基于FOX Hunting System技术的油菜抗逆基因筛选及功能验证

发布时间：2018-07-05 21:00

本文选题：油菜 + 非生物胁迫抗逆性　；参考：《河南大学》2017年硕士论文

【摘要】：油菜是世界上最重要的油籽作物之一。随着全球植物油的消费量稳步增长,年增长率约为5%,而油菜产油量占据全球植物油总产量的16%,是北欧、加拿大及中国的主要油料作物。在众多经济作物中,油菜极易遭受到干旱、盐害等非生物胁迫的影响。面对全球植物油消费量的稳步增长和气候变化引起的作物生长条件的恶化,不利环境条件下开发油菜的抗逆性以保持产量稳定性是当下紧迫的任务。在过去的几十年,通过转基因方法,各种胁迫应答基因已被用于探索改善植物的胁迫耐受性。本研究前期通过胁迫处理油菜材料,获得转录本并对其进行高通量测序分析,筛选出差异表达基因并克隆其全长cDNA。通过FOX hunting system(Full-length cDNA Over Expressor gene hunting system)结合高通量的Gateway技术构建了包含70个油菜干旱胁迫响应基因的植物表达文库并转入拟南芥,获得油菜抗逆相关基因FOX拟南芥超表达库。对超表达库材料用不同胁迫处理筛选,选出125个有表型的株系进行测序并进行进一步的表型确认和功能初步分析。1.对125个初筛有表型的株系进行纯合体鉴定,在T3代或T4代共获得纯合系42个。2.对所得42个纯合体株系进行抗逆性分析和生长表型监测,对照初筛表型,结合已报道相关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个基因,进行进一步的功能研究。3.通过转录激活活性分析和亚细胞定位,发现BnCOL2具有转录激活活性,定位于细胞核;组织表达分析表明,BnCOL2在油菜真叶和子叶中优势表达;诱导表达分析表明BnCOL2受NaCl、PEG、ABA、热和冷的胁迫诱导表达,其中PEG、ABA处理下诱导表达变化更为显著。通过对BnCOL2超表达拟南芥植株萌发及萌发后幼苗阶段的抗逆性分析,结果表明,BnCOL2超表达拟南芥植株在多种逆境条件下相比野生型植株呈现出敏感表型,干旱处理后死亡率、叶片失水率、气孔密度均高于WT,远红外成像结果显示BnCOL2超表达拟南芥叶温低于WT,BnCOL2超表达拟南芥叶绿素含量较WT高,抽薹时间提前。4.转录激活活性分析表明BnNAC1-1、BnNAC5-11、BnNAC18蛋白均具有转录激活活性。3个基因编码的蛋白主要定位于细胞核;组织表达分析表明,3个基因在油菜各个组织中都有表达,BnNAC1-1在根和子叶中优势表达;BnNAC5-11子叶中表达量最高;BnNAC18主要在子叶和真叶中表达。诱导表达分析显示BnNAC1-1、BnNAC5-11、BnNAC18受NaCl、PEG、ABA、热和冷等胁迫诱导表达。5.构建了4个基因的超表达载体和CRISPR/Cas9载体,转化油菜,均已获得转化体。
[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.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S565.4

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