花生ABA途径抗逆基因AhLOS5克隆与抗逆性研究

发布时间：2018-01-16 23:31

本文关键词：花生ABA途径抗逆基因AhLOS5克隆与抗逆性研究　出处：《山东农业大学》2017年博士论文　论文类型：学位论文

更多相关文章：花生 干旱胁迫 盐胁迫 脱落酸 AhLOS5

【摘要】：花生(Arachis hypogaea L.)是我国重要的油料作物和经济作物,以其扎根深、开花量大、花期长、无明显的需水临界期以及水分利用率较高等特点,具有较强的抗旱性,是发展旱作农业和旱薄地开发的理想作物。干旱往往伴随土壤荒漠化、盐渍化的加重,给农业生产带来多种因素的制约。在全球范围内,有2/3的花生分布在旱作地区,缺少灌溉条件加之瘠薄的砂质土壤,使干旱和高盐危害非常普遍,已成为花生生产上分布最广、危害程度最大的限制因素。在我国,常年有70%的花生遭受不同程度的干旱和高盐胁迫,干旱和高盐引起的荚果减产率平均在20%以上。植物对非生物逆境的响应伴随着对生长发育过程很多基因的调控。植物激素脱落酸(abscisic acid,ABA)能参与植物对许多环境胁迫如干旱、冷害、盐害等的生理反应和分子生物学效应。目前ABA已经公认为能够诱导植物产生对不良环境的抗性(抗旱性、抗寒性、抗病性、耐盐性等),是植物的“抗逆诱导因子”。目前公认的高等植物体内的ABA的生物合成主要为间接途径,反应过程中的玉米黄质环氧化酶(ZEP)、9-顺式环氧类胡萝卜素双加氧酶(NCED)、ABA醛氧化酶(AAO)及钼辅因子硫化酶(MCSU)是关键调控酶。本研究从花生栽培品种‘丰花1号’中克隆了钼辅因子硫酸化酶基因AhLOS5,进行了抗逆性鉴定和基因功能验证。构建了含AhLOS5基因的正义和反义植物表达载体,农杆菌介导分别转化三生烟草,获得了不同AhLOS5表达水平的转基因烟草植株,并对这些转基因株系的T1代植株进行了生物学功能分析。通过生理分析、遗传鉴定、分子生物学和生物化学的研究确定AhLOS5应答非生物逆境的机制。具体结果如下:(1)选取了黄淮海花生产区4个主推品种,进行抗旱性鉴定。抗旱性能排名为‘花育20号’‘丰花1号’‘海花1号’‘白沙1016’。(2)在黄淮海花生主产区种植面积较大,同时表现高抗旱性的花生品种‘丰花1号’中成功克隆花生AhLOS5基因。全长2731个碱基,包括2451bp的开放阅读框,115bp的5′非编码区,165bp的3′非编码区,ORF编码816个氨基酸。该基因编码钼辅因子硫酸化酶,属于MOSC超家族。同源性分析证明,我们克隆得到的‘丰花1号’AhLOS5与花生二倍体野生种A.duranensis中的AdLOS5同源性最高,达到94.7%,与AiLOS5同源性为92.2%。与其他高等植物相比,AhLOS5与同属豆科的狭叶羽扇豆、矮沙冬青、木豆、同源性较高,分别为77.7%、73.7%和72.9%,与属十字花科的拟南芥同源性略低,为59.9%。(3)以AhLOS5 cDNA核苷酸片段制备探针对4个主推品种中AhLOS5的表达特性进行了分析。结果显示,AhLOS5在4个主推品种中表达量顺序为‘花育20号’‘丰花1号’‘海花1号’‘白沙1016’。我们发现,4个品种间的AhLOS5基因表达量排序与其抗旱性排序一致。验证了该基因在花生中的抗旱性。(4)采用Northern杂交的方法对‘丰花1号’中AhLOS5的表达特性进行了分析。结果表明,AhLOS5在花生中组成型表达,花生各器官中表达量顺序为花成熟叶幼果果针根茎,在叶片中的表达量随着叶龄的增加而增加,在叶片展开第50d达到峰值。AhLOS5的表达还受干旱和盐胁迫诱导,诱导作用明显。(5)构建了2种植物表达载体,即含正义AhLOS5的载体pBI-LOS5和含反义AhLOS5的载体pBI-R-LOS5。以三生烟草为转化受体,采用农杆菌介导的方法,将上述2种载体分别转化。转不同载体的转基因烟草当代(T0代)所结的种子,经卡那霉素筛选,并结合转基因株系的T1代植株PCR鉴定结果,初步确定,含反义AhLOS5的转基因植株有20株,含正义AhLOS5的转基因植株有52株,并选取部分不同类型的转基因植株进行了Northern杂交分析。(6)通过转基因烟草植株种子根长、叶绿素含量、相对含水量、相对电导率、丙二醛(MDA)、抗坏血酸(ASA)、抗氧化酶活性(SOD,POD,CAT,APX)、内源ABA、脯氨酸等生理生化指标检测发现,在干旱和盐胁迫下,过表达AhLOS5的转基因烟草转基因植株体内内源ABA含量显著升高,抗氧化酶活性明显升高,膜脂完整性好,渗透调节物质含量高。过表达AhLOS5能够明显的提高转基因株系对干旱和盐胁迫的抗性。(7)用Northern blot方法对转基因烟草中另外3个抗逆相关基因DREB2B,RD22,P5CS表达进行检测。结果显示,在干旱和盐胁迫下,正义转基因和野生型对照植株的3个基因DREB2B,RD22,P5CS都诱导表达;而反义转基因植株中只有不依赖ABA的抗逆基因DREB2B诱导表达,依赖ABA的抗逆基因RD22和P5CS都不诱导表达。这证明了AhLOS5参与的植物抗逆途径属于依赖ABA途径,过表达AhLOS5转基因烟草获得的抗逆性是由胁迫诱导产生的内源ABA调控的。
[Abstract]:Peanut (Arachis hypogaea L.) is China's important oil crops and economic crops, with its deep rooted flowering, large quantity, long flowering period, no obvious characteristics of a higher rate of using the critical period of water requirement and water, has strong drought resistance, is the ideal development of dryland agriculture and dryland crops drought development. Often accompanied by soil desertification, salinization of agricultural production increase, to bring a variety of factors. In the global scope, the 2/3 distribution in peanut dry farming area, lack of sandy soil and irrigation conditions in the barren, drought and high salt harm is common, has become the