转AtCBF1基因提高紫花苜蓿抗寒性研究

发布时间：2018-05-06 09:25

本文选题：紫花苜蓿 + AtCBF1基因　；参考：《内蒙古农业大学》2016年博士论文

【摘要】：紫花苜蓿(Medicago sativa L.)为豆科苜蓿属多年生草本植物,是世界上最重要的牧草资源,具有极高的饲用及生态价值。近些年,随着紫花苜蓿栽培面积的不断扩大,抗寒性弱成为限制紫花苜蓿向北方地区推广的主要因素,转入抗寒基因提高紫花苜蓿抗寒性是解决该问题的高效育种方法之一。本研究从拟南芥中克隆得到冷诱导转录因子AtCBFl基因,采用农杆菌介导法转入到优良紫花苜蓿品种中,通过转化植株分子检测、抗寒性及田间鉴定形成株系,筛选出了转AtCBFl基因抗寒高产的转基因紫花苜蓿新材料。主要研究结果如下：1.研究了紫花苜蓿品种、外植体以及激素组合配比等影响组培的再生因子,结果表明,组织培养最佳基因型为猎人河苜蓿；最佳外植体为下胚轴；最佳愈伤组织诱导培养基为SH+2.0mg · L-12,4-D+0.5 mg · L11 6-BA,愈伤组织继代培养基为MSO+0.5mg · L-1 NAA+1.0mg · L-1 6-BA+1.0mg · L-1 AgNO3和MS+0.3mg·L-1TDZ分化培养基为MS+0.2mg · L-1 KT。2.克隆了拟南芥冷诱导转录因子AtCBF1基因并构建了适于紫花苜蓿农杆菌遗传转化的植物表达载体PBI121-CBF1。3.紫花苜蓿遗传转化因子研究确定出最佳农杆菌抑制剂为350mg·L-1 Carb:最佳Kan筛选浓度为60mg·L-1；最佳受体基因型为猎人河苜蓿；最佳外植体为下胚轴；最适宜农杆菌菌液浓度OD600为0.4～0.6,侵染时间10min；AS最适浓度为10mg·L-1。4.利用农杆菌介导法将AtCBF1基因转入紫花苜蓿,获得了抗Kan的转基因紫花苜蓿植株。经PCR检测,部分转化植株扩增出了大小为650bp左右特异带,初步表明目的基因AtCBF1已经整合到紫花苜蓿基因组,进一步通过RT-PCR检测,结果显示外源基因AtCBF1在部分转化植株的转录水平上表达。5.抗寒性生理研究测定了转基因苜蓿相对电导率、脯氨酸含量、可溶性糖含量及丙二醛含量,结果显示转AtCBF1基因紫花苜蓿植株较对照抗寒性有所提高,转基因株系以T9、T11和T3的抗寒性相对较高,且T9株系抗寒性最强。6.研究了转基因苜蓿人工模拟冷冻天气抗寒性,叶片受害及恢复生长结果表明转基因紫花苜蓿植株表现出较强的耐低温胁迫能力,即过量表达AtCBF1基因的转基因紫花苜蓿的抗寒性获得了提高。7.转基因紫花苜蓿形态、产量、再生速度及品质方面研究显示,转基因株系与对照相比在形态上没有明显生长延缓、植株矮小等不良变化,只是叶片长度较对照植株稍短;在产量和再生速度上,转基因各株系有的高于对照,有的低于对照,T11和T9株系在产量及再生速度上均显著高于对照和其他株系；营养成分转基因株系均表现出高于对照的粗蛋白含量,营养品质有所提高。综合研究结果,T9和T11株系为表现最优的转基因株系。
[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.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S541.9

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