: 1), in the laboratory, the seeds germinated and the early seedling growth experiments were carried out under the water solution stress of salt (NaCl), alkali salt (NaHCO3: Na2CO3=9:1) and mixed salt base [NaCl+ (NaHCO3:Na2CO3=9:1)) in the laboratory of Transgenic Wheat; the seed germination and the early young were in the nutrient soil and the saline alkali soil. The growth and development experiment of seedling.2), in the ordinary soil and saline alkali soil test fields, the morphological characteristics of the two groups of transgenic lines were observed, the physical and chemical indexes and the agronomic traits were recorded, and the yield correlation was analyzed by.3). In the gradient salt pool and the alkali pond, the transgenic lines of the T4 generation were introduced into the transgenic lines. The correlation analysis of agronomic traits and yield. 1, 1, in the laboratory, in the germination period, the two groups of transgenic lines treated by NaCl aqueous solution showed better salt tolerance than their parents; the difference was not significant under the alkaline saline solution; the two groups were treated by the mixed saline alkali [25 mM NaCl+50 mM (NaHCO3:Na2CO3=9:1)] solution treatment. The growth state of the gene lines was not significantly different from those of their parents; the relative germ length of the two groups of transgenic lines in the mixed saline alkali [50 mM NaCl+25 mM (NaHCO3: Na2CO3=9:1) treatment group was larger than the parent control. In the seedling stage, the transgenic lines treated by the NaCl aqueous solution showed the strong salt tolerance of the two groups. In the seedling stage, the transgenic lines showed higher resistance to mixed salt stress.2 than their parent controls. In the laboratory, seeds in the nutrient soil began to germinate and grow quickly after 24 h, and the seeds germinated almost all after the germination to fourth days. Seeds in saline alkali soil began to germinate after 48 h, and then several days later. The germination and germination rates were lower than that of the former, and the germination and germination rate of the two transgenic lines were better than those of their parents. The growth of transgenic plants in the 20 days of the vegetative soil was not significantly different from those of their parents, and the transgenic lines growing in saline alkali soil were compared with their parents, and the total of the transgenic lines in the saline alkali soil was significantly lower than that in the two groups. In the nutrient soil,.3, through the analysis of the physical and chemical indexes of the leaves and roots of two groups of transgenic lines growing in saline alkali soil, found that the K+/Na+ value of each development period (except for the root of the green period) was significantly higher than that of the parent control, and the leaves were higher than the root. The MDA content of the leaves and roots of the two groups of transgenic lines were significantly lower than those of the parents. Different from the K+/Na+ value, the MDA content in the leaves and roots increased obviously with the development process, and the yield of two transgenic lines obtained by the stem tip transformation method increased by 3.37% and 4.53% respectively in the ordinary soil test field, but compared with the parent Zhou 22 of the transgenic lines obtained by the callus method. There was no significant difference in the agronomic traits, the number of spikelet and the number of tillers were the most related to the yield per mu. In the saline alkali soil test field, the yield of the two transgenic lines was higher than that of their respective parents; the agronomic traits, the plant height, the flag leaf length and the number of spikes were the most related to the yield, and the plant height was negatively correlated, and the latter two were positively correlated.5, to the ladder. The agronomic characteristics of the salt pond and alkali pond showed that the wheat yield decreased with the increase of the concentration of salt (0-0.8%NaCl) and alkali salt (0-80 mM (NaHCO3:Na2CO3=9:1)), but when salt, alkali salt treatment concentration reached a greater value of [0.8%NaCl, 80 mM alkali pool (NaHCO3:Na2CO3=9:1), the transgenic line The yield of Zhou 22 was higher than that of parental control. At this time, the yield of three transgenic lines increased by 0.03,0.37 and 0.16 times in the 0.8% NaCl salt pond respectively. In the 80 mM (NaHCO3:Na2CO3=91) alkali pool, the 1000 grain weight of three transgenic lines increased by 0.18,0.37 and 0.57 times respectively compared with the parental control. In the laboratory, two groups under saline alkali stress were in the laboratory. The transgenic lines were better than their parents in the seed germination and seedling growth. In the saline alkali soil test fields, the two groups of transgenic lines protected the plasmalemma through ion regulation and oxidase activity to protect the normal growth of the plants. The results could be used to explore the salt tolerance mechanism of the wheat transgenic lines. The cable provides the basis and the technical route that can be referenced.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S512.1

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