抗冻基因转拟南芥抗冻机理及在玉米自交系中表达初步研究

发布时间：2018-08-01 08:34

【摘要】：适宜温度是作物生长、产量和品质形成的重要环境因子。我国幅员辽阔,地形复杂,冷害和冻害常年发生,作物生产常因冻害而遭受巨大损失。玉米是全国乃至世界第一大粮食作物,提高其抗冻能力对于增加玉米产量和改善玉米品质意义重大。利用基因工程技术,将已有的抗冻基因直接转入植物体内使其过量表达,能够直接有效的提高其抗冻能力,较传统育种技术,周期短,见效快。本试验在前人研究基础上,选择7个抗逆候选基因的过表达拟南芥材料(总计22个株系)进行抗冻性鉴定,以哥伦比亚野生型和空载体型拟南芥作为对照,筛选出抗冻性较强的转AT1G07890基因拟南芥株系,研究其基因对拟南芥的生长发育和生理代谢等的影响。拟南芥AT1G07890基因在玉米中的同源基因为GRMZM2G137839,通过对玉米自交系进行q RT-PCR,来观察GRMZM2G137839基因在玉米抗低温自交系(KR701、B125)以及玉米低温敏感自交系(黑8834、昌7-2)冻害胁迫处理前后的基因表达情况,为开展玉米抗冻基因工程来提供有力理论的依据。试验结果表明:(1)在7个抗逆候选基因的过表达拟南芥的材料中,转AT1G07890基因型比其它转基因型、野生型和空载体型拟南芥株系在冻害胁迫处理后存活率高,电导率和含水量低,说明其抗冻能力强。(2)在正常生长条件下,转AT1G07890基因型与野生型和空载体型拟南芥的叶片及根系生长发育无显著差异,说明转AT1G07890基因对拟南芥的正常生长没有影响。(3)转AT1G07890基因型在4℃和0℃的低温萌发试验中均比野生型和空载体型拟南芥萌发更早,萌发率更高。(4)转AT1G07890基因型叶绿素的含量在正常生长条件下与野生型和空载体型拟南芥幼苗无明显的差异,冻害胁迫后转基因型的叶绿素含量与之相比更高。(5)转AT1G07890基因型MDA的含量在正常生长条件下与野生型和空载体型拟南芥幼苗无明显的差异,冻害胁迫后转基因型MDA的含量低于野生型和空载体型拟南芥幼苗的,说明其细胞膜受到的损害较轻。(6)转AT1G07890基因型SOD和POD的活性在冻害胁迫处理前与野生型和空载体型拟南芥幼苗无显著差异,胁迫后转基因型SOD和POD的活性明显高于野生型与空载体型拟南芥幼苗的,因为抗氧化酶系统可以提高植株抗冻的能力,说明转AT1G07890基因提高了拟南芥幼苗抗冻的能力。(7)转AT1G07890基因型可溶性糖和Pro的含量在冻害胁迫处理前与野生型和空载体型拟南芥幼苗是无显著差异的,胁迫后转基因型可溶性糖和Pro的含量明显高于野生型与空载体型拟南芥幼苗的,因为渗透调节物质可以提高植株抗冻的能力,说明转AT1G07890基因提高了拟南芥幼苗抗冻的能力。(8)玉米自交系在冻害胁迫处理前,GRMZM2G137839基因在KR701、B125、黑8834和昌7-2玉米自交系幼苗中的表达量无明显差异,KR701较高。冻害胁迫后,GRMZM2G137839在4份玉米自交系幼苗的表达量都升高了,抗冻玉米自交系品种KR701和B125上升幅度大,敏感玉米自交系品种黑8834和昌7-2上升幅度低,可见GRMZM2G13783基因在玉米自交系幼苗受到冻害胁迫时上调表达。
[Abstract]:Suitable temperature is an important environmental factor for crop growth, yield and quality formation. China has a vast territory, complex terrain, cold and frost damage, and crop production often suffers from great loss due to freezing damage. Corn is the largest grain crop in the whole country and in the world. It is significant to increase its frost resistance to increase corn yield and improve the quality of corn. It is important to use genetic engineering technology to overexpress the existing antifreeze gene directly into the plant and improve its anti freezing ability directly and effectively. Compared with the traditional breeding technology, it has short cycle and fast effect. On the basis of previous studies, this experiment selected 7 anti inversion candidates for over expression of Arabidopsis material (total 22 strains). Antifreeze identification, using Columbia wild type and no-load Arabidopsis as control, to screen out the anti freeze AT1G07890 transgenic Arabidopsis strain, and study the effect of its gene on the growth and physiological metabolism of Arabidopsis. The homologous gene of Arabidopsis AT1G07890 gene in maize is GRMZM2G137839, through the self inbreeding of corn. Q RT-PCR was carried out to observe the gene expression of GRMZM2G137839 gene before and after freezing injury of maize low temperature inbred line (KR701, B125) and maize low temperature sensitive inbred line (black 8834, 7-2), which provided a powerful theory for the development of maize frost resistance gene engineering. The results showed: (1) over 7 anti adverse candidate genes. In the materials expressing Arabidopsis, the AT1G07890 