霸王ZxABCG11的功能验证及其对紫花苜蓿（Medicago sativa L.）的遗传转化

发布时间：2018-03-01 06:28

本文关键词： 转基因紫花苜蓿角质层 ZxABCG11基因抗旱性安全性　出处：《兰州大学》2017年硕士论文　论文类型：学位论文

【摘要】：非生物胁迫如干旱、高温、紫外、霜冻等已对全球农牧业发展和生态环境构成了严重威胁。长期处于恶劣生境的荒漠旱生植物在长期的进化过程中,形成了独特的抗旱机制,对其抗旱机理的研究及抗旱相关功能基因的发掘,进而利用基因工程对牧草和农作物进行遗传改良,已成为在传统育种受限下的必然手段。特别是对在我国西北地区广泛种植的具有良好饲用及生态环境治理价值的“牧草之王”紫花苜蓿(Medicago sativa L.)来说,这一手段显得格外的重要。因为苜蓿长期在良好栽培条件下种植,其遗传潜力已慢慢退化,传统育种很难提高其抗逆性。而荒漠旱生植物霸王(Zygophyllum xanthoxylum)不一样,其在长期适应荒漠极端环境中形成了一系列抗逆机制,富含优异的抗逆基因,适合用于紫花苜蓿的遗传改良。前期研究中我们课题组已用霸王Na+区域化功能基因Zx NHX和Zx VP1-1对紫花苜蓿进行了耐盐、耐贫瘠和抗旱方面的改良,这主要是利用霸王渗透调节方面的能力。而利用霸王表皮发达的角质层形成相关基因ZxABCG11来改良紫花苜蓿的研究尚未见报道。因此本研究在已克隆霸王角质层脂质转运蛋白编码基因ZxABCG11以及驱动该基因在植物地上部表皮特异表达的拟南芥(Arabidopsis thaliana)At CER6和蒺藜苜蓿(Medicago truncatula)Mt ML1启动子的基础上,通过构建霸王ZxABCG11基因的表达载体、在拟南芥中对其功能进行验证、并经农杆菌GV3101介导将其转入紫花苜蓿,取得如下主要结果:1.通过PCR的方法将ZxABCG11与FLAG标签融合,获得了融合基因ZxABCG11-FLAG;通过双酶切的方法成功将ZxABCG11-FLAG融合基因、安全标记基因pmi、表皮特异性启动子At CER6及Mt ML1顺次导入了p CAMBIA1302中,获得了安全型植物表达载体At CER6::ZxABCG11-FLAG::35s::pmi和Mt ML1::ZxABCG11-FLAG::35s::pmi。2.将所构植物表达载体导入拟南芥中,经甘露糖浓度为3.5 m M的1/2 MS培养基反复筛选及PCR、RT-PCR检测,获得了转ZxABCG11基因拟南芥纯合系。以此纯合系进行周期性间歇干旱,发现转基因株系AAP1、AAP2、MAP1、MAP2(AAP:转At CER6::ZxABCG11-FLAG::35s::pmi拟南芥;MAP:转Mt ML1::ZxABCG11-FLAG::35s::pmi拟南芥)的生物量、净光合速率(Pn)、水分利用效率(WUE)、叶绿素含量、叶片相对含水量(RWC)均高于野生型,而离体叶片脱水速率、蒸腾速率(Tr)、叶片相对质膜透性均低于野生型,且AAP1和AAP2的生长状态要优于MAP1和MAP2;同时,对3个周期性间歇干旱结束时的株高、角果数及苔干鲜重分析显示,转基因株系前期集中于营养生长,后期集中于生殖生长;此外,长期性持续干旱下,野生型(WT)全部死亡(0/45),而MAP1、MAP2、AAP1、AAP2的存活率分别为27.3%(12/44)、28.9%(13/45)、66.7%(30/45)、33.3%(15/45);上述研究结果表明霸王ZxABCG11基因是一个优异的抗旱基因,同时证明我们所构载体具有活性,可用于紫花苜蓿的遗传改良。3.对紫花苜蓿的遗传转化,我们确定了在各选择阶段筛选剂D-甘露糖与蔗糖的浓度配比,结果为体胚诱导阶段的最佳选择压为25/5 g·L-1,体胚来源的幼苗生长阶段的最佳选择压为15/15 g·L-1;使用农杆菌GV3101介导的紫花苜蓿遗传体系,通过延迟筛选和幼苗筛选,最终获得了59个抗性系列。综上所述,本研究为后期获得具备霸王发达角质层特性的转基因紫花苜蓿新品系(种)奠定了基础;对充分利用我国北方干旱、盐渍及瘠薄的边际土地具有重要的现实意义。
[Abstract]:Abiotic stresses such as drought, high temperature, UV, frost and other has been the development of global agriculture and animal husbandry and ecological environment pose a serious threat. In the long-term bad habitat desert xerophytic plants in the long process of evolution, the formation mechanism of drought resistance and drought resistance of unique, exploring genes related to the drought resistance mechanism of the then, for the genetic improvement of forage crops and using genetic engineering has become an inevitable way in traditional breeding is limited. Especially the widely cultivated in Northwest China has good forage and ecological environment value of "the king of grass" alfalfa (Medicago sativa L.), this method is particularly because an alfalfa planted in good long-term cultivation conditions, its genetic potential has been slowly degraded, traditional breeding is difficult to improve the resistance. The desert xerophils (Zygophyllum Xanthoxylum) Is not the same, in the long-term adaptation in desert environments has formed a series of resistance mechanisms, with excellent resistance gene, suitable for genetic improvement of alfalfa. In previous research, our research group has used fighter Na+ regional gene Zx NHX and Zx VP1-1 on Alfalfa for improved salt tolerance, resistance barren and drought areas, which is the main osmotic adjustment ability of the overlord. And based on the stratum corneum epidermis developed fighter