【摘要】：1[目的]利用超聲波輔花粉介導(dǎo)法將花青素基因NtAn2導(dǎo)入玉米自交系鄭58。優(yōu)化超聲波輔助花粉介導(dǎo)基因轉(zhuǎn)化方法的遺傳轉(zhuǎn)化體系。[方法]以新鮮的鄭58玉米花粉為試驗(yàn)材料,檢測(cè)其在不同濃度的蔗糖溶液中的體外萌發(fā)率;通過正交設(shè)計(jì)方法,對(duì)超聲波處理功率、處理時(shí)間、處理次數(shù)3個(gè)因素進(jìn)行優(yōu)化;通過正交試驗(yàn)對(duì)農(nóng)桿菌菌液濃度、超聲波處理參數(shù)、蔗糖用液濃度、AS濃度四個(gè)因素進(jìn)行優(yōu)化確定最有效的轉(zhuǎn)化組合。然后將NtAN2通過花粉介導(dǎo)法轉(zhuǎn)化至玉米自交系鄭58中。對(duì)收獲的籽粒的植株后代進(jìn)行PCR檢測(cè)、Dot blot雜交分析、southern blot雜交分析以及轉(zhuǎn)基因植株花青素含量的測(cè)定比較與分析[結(jié)果]玉米自交系鄭58的花粉最適宜的蔗糖溶液濃度為20%;超聲波處理最佳參數(shù)為:處理功率為150w,處理時(shí)間為5s/次,處理次數(shù)為6次。綜合考慮轉(zhuǎn)化效率和結(jié)實(shí)效率轉(zhuǎn)化中農(nóng)桿菌菌液最適濃度OD600為0.6、AS最適濃度為100μmol/L、蔗糖溶液最適濃度為20%、超聲波處理參數(shù)為處理功率為150w,處理時(shí)間為5s/次,處理次數(shù)為6次。收獲的籽粒中兩粒呈紫色,經(jīng)過PCR、southern鑒定成陽(yáng)性,初步證實(shí)NtAN2已被整合到玉米基因組中,并且可以被用于對(duì)轉(zhuǎn)化子的直觀篩選。隨后對(duì)其T2代植株進(jìn)行檢測(cè)的結(jié)果也均成陽(yáng)性,證實(shí)外源基因可以穩(wěn)定的遺傳本研究確立了超聲波輔助花粉介導(dǎo)法遺傳轉(zhuǎn)化的最佳體系,為提高該植物基因轉(zhuǎn)化方法的效率奠定了基礎(chǔ)。2[目的]大蒜是一種現(xiàn)食藥兩用的植物,備受關(guān)注,它在植物、動(dòng)物、人類醫(yī)學(xué)界都有重要地位,尤其是它在植物中的抑菌作用,本研究利用這一性質(zhì)探究它對(duì)玉米病菌的抑菌效果如何,并且有研究表明半胱氨酸合成酶的產(chǎn)物半胱氨酸是大蒜中含硫氨基酸豐富是植物中含硫氨基酸的重要來源,研究大蒜半胱氨酸合成酶的性質(zhì)及生物學(xué)功能以明確其在大蒜硫代謝中的作用。[方法]利用涂布打孔發(fā)法比較分析大蒜粗酶提取液、大蒜素、蒜氨酸酶對(duì)玉米的霉菌、絲黑穗菌、黑粉病菌的抑制情況。利用生物信息學(xué)技術(shù)分析從NCBI數(shù)據(jù)庫(kù)中獲得的3個(gè)大蒜的半胱氨酸合成酶基因(ASGCS2、ASGCS3和ASGCS4)的開放閱讀框,預(yù)測(cè)其蛋白序列、分子量大小、蛋白質(zhì)特性、亞細(xì)胞定位、系統(tǒng)進(jìn)化等特征;通過熒光定量PCR分析了3個(gè)半胱氨酸合成酶的組織表達(dá)特性。[結(jié)果]大蒜素及粗酶提取液對(duì)玉米的三種病菌都以抑制作用。3個(gè)大蒜的半胱氨酸合成酶基因ASGCS2、ASGCS3和ASGCS4的開放態(tài)讀碼框長(zhǎng)度分別為1152bp,1296bp和1236bp;其編碼蛋白的理論相對(duì)分子量分別為40.6KD,34.1KD和36.1KD。ASGCS2被亞細(xì)胞定位于葉綠體中,而ASGCS3和ASGCS4均被定位于細(xì)胞質(zhì)中。氨基酸比對(duì)結(jié)果表明,ASGCS2與ASGCS3相似度為70%,與ASGCS4相似度為59%;而ASGCS3與ASGCS4相似度為68%。進(jìn)化分析表明,AsGCS2屬于Bsas2亞家族,AsGCS3屬于Bsas1亞家族,AsGCS4屬于Bsas6亞家族。熒光定量PCR結(jié)果表明,大蒜不同CSase的組織特異性是不同的,ASGCS2在葉中表達(dá)量最高,ASGCS3在根中表達(dá)量最高,而ASGCS4在葉和根中都有較高的表達(dá)量。
[Abstract]:1[Objective] the anthocyanin gene NtAn2 was introduced into the maize inbred line Zheng 58. by ultrasonic assisted pollen mediating method to optimize the transformation system of ultrasonic assisted pollen mediated gene transformation. [method] the germination rate of fresh Zhengzhou 58 maize pollen was tested in different concentration of sucrose solution, and the orthogonal design was designed by orthogonal design. Methods 3 factors such as ultrasonic power, processing time and processing times were optimized, and the most effective conversion combination was determined by orthogonal test on the concentration of Agrobacterium tumefaciens, the ultrasonic treatment parameters, the concentration of sucrose solution and the concentration of AS, and then transformed into the maize inbred Zheng 58 by the method of NtAN2. The progeny of the harvested grain was detected by PCR, Dot blot hybridization analysis, Southern blot hybridization analysis and the determination of anthocyanin content in transgenic plants. The optimum concentration of sucrose solution in Maize Inbred Line Zheng 58 was 20%, and the optimum parameters for ultrasonic treatment