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基因枪介导抗逆相关基因转化小麦的研究

标题: 基因枪介导抗逆相关基因转化小麦的研究
英文标题: Study on the Gene Transferring of Resistance-related Genes into Wheat by Bombardment
作者: 郭丽羡
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: 小麦,基因枪,遗传转化,抗逆转录因子
英文关键词: Wheat,Bombardment,Genetic transformation,Resistance-related Genes
论文级别: 硕士
学位: 学位论文
导师: 刘桂茹
专业: 植物资源学
提交时间: 2012
摘要: 干旱、盐渍和低温等非生物逆境对农业生产有着极其严重的影响.小麦是我国最重要的粮食作物之一,其抗逆性亟待提高.目前利用基因工程进行作物改良是较为有效的途径,而植物对干旱、盐渍及低温耐性的强弱往往不取决于某一单个因子,其性状大都是多基因控制的数量性状,受到许多因子的影响.利用单一基因,虽能在一定程度上改善植物的抗旱性或耐盐性,但不能使植物的抗逆性得到较为理想的综合改良.因此,从改良或增强一个关键的转录因子调控着手,是使植物抗逆性得到综合改良的有效的途径和方法.本文研究了利用基因枪对抗逆相关转录因子OsDREB2.2、EeAP2-1.1进行小麦遗传转化,以期获得转基因植株,为培育抗旱小麦品种奠定基础.主要研究结果如下:1.优化了小麦基因枪转化再生体系.通过对比9个不同冬小麦基因型的不同外植体(幼胚和幼穗)在不同诱导、分化和生根培养基下分化及成苗的差异,优化了小麦组织培养高效再生体系.高效的再生体系是基因转化的基本保障.以幼穗作为外植体,愈伤组织诱导率与幼胚相比无明显差异均接近100%,但是分化率明显低于幼胚,因此幼胚更适宜作为转化受体.本研究所用受体品种组培表现最好的为河农827,分化率为77.48%,最差的为良星99,分化率为31.20%.对于幼胚愈伤组织诱导来说,2 mg/L的2,4-D、0.5 mg/L的ABA、10 mg/L的VB1是必须的,谷氨酰胺作用不明显.愈伤组织分化过程ZT较KT作用好,ZT使用量以2 mg/L为佳.生根培养基中NAA必不可少,VB2并非必须.2.优化了基因枪转化体系.本研究在不同金粉用量(50μg、100μg、250μg、500μg)、轰击压力(1100 psi、1350 psi)轰击距离(6 cm、9 cm、12 cm)、轰击次数及轰击后恢复培养、干燥处理等条件下,用基因枪PDS-1000/He将OsDREB2.2基因转入不同基因型小麦幼胚愈伤组织,明确了以250μg/枪金粉、1100 psi轰击压力、9 cm轰击距离、轰击1次、恢复培养2周、干燥处理12 h,卡那霉素筛选浓度15 mg/L为本实验所选品种的较适宜基因枪转化体系.3.获得了转基因植株,并初步进行了遗传分析.利用所建体系进行了小麦基因枪OsDREB2.2、EeAP2-1.1基因的转化.得到OsDREB2.2及EeAP2-1.1转基因植株及后代.得到转OsDREB2.2基因小麦T0代植株19株,T1代35株,T2代20株行,得到转EeAP2-1.1基因植株T0代16株.OsDREB2.2基因可以遗传给后代,后代分离比率不完全符合孟德尔遗传定律,分离比为(0-1.31):1,小于孟德尔分离比3:1.
英文摘要: Drought, salinity, low temperature, all these abiotic stresses have adverse effects on agricultural production. Wheat is one of the most important food crops in China, and their resistance needs to be improved. Using genetic engineering for crop improvement is one of the effective way, The tolerance to drought, salinity, low temperature of plants does not only depend on single factor but multiple genes which controll the quantitative traits. Although, using single gene is able to improve drought resistance or salt tolerance to a certain extent, but it can't make comprehensive improvement resistance of plants .Therefore, improving or enhancing a key transcription factors is the effective ways for the comprehensive improvement of plant to biotic stresses. This study is on the transferring of resistance-related genes into wheat by bombardment, and the establishment of a transformation system for wheat regeneration system and bombardment.In this study, we have the following results:1. The optimization of the transformation system for wheat regeneration system. By comparing the nine winter wheat genotypes of different explants (immature embryos and immature tassel) in the different induction medium, differentiation and rooting medium, obsever the differentiation and seedling differences, we established an efficient wheat tissue culture regeneration system. Efficient regeneration system is the basic guarantee for genetic transformation.The immature tassels'callus induction rate is close to 100%,which is similar to immature embryo , but the differentiation rate of the two receptors is different ,the immature tassels is less than immature embryo, so the immature tassels are not suitable for conversion of the receptor, immature embryo can be used as the conversion of the receptor. Henong 827 had the best tissue cultural performance, the differentiation rate is 77.48%, the worst differentiation rate is 31.20% for the liangxin 99. 2 mg/L 2,4-D, 0.5 mg/L ABA, 10 mg/L VB1 are necessity for the immature embryo callus induction, while glutamine has little effect on it .In the process of callus differentiation, ZT is better than KT. 2 mg/L ZT is the proper concentration. NAA is essential to rooting medium, while VB2 is not the must. Kanamycin screening concentration should be at 15 mg/L and differentiation at the same time. 2. The establishment of bombardment transformation system In this study, we use different powder dosage (50μg, 100μg, 250μg, 500μg), bombardment pressure (1100 psi, 1350 psi) bombardment distance (6 cm, 9 cm, 12 cm), the times of bombardment, resumed culturing, dry culturing, etc. All these processing conditions used with Bombardment (PDS-1000/He) for gene-OsDREB2.2 transferred into immature embryo callus of different genotypes of wheat, According to the rate of green dot after the screening we established and optimized the bombardment transformation system. The results are as follows: 250μg/gun, 1100 psi, 9 cm, bombarding for 1time, a restored culture for two weeks, selected varieties of dried for 12 h.3. Obtained the transgenic plants and the genetic analysis of the OsDREB2.2 Using the system we have built, we obtained the OsDREB2.2 and EeAP2-1.1 transgenic plants.The number of the T0 period plants of OsDREB2.2 is 19, T1 period plants is 35 , lines of the T2 period is 20; the number of the T0 period plants of EeAP2-1.1 is 16. The OsDREB2.2 could transmit to their offsprings, the separation ratio of offspring does not meet the Mendel's principles, the separation ratio is (0-1.31):1, less than the Mendel's segregation ratio of 3:1.