most widely distributed peanut production, the largest limiting factors of hazards. In our country, there are 70% perennial peanut suffered varying degrees of drought and high salt stress, drought and high salt induced pod yield reduction rate of 20% on average. The plant response to abiotic stress along with the growth of During the development of regulation of many genes. The plant hormone abscisic acid (abscisic acid, ABA) can participate in many plants to environmental stresses such as drought, chilling injury, physiological and molecular responses of salt damage and so on. At present ABA has been recognized as to induce resistance to the adverse environment (drought resistance, cold resistance, disease resistance. The salt tolerance of plants, etc.) is the "resistance inducing factor". The biosynthesis recognized in higher plants ABA mainly indirectly in the reaction process of zeaxanthin epoxidase (ZEP), 9- CIS epoxy carotenoid dioxygenase (NCED), ABA aldehyde oxidase (AAO) and the molybdenum cofactor sulfurase (MCSU) is a key regulatory enzyme. This study from the peanut cultivar "Fenghua 1" cloned molybdenum cofactor sulfurylase gene AhLOS5, the resistance identification and gene function. The construct containing AhLOS5 gene and anti justice Antisense plant expression vector and Agrobacterium mediated transformation respectively three tobacco, obtained a different AhLOS5 expression level in transgenic tobacco plants, and the transgenic plants of the T1 generation of biological function analysis, genetic identification by physiology, biochemistry and molecular biology research to determine the mechanism of non biological stress response AhLOS5. The main results are as follows: (1) the flower production area of Huanghuaihai 4 main varieties, the drought resistance of drought resistance identification. Ranking as "Huayu 20" and "Fenghua 1" Haihua No. 1 "and" 1016 white ". (2) in the larger planting peanut production area of Huanghuaihai area. At the same time high drought resistance of peanut varieties" Fenghua 1 "in the successful cloning of AhLOS5 gene from peanut. A total length of 2731 nucleotides, including an open reading frame of 2451bp, 115bp 5 '- 3' of the 165bp encoding region, non encoding region of ORF encoding 816 amino acids. The gene encoding molybdenum cofactor sulfurylase, belonging to the MOSC superfamily. Homology analysis, we cloned the "Fenghua 1" AhLOS5 and peanut wild diploid species in A.duranensis AdLOS5 the highest homology reached 94.7%, compared to 92.2%. and AiLOS5 homology with other higher plants, and AhLOS5 belong to the legume narrow leaved lupine, Ammopiptanthus nanus, Dal, homology, respectively 77.7%, 73.7% and 72.9% homology with the Arabidopsis Cruciferae family of slightly lower, 59.9%. (3) to AhLOS5 cDNA nucleotide fragment probe preparation was analyzed in 4 main varieties in the expression of