摘要
以转反义TrxS基因株系01TY18、01TY70、01TY34和00T89的T5代和非转基因品种为材料,运用PCR和相关生理生化指标测定的方法,研究了外源基因在不同株系中的整合、遗传稳定性及转基因小麦穗发芽的特性。在此基础上,选用纯合株系,系统研究了转基因小麦品质特性以及转基因小麦在重金属锌离子胁迫下的抗逆性。主要研究结果如下:
1转反义Trxs基因后代的分子鉴定及遗传分析
利用PCR检测方法,对转基因小麦01TY18、01TY70、01TY34 T4代植株检测呈阳性的23个转基因株系跟踪检测,共有21个株系的单株出现了与质粒片段大小相同的条带。经对检测有目标片段的21个T5代转基因株系1000个单株的PCR检测表明,阳性率占到85%以上,说明,外源TrxS基因已经整合到小麦基因组中,并且能够稳定的遗传到后代。
2人工模拟降雨条件下转基因株系穗发芽抗性研究
人工模拟降雨发芽试验结果证明,反义TrxS基因使转基因小麦01TY18、01TY70、01TY34大部分株系的穗发芽率都较对照有所降低,平均分别比对照下降了33.2%、20.7%和44.6%。延缓了转基因麦穗及籽粒的发芽时间,其抑制效应主要体现在发芽前期,且效应随着萌发时间延长而降低,萌发后期对照与转基因穗发芽差异主要表现在发芽度上。
3转基因对中筋小麦的品质有改善作用
转基因小麦01TY18、01TY70、01TY34和对照间清蛋白、球蛋白、醇溶蛋白含量差异较小,而谷蛋白含量差异显著。转基因使豫麦18和豫麦70的延伸性及拉伸阻力与对照相比有所提高,而弱化度则显著减小;而01TY34与非转基因材料相比延伸性及拉伸阻力反而有所下降,说明转基因能使中筋小麦豫麦18和豫麦70的延伸性及拉伸阻力有所提高,对强筋小麦豫麦34反而有使其延伸性和抗拉伸阻力减小的作用。
4锌离子胁迫下转基因小麦具有一定的抗性
重金属锌离子胁迫处理下,转基因小麦的芽长、根长、根条数都比对照表现好;转基因小麦氧自由基活性明显低于对照;转基因小麦籽粒在萌发阶段受重金属离子胁迫后反义Trxh表达能引起过氧化物酶活性升高;从萌发6h开始到96h转基因籽粒淀粉酶活性高于对照,说明在重金属胁迫后转基因小麦的萌动状态高于对照;经重金属锌离子胁迫处理的转基因和对照小麦籽粒在萌发过程谷胱甘肽过氧化物酶活性都呈现先平稳后突然升高再降低的趋势,但在萌发120h期间转基因酶活始终高于对照;在胁迫24h之后转基因材料的硫氧还蛋白活性一直大于对照,说明转基因材料在重金属锌离子胁迫环境中的抗逆作用大于对照材料。
Take the foreign antisense-trxs gene in different transgenic line 01TY18, 01TY70, 01TY34 ,00T89 T5 generation and their non-transgene variety as the material, Using PCR and related physiological biochemistry indicators as the determination method. We have studied the germinates characteristic , conformity and heredity stability of the exogenous gene in different transgene line。Based on the above result ,the quality character of antisence-Trxs gene transgenetic wheat and the change of seed physiology characteristic in Zn2+stress in transgenic homozygous line and non-transgenic seeds of wheat were systematically studied. The main results were summerized as followings:
1. Molecular detection and genetic analysis of transgenic progenies
Useing the PCR examination method,We assume the masculine to the transgene wheat 01TY18、01TY70、01TY34 T4 generation of adult plant examination 23 transgene strain track examination. There were 21 strain's adult plant appeared with the material particle fragment size same banding.We had interese fragment 21 T5 forward on behalf of someone gene strain 1000 single to the examination to carry on the PCR examination,the masculine gender rate occupies 85% above,which indicated that extraneous source TrxS gene already conformity to wheat genome team, and can stabilize heredity descendant。
2. Under the artificial simulation rainfall condition the resistant germination characteristic research of transgene strain
Artificial simulation rainfall germination test result proof that the antisense-trxs gene causes majority of strain transgene wheat 01TY18, 01TY70 and 01TY34 germination percentage to have reduced with their wild types,dropped 33.2%,20.7% and 44.6% separately equally compared to the comparison, delayed the germinating time of transgene wheat ear and the grain,Its inhibitory action mainly manifests the germination earlier period and the effect reduces along with the germination time expand. The difference germinates of compares and transgene in the germination later period mainly to display on germination.
3.Transgene has improvement function to the strong muscle wheat's quality
Transgene wheat 01TY18, 01TY70, 01TY34 and the comparisons the albumen, the globulin, the prolamin content difference is small but the prolamine content achieves the remarkable difference. The transgene caused the extensibilities and the attenuation of Yumai 18 and Yumai70 improve compared with the wild type though the attenuation obviously reduces. 01TY34 compared the extensibilities and the attenuation with the wild type material instead to drop. Explained that the transgene can cause the extensibility and the stretch resistance improves to the muscle wheat Yumai 18 and Yumai70. The transgene can cause the function which make the strong muscle wheat Yumai 34 extensibility and the anti-stretch resistance reduce.
4.The transgene wheat has certain resistance to the zinc ion coercion
Under heavy metal zinc ion coercion processing the transgene wheat's bud, root and root displays good compared with the comparison. Transgene wheat oxygen free radical activeness is lower than the wild type obviously. The antisense-trxs gene expression can improve the peroxide enzyme activity in the germination stage transgene wheat grain under the heavy metal zinc ion coercion. From sprouts 6 hours to start to the 96 hours transgene grain starch enzyme activity to be higher than the wild type. Explanation after heavy metal coercion the transgene wheat's germination is higher than the wild type. After heavy metal zinc ion coercion processing the Guguangantai peroxide enzyme activity of the transgene and the wild type wheat grain is first steady then suddenly elevates and then reduces in the germination process but the enzyme activity of the transgene wheat is higher than the wild type throughout the sprouting process of 120 hours ; After forcing 24 hours, the thioredoxin activeness of transgene material has also been higher than the wild type. Explained that the resistance function of transgene material is higher than the wild type material in heavy metal zinc ion coercion environment.
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