本文選題：紫花苜蓿 + AtCBF1基因　； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：紫花苜蓿(Medicago sativa L.)為豆科苜蓿屬多年生草本植物,是世界上最重要的牧草資源,具有極高的飼用及生態(tài)價(jià)值。近些年,隨著紫花苜蓿栽培面積的不斷擴(kuò)大,抗寒性弱成為限制紫花苜蓿向北方地區(qū)推廣的主要因素,轉(zhuǎn)入抗寒基因提高紫花苜�？购允墙鉀Q該問題的高效育種方法之一。本研究從擬南芥中克隆得到冷誘導(dǎo)轉(zhuǎn)錄因子AtCBFl基因,采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)入到優(yōu)良紫花苜蓿品種中,通過轉(zhuǎn)化植株分子檢測、抗寒性及田間鑒定形成株系,篩選出了轉(zhuǎn)AtCBFl基因抗寒高產(chǎn)的轉(zhuǎn)基因紫花苜蓿新材料。主要研究結(jié)果如下：1.研究了紫花苜蓿品種、外植體以及激素組合配比等影響組培的再生因子,結(jié)果表明,組織培養(yǎng)最佳基因型為獵人河苜蓿；最佳外植體為下胚軸；最佳愈傷組織誘導(dǎo)培養(yǎng)基為SH+2.0mg · L-12,4-D+0.5 mg · L11 6-BA,愈傷組織繼代培養(yǎng)基為MSO+0.5mg · L-1 NAA+1.0mg · L-1 6-BA+1.0mg · L-1 AgNO3和MS+0.3mg·L-1TDZ分化培養(yǎng)基為MS+0.2mg · L-1 KT。2.克隆了擬南芥冷誘導(dǎo)轉(zhuǎn)錄因子AtCBF1基因并構(gòu)建了適于紫花苜蓿農(nóng)桿菌遺傳轉(zhuǎn)化的植物表達(dá)載體PBI121-CBF1。3.紫花苜蓿遺傳轉(zhuǎn)化因子研究確定出最佳農(nóng)桿菌抑制劑為350mg·L-1 Carb:最佳Kan篩選濃度為60mg·L-1；最佳受體基因型為獵人河苜蓿；最佳外植體為下胚軸；最適宜農(nóng)桿菌菌液濃度OD600為0.4～0.6,侵染時(shí)間10min；AS最適濃度為10mg·L-1。4.利用農(nóng)桿菌介導(dǎo)法將AtCBF1基因轉(zhuǎn)入紫花苜蓿,獲得了抗Kan的轉(zhuǎn)基因紫花苜蓿植株。經(jīng)PCR檢測,部分轉(zhuǎn)化植株擴(kuò)增出了大小為650bp左右特異帶,初步表明目的基因AtCBF1已經(jīng)整合到紫花苜�；蚪M,進(jìn)一步通過RT-PCR檢測,結(jié)果顯示外源基因AtCBF1在部分轉(zhuǎn)化植株的轉(zhuǎn)錄水平上表達(dá)。5.抗寒性生理研究測定了轉(zhuǎn)基因苜蓿相對電導(dǎo)率、脯氨酸含量、可溶性糖含量及丙二醛含量,結(jié)果顯示轉(zhuǎn)AtCBF1基因紫花苜蓿植株較對照抗寒性有所提高,轉(zhuǎn)基因株系以T9、T11和T3的抗寒性相對較高,且T9株系抗寒性最強(qiáng)。6.研究了轉(zhuǎn)基因苜蓿人工模擬冷凍天氣抗寒性,葉片受害及恢復(fù)生長結(jié)果表明轉(zhuǎn)基因紫花苜蓿植株表現(xiàn)出較強(qiáng)的耐低溫脅迫能力,即過量表達(dá)AtCBF1基因的轉(zhuǎn)基因紫花苜蓿的抗寒性獲得了提高。7.轉(zhuǎn)基因紫花苜蓿形態(tài)、產(chǎn)量、再生速度及品質(zhì)方面研究顯示,轉(zhuǎn)基因株系與對照相比在形態(tài)上沒有明顯生長延緩、植株矮小等不良變化,只是葉片長度較對照植株稍短;在產(chǎn)量和再生速度上,轉(zhuǎn)基因各株系有的高于對照,有的低于對照,T11和T9株系在產(chǎn)量及再生速度上均顯著高于對照和其他株系；營養(yǎng)成分轉(zhuǎn)基因株系均表現(xiàn)出高于對照的粗蛋白含量,營養(yǎng)品質(zhì)有所提高。綜合研究結(jié)果,T9和T11株系為表現(xiàn)最優(yōu)的轉(zhuǎn)基因株系。
[Abstract]:Medicago sativa L. Alfalfa perennial herbaceous plant is the most important forage resource in the world and has high fodder and ecological value. In recent years, with the continuous expansion of alfalfa cultivation area, the weak cold resistance has become the main factor limiting alfalfa extension to the north, and the transformation of cold resistance gene to improve alfalfa cold resistance is one of the efficient breeding methods to solve this problem. In this study, the cold-induced transcription factor AtCBFl gene was cloned from Arabidopsis thaliana and transferred into the superior alfalfa varieties by Agrobacterium tumefaciens. A new transgenic alfalfa material with high cold resistance and high yield of AtCBFl gene was screened out. The main results are as follows: 1. The regeneration factors affecting tissue culture of alfalfa varieties, explants and hormone combinations were studied. The results