genotypes were higher than those of other transgenic, wild and unloaded Arabidopsis plants after freezing stress treatment, and their electrical conductivity and water content were low. (2) under normal growth conditions, the leaves and roots of Arabidopsis thaliana were transferred from AT1G07890 to wild and unloaded. There was no significant difference in growth and development, indicating that the transgenic AT1G07890 gene had no effect on the normal growth of Arabidopsis. (3) the transformation of AT1G07890 genotype at low temperature germination test at 4 and 0 C was earlier than that of wild and unloaded Arabidopsis, and the germination rate was higher. (4) the content of AT1G07890 based type chlorophyll was in normal growth conditions and field. There was no significant difference between the seedlings of Arabidopsis thaliana seedlings in the form of raw and unloaded, and the chlorophyll content of the transgenic type was higher than that of it after the frost stress. (5) the content of the transgenic AT1G07890 genotype MDA was not significantly different from the wild type and the empty type Arabidopsis seedlings under normal growth conditions, and the content of the transgenic MDA was lower than that of the wild type after the frost stress. (6) the activity of AT1G07890 genotype SOD and POD had no significant difference between the wild and no-load Arabidopsis seedlings before freezing stress treatment. The activity of transgenic SOD and POD after stress was significantly higher than that of wild type and unloaded Arabidopsis, because of resistance to the wild type and POD. The oxidase system could improve the anti freezing ability of the plant, indicating that the transgenic AT1G07890 gene enhanced the anti freezing ability of the Arabidopsis seedlings. (7) the content of AT1G07890 genotype soluble sugar and Pro had no significant difference between the wild type and the empty Arabidopsis thaliana seedlings before the frost stress treatment, and the content of transgenic soluble sugar and Pro after stress was not significant. The amount was significantly higher than that of the wild and unloaded Arabidopsis thaliana seedlings, because the osmotic regulation could improve the ability of the plant to resist freezing. It indicated that the transgenic AT1G07890 gene enhanced the anti freezing ability of the Arabidopsis seedlings. (8) the GRMZM2G137839 gene was in the seedlings of KR701, B125, black 8834 and chang7-2 maize inbred lines before the treatment of frost stress. There was no obvious difference in the expression of KR701. After the frost stress, the expression of GRMZM2G137839 in the 4 maize inbred lines increased, the KR701 and B125 of the inbred lines of the anti freezing maize increased greatly, the sensitive maize inbred lines black 8834 and the Chang 7-2 rose low, so the GRMZM2G13783 gene in the maize inbred lines was affected by the frost damage. The expression is up-regulated.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S513

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