AhLOS5 were investigated. According to AhLOS5, in the 4 main varieties in order for the expression of "Huayu 20" and "Fenghua 1" Haihua No. 1 "Baisha 1016 '. We found that the AhLOS5 gene, 4 varieties of drought resistance and expression of sort order is the same. The validation of the gene Drought resistance in peanut. (4) using the method of Northern hybridization analyses' chongban expression characteristics of No. 1 "in AhLOS5. The results showed that the expression of AhLOS5 in peanut constitutively expressed in various organs of peanut order flowers mature leaves of young fruit needle roots, the expression in leaves and increased with the age increasing, the expression of 50D in leaf expansion reached the peak of.AhLOS5 is also affected by drought and salt stress induced induced obviously. (5) constructed 2 plant expression vector, the vector pBI-LOS5 containing AhLOS5 and justice containing antisense AhLOS5 vector pBI-R-LOS5. to three tobacco transformation, using the method of agricultural Agrobacterium mediated, the 2 vectors were transformed into different carriers. Contemporary transgenic tobacco (T0 generation) with seeds selected by kanamycin, preliminary determination combined with transgenic T1 plants PCR identification results, containing antisense AhL There were 20 strains of OS5 transgenic plants, transgenic plants containing just 52 strains were AhLOS5, and selected parts of the different types of transgenic plants were analyzed by Northern blot analysis. (6) by transgenic tobacco seed root length, chlorophyll content, relative water content, relative conductivity, malondialdehyde (MDA), ascorbic acid (ASA), anti oxidase activity (SOD, POD, CAT, APX), endogenous ABA, detection of proline and other physiological and biochemical indexes, under drought and salt stress, the expression of AhLOS5 in transgenic tobacco transgenic plants with endogenous ABA content significantly increased, antioxidant enzyme activity increased, membrane lipid integrity, osmotic adjustment substance content is high. Overexpression of AhLOS5 can significantly improve the transgenic lines resistant to drought and salt stress. (7) in the other 3 of the transgenic tobacco resistance related gene DREB2B, RD22 Northern blot method was used to detect the expression of P5CS. The results showed, Under drought and salt stress, 3 genes, DREB2B, transgenic and wild type controls were RD22, P5CS induced expression; antisense transgenic plants not only depend on the ABA resistance gene expression induced by DREB2B, ABA dependent resistance genes RD22 and P5CS are not induced. This proves that AhLOS5 participate in the plant resistance belongs to the ABA dependent pathway, overexpression of AhLOS5 in transgenic tobacco resistance is produced by the regulation of endogenous ABA induced by stress.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2;S565.2

【相似文献】