showed that the optimum genotypes of tissue culture were Huntsman River alfalfa, the best explants were Hypocotyl, and so on. The best medium for callus induction was SH 2.0mg L-124-D 0.5 mg L 11 6-BA. the subculture medium for callus was MSO 0.5mg L-1 NAA 1.0mg L-1 6-BA 1.0mg L-1 AgNO3 and MS 0.3mg L-1TDZ medium for differentiation. The AtCBF1 gene of Arabidopsis thaliana cold-induced transcription factor was cloned and the plant expression vector PBI121-CBF1.3was constructed. The genetic transformation factor of alfalfa confirmed that the best Agrobacterium inhibitor was 350mg L-1 Carb, the best concentration of Kan screening was 60mg L -1, the best receptor genotype was Huntsman River alfalfa, the best explant was Hypocotyl; The optimum concentration of Agrobacterium tumefaciens was 0.4 OD600 and 10mg L-1.4. The AtCBF1 gene was transferred into alfalfa by Agrobacterium tumefaciens and transgenic alfalfa plants resistant to Kan were obtained. The specific bands of 650bp were amplified from some transformed plants by PCR detection, which indicated that the target gene AtCBF1 had been integrated into alfalfa genome and further detected by RT-PCR. The results showed that the exogenous gene AtCBF1 was expressed at the transcriptional level of partial transformed plants. The relative electrical conductivity, proline content, soluble sugar content and malondialdehyde content of transgenic alfalfa were measured. The results showed that the cold resistance of transgenic alfalfa plants with AtCBF1 gene was higher than that of control. The chilling resistance of T9 T11 and T3 lines was relatively high, and that of T9 lines was the strongest. 6. The cold resistance of transgenic alfalfa was studied. The results showed that transgenic alfalfa plants showed strong tolerance to low temperature stress. The cold resistance of alfalfa with overexpression of AtCBF1 gene was improved by 7. 7%. The studies on morphology, yield, regeneration speed and quality of transgenic alfalfa showed that the transgenic lines had no obvious growth retardation in morphology and short plants, but the length of leaves was a little shorter than that of the control. In terms of yield and regeneration rate, some transgenic lines were higher than the control, others lower than the control T11 and T9 lines in yield and regeneration rate were significantly higher than the control and other lines. All the transgenic lines showed higher crude protein content and higher nutritional quality than the control. The results showed that T9 and T11 lines were the best transgenic lines.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S541.9

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