本文選題：紫花苜蓿　切入點(diǎn)：農(nóng)桿菌介導(dǎo)　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：紫花苜蓿(Medicago sativa L.)是世界上分布和種植面積最廣的一種多年生豆科牧草,其品質(zhì)優(yōu)良、營養(yǎng)成分豐富、適口性極佳,被譽(yù)為“牧草之王”。但是,反芻家畜鮮食過量苜蓿后容易引發(fā)臌脹病,嚴(yán)重時會導(dǎo)致家畜死亡,因而限制了這種優(yōu)良牧草在畜牧業(yè)生產(chǎn)中的應(yīng)用。研究發(fā)現(xiàn),紫花苜蓿中縮合單寧(condensed tannins,CT)含量較低是引發(fā)反芻家畜臌脹病的主要因素之一。因此,適當(dāng)提高紫花苜蓿中縮合單寧的含量,可以降低反芻家畜臌脹病發(fā)生幾率,具有重要的理論和實(shí)踐意義。本試驗通過農(nóng)桿菌介導(dǎo)法將編碼縮合單寧合成途徑中的關(guān)鍵酶(花青素還原酶)基因,即BAN基因轉(zhuǎn)入紫花苜蓿栽培品種‘甘農(nóng)3號’紫花苜�；蚪M中,并對T0代轉(zhuǎn)化植株花青素還原酶(ANR)活性和縮合單寧含量進(jìn)行了測定,為抗臌脹病轉(zhuǎn)基因苜蓿新品種的育成奠定了基礎(chǔ)。具體研究內(nèi)容及結(jié)果如下:1.高效再生體系的優(yōu)化。以‘甘農(nóng)3號’紫花苜蓿下胚軸為外植體,進(jìn)行了愈傷組織誘導(dǎo)、芽分化及生根的研究。結(jié)果表明:苜蓿愈傷組織誘導(dǎo)以3.0 mg/L 2,4-D和1.0 mg/L NAA配比處理為最佳,愈傷組織誘導(dǎo)率達(dá)84.67%;愈傷組織分化則以0.5 mg/L KT和0.5mg/L NAA配比處理為最佳,體胚誘導(dǎo)率最高,達(dá)92.00%,后期胚狀體出芽率也最高,達(dá)81.52%;芽生根時以1/2 MS培養(yǎng)基為基礎(chǔ)單獨(dú)添加0.5 mg/L NAA處理為最佳,誘根率達(dá)86.00%,且根系長、粗壯、須根發(fā)達(dá),有利于后期移栽。2.遺傳轉(zhuǎn)化體系的建立。以‘甘農(nóng)3號’紫花苜蓿下胚軸為外植體,以含有CPB-BAN-GFP載體的農(nóng)桿菌為介導(dǎo),優(yōu)化遺傳轉(zhuǎn)化體系。結(jié)果表明:最適草銨膦篩選濃度為2.0 mg/L,最佳篩選時間為愈傷組織分化階段;脫菌時抗生素選取濃度為300 mg/L頭孢霉素(Cef)為宜;農(nóng)桿菌侵染時間為15 min,菌液濃度OD600為0.6~0.8,共培養(yǎng)時間為3 d時有利于基因轉(zhuǎn)化。3.轉(zhuǎn)基因苜蓿的分子檢測及表達(dá)分析。以上述遺傳轉(zhuǎn)化和再生體系為基礎(chǔ),通過初步篩選共獲得8株抗草銨膦苜蓿植株。經(jīng)PCR檢測,4株抗性植株擴(kuò)增出了大小為1020 bp的特異性條帶,并通過Southern blot檢測證實(shí)BAN基因已經(jīng)整合到這4株陽性植株的基因組中。進(jìn)一步通過RT-PCR和qRT-PCR檢測,結(jié)果顯示外源BAN基因可在轉(zhuǎn)基因植株的轉(zhuǎn)錄水平上正常表達(dá)。4.轉(zhuǎn)基因苜蓿ANR活性和縮合單寧含量的測定。通過對已鑒定的4株轉(zhuǎn)基因苜蓿ANR活性和縮合單寧含量分析表明:與野生型植株相比,4株轉(zhuǎn)基因苜蓿ANR活性均顯著提高,而其中1株縮合單寧含量得到大幅度提高,并且可能因ANR酶活性的上升促進(jìn)了花青素的合成,導(dǎo)致該植株部分葉片呈現(xiàn)紫紅色,BAN基因過量表達(dá)。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S541.9

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