本文選題：油菜 + 非生物脅迫抗逆性��； 參考：《河南大學》2017年碩士論文

【摘要】：油菜是世界上最重要的油籽作物之一。隨著全球植物油的消費量穩(wěn)步增長,年增長率約為5%,而油菜產油量占據全球植物油總產量的16%,是北歐、加拿大及中國的主要油料作物。在眾多經濟作物中,油菜極易遭受到干旱、鹽害等非生物脅迫的影響。面對全球植物油消費量的穩(wěn)步增長和氣候變化引起的作物生長條件的惡化,不利環(huán)境條件下開發(fā)油菜的抗逆性以保持產量穩(wěn)定性是當下緊迫的任務。在過去的幾十年,通過轉基因方法,各種脅迫應答基因已被用于探索改善植物的脅迫耐受性。本研究前期通過脅迫處理油菜材料,獲得轉錄本并對其進行高通量測序分析,篩選出差異表達基因并克隆其全長cDNA。通過FOX hunting system(Full-length cDNA Over Expressor gene hunting system)結合高通量的Gateway技術構建了包含70個油菜干旱脅迫響應基因的植物表達文庫并轉入擬南芥,獲得油菜抗逆相關基因FOX擬南芥超表達庫。對超表達庫材料用不同脅迫處理篩選,選出125個有表型的株系進行測序并進行進一步的表型確認和功能初步分析。1.對125個初篩有表型的株系進行純合體鑒定,在T3代或T4代共獲得純合系42個。2.對所得42個純合體株系進行抗逆性分析和生長表型監(jiān)測,對照初篩表型,結合已報道相關同源基因的功能研究,克隆了油菜CONSTANS LIKE家族基因BnCONSTANS-LIKE2(BnCOL2)和NAC家族基因BnNAC18、BnNAC1-1、BnNAC5-11等4個基因,進行進一步的功能研究。3.通過轉錄激活活性分析和亞細胞定位,發(fā)現BnCOL2具有轉錄激活活性,定位于細胞核;組織表達分析表明,BnCOL2在油菜真葉和子葉中優(yōu)勢表達;誘導表達分析表明BnCOL2受NaCl、PEG、ABA、熱和冷的脅迫誘導表達,其中PEG、ABA處理下誘導表達變化更為顯著。通過對BnCOL2超表達擬南芥植株萌發(fā)及萌發(fā)后幼苗階段的抗逆性分析,結果表明,BnCOL2超表達擬南芥植株在多種逆境條件下相比野生型植株呈現出敏感表型,干旱處理后死亡率、葉片失水率、氣孔密度均高于WT,遠紅外成像結果顯示BnCOL2超表達擬南芥葉溫低于WT,BnCOL2超表達擬南芥葉綠素含量較WT高,抽薹時間提前。4.轉錄激活活性分析表明BnNAC1-1、BnNAC5-11、BnNAC18蛋白均具有轉錄激活活性。3個基因編碼的蛋白主要定位于細胞核;組織表達分析表明,3個基因在油菜各個組織中都有表達,BnNAC1-1在根和子葉中優(yōu)勢表達;BnNAC5-11子葉中表達量最高;BnNAC18主要在子葉和真葉中表達。誘導表達分析顯示BnNAC1-1、BnNAC5-11、BnNAC18受NaCl、PEG、ABA、熱和冷等脅迫誘導表達。5.構建了4個基因的超表達載體和CRISPR/Cas9載體,轉化油菜,均已獲得轉化體。
[Abstract]:Rape is one of the most important oilseed crops in the world. With global vegetable oil consumption growing steadily, the annual growth rate is about 5 percent, while rape oil production accounts for 16 percent of the world's total vegetable oil production and is the main oil crop in northern Europe, Canada and China. Among many cash crops, rapeseed is vulnerable to abiotic stresses such as drought, salt damage and so on. In the face of the steady increase of the global vegetable oil consumption and the deterioration of crop growth conditions caused by climate change, it is urgent to develop rapeseed resistance to maintain yield stability under adverse environmental conditions. In the past few decades, various stress response genes have been used to improve plant stress tolerance through transgenic methods. In this study, the transcripts were obtained and analyzed by high-throughput sequencing. The differentially expressed genes were screened out and the full-length cDNAs were cloned. The plant expression library containing 70 drought stress response genes of rape was constructed by Fox hunting system (Full-length cDNA over Expressor gene hunting system) combined with high-throughput Gateway technique and transferred into Arabidopsis thaliana to obtain the overexpression library of rape stress-related gene Fox Arabidopsis thaliana (Arabidopsis thaliana). 125 lines with phenotype were selected and sequenced, and further phenotypic identification and functional analysis were carried out. A total of 42 homozygous lines were obtained in T3 generation or T4 generation by homozygous identification of 125 lines with phenotypic phenotype. The resistance and growth phenotype of 42 homozygous lines were analyzed and compared. Four genes, BnCONSTANS-LIKE2 (BnCOL2) and BnNAC18BnNAC1-1 (BnNAC5-11), were cloned from rape (Brassica napus) family BnSTANNS-LIKE2 (BnCOL2) and NAC family gene (BnNAC18BnNAC1-1, BnNAC5-11), combined with functional studies of reported homologous genes. Further functional study. Through transcriptional activation activity analysis and subcellular localization, it was found that BnCOL2 had transcriptional activation activity and was located in the nucleus, and the tissue expression analysis showed that BnCOL2 was predominantly expressed in the true leaves and cotyledon of Brassica napus. The induced expression analysis showed that BnCOL2 was induced by NaCl-PEGG ABA, heat and cold stress, and the induced expression of BnCOL2 was more obvious under the treatment of PEGN ABA. The stress resistance of Arabidopsis thaliana plants with overexpression of BnCOL2 in germination and seedling stage after germination was analyzed. The results showed that the overexpression of BnCOL2 in Arabidopsis thaliana plants showed sensitive phenotype compared with wild-type plants under various stress conditions, and the mortality after drought treatment was higher than that of wild type plants. The leaf water loss rate and stomatal density were higher than those of WT.The results of far-infrared imaging showed that the leaf temperature of BnCOL2 was lower than that of WT-BnCOL2, and the chlorophyll content of Arabidopsis thaliana was higher than that of WT, and the bolting time was earlier than that of WT. Analysis of transcriptional activation activity showed that BnNAC1-1, BnNAC5-11 and BnNAC18 proteins all had transcriptional activation activities. Tissue expression analysis showed that BnNAC1-1 was expressed mostly in root and cotyledon. The highest expression level of BnNAC5-11 cotyledons was found in cotyledons. BnNAC18 was mainly expressed in cotyledons and true leaves. The induced expression analysis showed that BnNAC1-1, BnNAC5-11 and BnNAC18 were induced by NaCl-PEGG ABA, heat and cold stress. Four gene superexpression vectors and CRISPRR / Cas9 vector were constructed and transformed into rapeseed.
【學位授予單位】：河南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2;S565.4

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相關期刊論文 前1條

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