were 150W and 5s/ time. The optimum concentration of Agrobacterium tumefaciens was 0.6, the optimum concentration of OD600 was 0.6, the optimum concentration of AS was 100 mu mol/L, the optimum concentration of the sucrose solution was 20%, the ultrasonic treatment parameters were 150W, the treatment time was 5s/ and the processing times were 6 times. The two grains in the harvest were purple, PCR, sou Thern was identified as positive. It was preliminarily confirmed that NtAN2 had been integrated into the maize genome and could be used to screen the transformants. Then the results of the T2 generation were also positive. It was proved that the stable genetic study of foreign genes established the best system of ultrasonic assisted pollen mediated genetic transformation. In order to improve the efficiency of the gene transformation method, garlic is a basic.2[Objective] garlic is a kind of dual use plant, which has been paid much attention to. It has an important position in plant, animal and human medical field, especially its bacteriostasis in plants. This study uses this nature to explore how it has the effect on the Bacteriostasis of corn germs. Cysteine, the product of cysteine synthase, is an important source of sulphur containing amino acids in garlic. The study of the properties and biological functions of garlic cysteine synthetase to clarify its role in the sulfur metabolism of garlic. [method] to compare and analyze the extract of garlic crude enzyme by using the coating method. The inhibition of allicin and alliinase on corn, smut, and powdery mildew. Using bioinformatics to analyze the open reading frame of 3 garlic cysteine synthetase genes (ASGCS2, ASGCS3 and ASGCS4) obtained from the NCBI database and predict its sequence, molecular weight, protein characteristics, subcellular localization, and system Evolution and other characteristics; analysis of the tissue expression characteristics of 3 cysteine synthetases by fluorescence quantitative PCR. [results] allicin and crude enzyme extract can inhibit the cysteine synthetase gene ASGCS2 of.3 garlic, ASGCS3 and ASGCS4, the length of the open reading frame of ASGCS3 and ASGCS4, respectively, 1152bp, 1296bp and 1236bp; The theoretical relative molecular weight of the code protein was 40.6KD, 34.1KD and 36.1KD.ASGCS2 were located in the chloroplasts, while ASGCS3 and ASGCS4 were located in the cytoplasm. The similarity between ASGCS2 and ASGCS3 was 70% and the similarity of ASGCS4 was 59%, while the similarity between ASGCS3 and ASGCS4 was 68%. evolutionary analysis showed that AsGCS2 genera In the Bsas2 subfamily, AsGCS3 belongs to the Bsas1 subfamily and AsGCS4 belongs to the Bsas6 subfamily. The fluorescence quantitative PCR results show that the tissue specificity of garlic in different CSase is different, the highest expression of ASGCS2 in the leaves, the highest expression of ASGCS3 in the root, and the higher expression of ASGCS4 in the leaves and roots.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S513

