农杆菌介导的BAN基因在紫花苜蓿中的转化和表达的研究

发布时间：2018-03-24 15:07

本文选题：紫花苜蓿　切入点：农杆菌介导　出处：《甘肃农业大学》2017年硕士论文

【摘要】：紫花苜蓿(Medicago sativa L.)是世界上分布和种植面积最广的一种多年生豆科牧草,其品质优良、营养成分丰富、适口性极佳,被誉为“牧草之王”。但是,反刍家畜鲜食过量苜蓿后容易引发臌胀病,严重时会导致家畜死亡,因而限制了这种优良牧草在畜牧业生产中的应用。研究发现,紫花苜蓿中缩合单宁(condensed tannins,CT)含量较低是引发反刍家畜臌胀病的主要因素之一。因此,适当提高紫花苜蓿中缩合单宁的含量,可以降低反刍家畜臌胀病发生几率,具有重要的理论和实践意义。本试验通过农杆菌介导法将编码缩合单宁合成途径中的关键酶(花青素还原酶)基因,即BAN基因转入紫花苜蓿栽培品种‘甘农3号’紫花苜蓿基因组中,并对T0代转化植株花青素还原酶(ANR)活性和缩合单宁含量进行了测定,为抗臌胀病转基因苜蓿新品种的育成奠定了基础。具体研究内容及结果如下:1.高效再生体系的优化。以‘甘农3号’紫花苜蓿下胚轴为外植体,进行了愈伤组织诱导、芽分化及生根的研究。结果表明:苜蓿愈伤组织诱导以3.0 mg/L 2,4-D和1.0 mg/L NAA配比处理为最佳,愈伤组织诱导率达84.67%;愈伤组织分化则以0.5 mg/L KT和0.5mg/L NAA配比处理为最佳,体胚诱导率最高,达92.00%,后期胚状体出芽率也最高,达81.52%;芽生根时以1/2 MS培养基为基础单独添加0.5 mg/L NAA处理为最佳,诱根率达86.00%,且根系长、粗壮、须根发达,有利于后期移栽。2.遗传转化体系的建立。以‘甘农3号’紫花苜蓿下胚轴为外植体,以含有CPB-BAN-GFP载体的农杆菌为介导,优化遗传转化体系。结果表明:最适草铵膦筛选浓度为2.0 mg/L,最佳筛选时间为愈伤组织分化阶段;脱菌时抗生素选取浓度为300 mg/L头孢霉素(Cef)为宜;农杆菌侵染时间为15 min,菌液浓度OD600为0.6~0.8,共培养时间为3 d时有利于基因转化。3.转基因苜蓿的分子检测及表达分析。以上述遗传转化和再生体系为基础,通过初步筛选共获得8株抗草铵膦苜蓿植株。经PCR检测,4株抗性植株扩增出了大小为1020 bp的特异性条带,并通过Southern blot检测证实BAN基因已经整合到这4株阳性植株的基因组中。进一步通过RT-PCR和qRT-PCR检测,结果显示外源BAN基因可在转基因植株的转录水平上正常表达。4.转基因苜蓿ANR活性和缩合单宁含量的测定。通过对已鉴定的4株转基因苜蓿ANR活性和缩合单宁含量分析表明:与野生型植株相比,4株转基因苜蓿ANR活性均显著提高,而其中1株缩合单宁含量得到大幅度提高,并且可能因ANR酶活性的上升促进了花青素的合成,导致该植株部分叶片呈现紫红色,BAN基因过量表达。
[Abstract]:Medicago sativa L.) is one of the most widely distributed and planted perennial legumes in the world. Its quality, nutrition and palatability are very good. Regurgitating livestock after fresh eating alfalfa is prone to cause distention disease, which can lead to animal death in serious cases, thus limiting the application of this fine forage in animal husbandry. The low content of condensed tannin in alfalfa is one of the main factors causing ruminant distention. Therefore, increasing the content of condensed tannin in alfalfa can reduce the probability of ruminant swelling. It is of great theoretical and practical significance. The key enzyme (anthocyanin reductase) gene in the pathway of condensation tannin synthesis was encoded by Agrobacterium tumefaciens mediated by Agrobacterium tumefaciens. The BAN gene was transferred into alfalfa genome of 'Gannon 3', and the anthocyanin reductase activity and condensate tannin content were determined. The research contents and results are as follows: 1. Optimization of efficient regeneration system. Hypocotyl of 'Gannong 3' alfalfa was used as explant to induce callus. The results showed that 3.0 mg/L 24-D and 1.0 mg/L NAA were the best treatments for callus induction, and the callus induction rate was 84.67%, while 0.5 mg/L KT and 0.5mg/L NAA were the best for callus differentiation. The induction rate of somatic embryos was the highest, 92.00% and 81.52% of embryoid buds were obtained in the later stage, the best one was treated with 0.5 mg/L NAA on the basis of 1 / 2 MS medium for rooting, the rate of inducing roots was 86.00%, and the root system was long, strong, and fibrous root was developed. It is beneficial to the later transplanting. 2. The establishment of genetic transformation system. Hypocotyl of alfalfa was used as explant and Agrobacterium tumefaciens containing CPB-BAN-GFP vector was used as explant. The results showed that the optimal concentration of phosphine oxalate was 2.0 mg / L, the optimal screening time was at the stage of callus differentiation, and the optimal concentration of antibiotics was 300 mg/L cef. during sterilization. The infection time of Agrobacterium tumefaciens was 15 min, the concentration of OD600 was 0.6%, and the co-culture time was 3 days. The molecular detection and expression analysis of transgenic alfalfa were based on the above genetic transformation and regeneration system. A total of 8 resistant alfalfa plants were obtained by preliminary screening. The specific bands of 1020 BP were amplified from 4 resistant plants by PCR. Southern blot analysis confirmed that the BAN gene had been integrated into the genomes of the four positive plants. Further, the BAN gene was detected by RT-PCR and qRT-PCR. The results showed that the exogenous BAN gene could be expressed normally at the transcriptional level of transgenic plants. The activity of ANR and the content of condensed tannin in transgenic alfalfa were determined. The ANR activity and condensed tannin content of 4 transgenic alfalfa strains were identified. The results showed that the ANR activity of 4 transgenic alfalfa plants was significantly higher than that of wild type alfalfa plants. However, the content of condensed tannin in one of the plants was significantly increased, and the increased activity of ANR could promote the synthesis of anthocyanins, which led to the over-expression of the gene in some leaves of the plant.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S541.9

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