花粉介导玉米转花青素基因NtAN2的研究及大蒜半胱氨酸合成酶性质的研究

发布时间：2018-07-31 15:58

【摘要】：1[目的]利用超声波辅花粉介导法将花青素基因NtAn2导入玉米自交系郑58。优化超声波辅助花粉介导基因转化方法的遗传转化体系。[方法]以新鲜的郑58玉米花粉为试验材料,检测其在不同浓度的蔗糖溶液中的体外萌发率;通过正交设计方法,对超声波处理功率、处理时间、处理次数3个因素进行优化;通过正交试验对农杆菌菌液浓度、超声波处理参数、蔗糖用液浓度、AS浓度四个因素进行优化确定最有效的转化组合。然后将NtAN2通过花粉介导法转化至玉米自交系郑58中。对收获的籽粒的植株后代进行PCR检测、Dot blot杂交分析、southern blot杂交分析以及转基因植株花青素含量的测定比较与分析[结果]玉米自交系郑58的花粉最适宜的蔗糖溶液浓度为20%;超声波处理最佳参数为:处理功率为150w,处理时间为5s/次,处理次数为6次。综合考虑转化效率和结实效率转化中农杆菌菌液最适浓度OD600为0.6、AS最适浓度为100μmol/L、蔗糖溶液最适浓度为20%、超声波处理参数为处理功率为150w,处理时间为5s/次,处理次数为6次。收获的籽粒中两粒呈紫色,经过PCR、southern鉴定成阳性,初步证实NtAN2已被整合到玉米基因组中,并且可以被用于对转化子的直观筛选。随后对其T2代植株进行检测的结果也均成阳性,证实外源基因可以稳定的遗传本研究确立了超声波辅助花粉介导法遗传转化的最佳体系,为提高该植物基因转化方法的效率奠定了基础。2[目的]大蒜是一种现食药两用的植物,备受关注,它在植物、动物、人类医学界都有重要地位,尤其是它在植物中的抑菌作用,本研究利用这一性质探究它对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产物半胱氨酸是大蒜中含硫氨基酸丰富是植物中含硫氨基酸的重要来源,研究大蒜半胱氨酸合成酶的性质及生物学功能以明确其在大蒜硫代谢中的作用。[方法]利用涂布打孔发法比较分析大蒜粗酶提取液、大蒜素、蒜氨酸酶对玉米的霉菌、丝黑穗菌、黑粉病菌的抑制情况。利用生物信息学技术分析从NCBI数据库中获得的3个大蒜的半胱氨酸合成酶基因(ASGCS2、ASGCS3和ASGCS4)的开放阅读框,预测其蛋白序列、分子量大小、蛋白质特性、亚细胞定位、系统进化等特征;通过荧光定量PCR分析了3个半胱氨酸合成酶的组织表达特性。[结果]大蒜素及粗酶提取液对玉米的三种病菌都以抑制作用。3个大蒜的半胱氨酸合成酶基因ASGCS2、ASGCS3和ASGCS4的开放态读码框长度分别为1152bp,1296bp和1236bp;其编码蛋白的理论相对分子量分别为40.6KD,34.1KD和36.1KD。ASGCS2被亚细胞定位于叶绿体中,而ASGCS3和ASGCS4均被定位于细胞质中。氨基酸比对结果表明,ASGCS2与ASGCS3相似度为70%,与ASGCS4相似度为59%;而ASGCS3与ASGCS4相似度为68%。进化分析表明,AsGCS2属于Bsas2亚家族,AsGCS3属于Bsas1亚家族,AsGCS4属于Bsas6亚家族。荧光定量PCR结果表明,大蒜不同CSase的组织特异性是不同的,ASGCS2在叶中表达量最高,ASGCS3在根中表达量最高,而ASGCS4在叶和根中都有较高的表达量。
[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.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513

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