本文關(guān)鍵詞： 轉(zhuǎn)基因紫花苜蓿 角質(zhì)層 ZxABCG11基因 抗旱性 安全性　出處：《蘭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：非生物脅迫如干旱、高溫、紫外、霜凍等已對(duì)全球農(nóng)牧業(yè)發(fā)展和生態(tài)環(huán)境構(gòu)成了嚴(yán)重威脅。長(zhǎng)期處于惡劣生境的荒漠旱生植物在長(zhǎng)期的進(jìn)化過(guò)程中,形成了獨(dú)特的抗旱機(jī)制,對(duì)其抗旱機(jī)理的研究及抗旱相關(guān)功能基因的發(fā)掘,進(jìn)而利用基因工程對(duì)牧草和農(nóng)作物進(jìn)行遺傳改良,已成為在傳統(tǒng)育種受限下的必然手段。特別是對(duì)在我國(guó)西北地區(qū)廣泛種植的具有良好飼用及生態(tài)環(huán)境治理價(jià)值的“牧草之王”紫花苜蓿(Medicago sativa L.)來(lái)說(shuō),這一手段顯得格外的重要。因?yàn)檐俎ｉL(zhǎng)期在良好栽培條件下種植,其遺傳潛力已慢慢退化,傳統(tǒng)育種很難提高其抗逆性。而荒漠旱生植物霸王(Zygophyllum xanthoxylum)不一樣,其在長(zhǎng)期適應(yīng)荒漠極端環(huán)境中形成了一系列抗逆機(jī)制,富含優(yōu)異的抗逆基因,適合用于紫花苜蓿的遺傳改良。前期研究中我們課題組已用霸王Na+區(qū)域化功能基因Zx NHX和Zx VP1-1對(duì)紫花苜蓿進(jìn)行了耐鹽、耐貧瘠和抗旱方面的改良,這主要是利用霸王滲透調(diào)節(jié)方面的能力。而利用霸王表皮發(fā)達(dá)的角質(zhì)層形成相關(guān)基因ZxABCG11來(lái)改良紫花苜蓿的研究尚未見(jiàn)報(bào)道。因此本研究在已克隆霸王角質(zhì)層脂質(zhì)轉(zhuǎn)運(yùn)蛋白編碼基因ZxABCG11以及驅(qū)動(dòng)該基因在植物地上部表皮特異表達(dá)的擬南芥(Arabidopsis thaliana)At CER6和蒺藜苜蓿(Medicago truncatula)Mt ML1啟動(dòng)子的基礎(chǔ)上,通過(guò)構(gòu)建霸王ZxABCG11基因的表達(dá)載體、在擬南芥中對(duì)其功能進(jìn)行驗(yàn)證、并經(jīng)農(nóng)桿菌GV3101介導(dǎo)將其轉(zhuǎn)入紫花苜蓿,取得如下主要結(jié)果:1.通過(guò)PCR的方法將ZxABCG11與FLAG標(biāo)簽融合,獲得了融合基因ZxABCG11-FLAG;通過(guò)雙酶切的方法成功將ZxABCG11-FLAG融合基因、安全標(biāo)記基因pmi、表皮特異性啟動(dòng)子At CER6及Mt ML1順次導(dǎo)入了p CAMBIA1302中,獲得了安全型植物表達(dá)載體At CER6::ZxABCG11-FLAG::35s::pmi和Mt ML1::ZxABCG11-FLAG::35s::pmi。2.將所構(gòu)植物表達(dá)載體導(dǎo)入擬南芥中,經(jīng)甘露糖濃度為3.5 m M的1/2 MS培養(yǎng)基反復(fù)篩選及PCR、RT-PCR檢測(cè),獲得了轉(zhuǎn)ZxABCG11基因擬南芥純合系。以此純合系進(jìn)行周期性間歇干旱,發(fā)現(xiàn)轉(zhuǎn)基因株系A(chǔ)AP1、AAP2、MAP1、MAP2(AAP:轉(zhuǎn)At CER6::ZxABCG11-FLAG::35s::pmi擬南芥;MAP:轉(zhuǎn)Mt ML1::ZxABCG11-FLAG::35s::pmi擬南芥)的生物量、凈光合速率(Pn)、水分利用效率(WUE)、葉綠素含量、葉片相對(duì)含水量(RWC)均高于野生型,而離體葉片脫水速率、蒸騰速率(Tr)、葉片相對(duì)質(zhì)膜透性均低于野生型,且AAP1和AAP2的生長(zhǎng)狀態(tài)要優(yōu)于MAP1和MAP2;同時(shí),對(duì)3個(gè)周期性間歇干旱結(jié)束時(shí)的株高、角果數(shù)及苔干鮮重分析顯示,轉(zhuǎn)基因株系前期集中于營(yíng)養(yǎng)生長(zhǎng),后期集中于生殖生長(zhǎng);此外,長(zhǎng)期性持續(xù)干旱下,野生型(WT)全部死亡(0/45),而MAP1、MAP2、AAP1、AAP2的存活率分別為27.3%(12/44)、28.9%(13/45)、66.7%(30/45)、33.3%(15/45);上述研究結(jié)果表明霸王ZxABCG11基因是一個(gè)優(yōu)異的抗旱基因,同時(shí)證明我們所構(gòu)載體具有活性,可用于紫花苜蓿的遺傳改良。3.對(duì)紫花苜蓿的遺傳轉(zhuǎn)化,我們確定了在各選擇階段篩選劑D-甘露糖與蔗糖的濃度配比,結(jié)果為體胚誘導(dǎo)階段的最佳選擇壓為25/5 g·L-1,體胚來(lái)源的幼苗生長(zhǎng)階段的最佳選擇壓為15/15 g·L-1;使用農(nóng)桿菌GV3101介導(dǎo)的紫花苜蓿遺傳體系,通過(guò)延遲篩選和幼苗篩選,最終獲得了59個(gè)抗性系列。綜上所述,本研究為后期獲得具備霸王發(fā)達(dá)角質(zhì)層特性的轉(zhuǎn)基因紫花苜蓿新品系(種)奠定了基礎(chǔ);對(duì)充分利用我國(guó)北方干旱、鹽漬及瘠薄的邊際土地具有重要的現(xiàn)實(shí)意義。
[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.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S541.9

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本文編號(hào)：1550817