【相似文獻(xiàn)】

相關(guān)期刊論文 前2條

1 彭凌濤,王江,李琳,安林升,張景六;水稻谷氨酰半胱氨酸合成酶基因的結(jié)構(gòu)和表達(dá)分析[J];植物生理與分子生物學(xué)學(xué)報(bào);2004年05期

2 ;[J];;年期

相關(guān)會(huì)議論文 前5條

1 朱運(yùn)福;戴愛國(guó);胡瑞成;;支氣管哮喘豚鼠肺組織中絲裂原活化蛋白激酶對(duì)γ谷氨酰半胱氨酸合成酶的作用[A];中華醫(yī)學(xué)會(huì)第五次全國(guó)哮喘學(xué)術(shù)會(huì)議暨中國(guó)哮喘聯(lián)盟第一次大會(huì)論文匯編[C];2006年

2 劉明坤;劉關(guān)君;魏志剛;閻秀峰;曲春浦;王垠;劉桂豐;楊傳平;;西伯利亞蓼半胱氨酸合成酶基因的克隆與表達(dá)[A];第六屆全國(guó)林木遺傳育種大會(huì)論文集[C];2008年

3 朱運(yùn)福;戴愛國(guó);胡瑞成;;支氣管哮喘豚鼠肺組織中核因子E2相關(guān)因子2對(duì)γ谷氨酰半胱氨酸合成酶的作用[A];中華醫(yī)學(xué)會(huì)第五次全國(guó)哮喘學(xué)術(shù)會(huì)議暨中國(guó)哮喘聯(lián)盟第一次大會(huì)論文匯編[C];2006年

4 朱運(yùn)福;戴愛國(guó);胡瑞成;;支氣管哮喘患者痰炎性細(xì)胞中絲裂原活化蛋白激酶與核因子E2相關(guān)因子2對(duì)γ谷氨酰半胱氨酸合成酶的作用[A];中華醫(yī)學(xué)會(huì)第五次全國(guó)哮喘學(xué)術(shù)會(huì)議暨中國(guó)哮喘聯(lián)盟第一次大會(huì)論文匯編[C];2006年

5 戴愛國(guó);朱運(yùn)福;胡瑞成;蔣永亮;;絲裂原活化蛋白激酶與核因子E2相關(guān)因子2調(diào)節(jié)γ谷氨酰半胱氨酸合成酶對(duì)豚鼠支氣管哮喘的作用[A];中華醫(yī)學(xué)會(huì)第五次全國(guó)哮喘學(xué)術(shù)會(huì)議暨中國(guó)哮喘聯(lián)盟第一次大會(huì)論文匯編[C];2006年

相關(guān)博士學(xué)位論文 前1條

1 范翠麗;DL-2-氨基-△~2-噻唑林-4-羧酸誘導(dǎo)L-半胱氨酸合成酶的生產(chǎn)及酶學(xué)特性研究[D];華東理工大學(xué);2014年

相關(guān)碩士學(xué)位論文 前5條

1 郭天璐;花粉介導(dǎo)玉米轉(zhuǎn)花青素基因NtAN2的研究及大蒜半胱氨酸合成酶性質(zhì)的研究[D];山西大學(xué);2016年

2 郭燕蓉;紅霉素對(duì)吸煙大鼠肺內(nèi)轉(zhuǎn)化生長(zhǎng)因子-β_1和γ-谷氨酰胺半胱氨酸合成酶表達(dá)的影響[D];山西醫(yī)科大學(xué);2007年

3 孫麗娜;γ－谷氨酰半胱氨酸合成酶、轉(zhuǎn)化生長(zhǎng)因子β_1在吸煙大鼠肺組織中表達(dá)的研究[D];山西醫(yī)科大學(xué);2005年

4 鄭春麗;嗜酸氧化亞鐵硫桿菌硫同化途徑蛋白ATP硫酸化酶、APS還原酶、半胱氨酸合成酶的研究[D];中南大學(xué);2009年

5 吳冬梅;人谷氨酰半胱氨酸合成酶通過抑制JNK防止紫外刺激誘導(dǎo)的細(xì)胞死亡的研究[D];南京師范大學(xué);2005年

，

本文編號(hào)：2156077