formation related genes ZxABCG11 to improve alfalfa has not been reported. Therefore in this study cloned cuticle lipid transfer protein Rex encoding gene ZxABCG11 and drive the gene in plant shoot epidermal specific the expression of Arabidopsis (Arabidopsis thaliana) At CER6 (Medicago truncatula) and Medicago truncatula based Mt ML1 promoter, the expression vector of ZxABCG11 gene of t., To verify its function in Arabidopsis by Agrobacterium tumefaciens GV3101 transferred into alfalfa, the main results are as follows: 1. through PCR method ZxABCG11 and FLAG fusion tag, the fusion gene ZxABCG11-FLAG by double enzyme digestion method; the success of the ZxABCG11-FLAG fusion gene, safe marker gene PMI, epidermal characteristics promoter At CER6 and Mt ML1 in order to import the P CAMBIA1302, to obtain the security type At plant expression vector CER6:: ZxABCG11-FLAG:: 35s:: PMI and Mt ML1:: ZxABCG11-FLAG:: 35s:: pmi.2.. The plant expression vector into Arabidopsis thaliana, the mannose concentration of 3.5 m M 1/2 MS medium repeated screening and PCR, RT-PCR detection, the ZxABCG11 transgenic Arabidopsis homozygous lines. The homozygous lines of Intermittent Drought, found that the transgenic lines AAP1, AAP2, MAP1, MAP2 (AAP: At CER6:: ZxABCG11-FLAG:: 35s: PMI MAP: Mt: ML1: transgenic Arabidopsis thaliana; ZxABCG11-FLAG:: 35s:: PMI) of Arabidopsis thaliana biomass, net photosynthetic rate (Pn), water use efficiency (WUE), chlorophyll content, leaf relative water content (RWC) were higher than that of the wild type, while the in vitro leaf dehydration rate, transpiration rate (Tr), relative leaf the membrane permeability was lower than that of the wild type, and the growth state of AAP1 and AAP2 is better than MAP1 and MAP2; at the same time, the 3 periodic intermittent drought at the end of the plant height, fresh weight analysis showed that the pod number and dry moss, transgenic lines focus on earlier stages of vegetative growth and reproductive growth in later period; in addition, long term drought, wild type (WT) all died, and (0/45) MAP1, MAP2, AAP1, the survival rate of AAP2 was 27.3% (12/44), 28.9% (13/45), 66.7% (30/45), 33.3% (15/45); the results of the study showed that the fighter ZxABCG11 gene is an excellent drought resistance gene at the same time, we prove that constitutive vector has The activity can be used for genetic transformation of alfalfa genetic improvement of Alfalfa.3., we determined the concentration of screening agent D- mannose and sucrose in the selection stage, the best choice for the induction of somatic embryo stage pressure was 25/5 g L-1, the best choice of somatic embryos from seedling growth stage pressure is 15/15 g L-1; the use of alfalfa genetic system of Agrobacterium mediated by GV3101, through the delayed selection and seedling screening, finally obtained 59 resistant series. In summary, this study obtained transgenic alfalfa varieties have developed cuticle characteristics as overlord later (kind) laid the foundation; to make full use of drought in North China that has important practical significance and barren saline marginal land.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S541.9

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