抗凍基因轉(zhuǎn)擬南芥抗凍機理及在玉米自交系中表達初步研究
發(fā)布時間：2018-08-01 08:34
【摘要】：適宜溫度是作物生長、產(chǎn)量和品質(zhì)形成的重要環(huán)境因子。我國幅員遼闊,地形復(fù)雜,冷害和凍害常年發(fā)生,作物生產(chǎn)常因凍害而遭受巨大損失。玉米是全國乃至世界第一大糧食作物,提高其抗凍能力對于增加玉米產(chǎn)量和改善玉米品質(zhì)意義重大。利用基因工程技術(shù),將已有的抗凍基因直接轉(zhuǎn)入植物體內(nèi)使其過量表達,能夠直接有效的提高其抗凍能力,較傳統(tǒng)育種技術(shù),周期短,見效快。本試驗在前人研究基礎(chǔ)上,選擇7個抗逆候選基因的過表達擬南芥材料(總計22個株系)進行抗凍性鑒定,以哥倫比亞野生型和空載體型擬南芥作為對照,篩選出抗凍性較強的轉(zhuǎn)AT1G07890基因擬南芥株系,研究其基因?qū)M南芥的生長發(fā)育和生理代謝等的影響。擬南芥AT1G07890基因在玉米中的同源基因為GRMZM2G137839,通過對玉米自交系進行q RT-PCR,來觀察GRMZM2G137839基因在玉米抗低溫自交系(KR701、B125)以及玉米低溫敏感自交系(黑8834、昌7-2)凍害脅迫處理前后的基因表達情況,為開展玉米抗凍基因工程來提供有力理論的依據(jù)。試驗結(jié)果表明:(1)在7個抗逆候選基因的過表達擬南芥的材料中,轉(zhuǎn)AT1G07890基因型比其它轉(zhuǎn)基因型、野生型和空載體型擬南芥株系在凍害脅迫處理后存活率高,電導(dǎo)率和含水量低,說明其抗凍能力強。(2)在正常生長條件下,轉(zhuǎn)AT1G07890基因型與野生型和空載體型擬南芥的葉片及根系生長發(fā)育無顯著差異,說明轉(zhuǎn)AT1G07890基因?qū)M南芥的正常生長沒有影響。(3)轉(zhuǎn)AT1G07890基因型在4℃和0℃的低溫萌發(fā)試驗中均比野生型和空載體型擬南芥萌發(fā)更早,萌發(fā)率更高。(4)轉(zhuǎn)AT1G07890基因型葉綠素的含量在正常生長條件下與野生型和空載體型擬南芥幼苗無明顯的差異,凍害脅迫后轉(zhuǎn)基因型的葉綠素含量與之相比更高。(5)轉(zhuǎn)AT1G07890基因型MDA的含量在正常生長條件下與野生型和空載體型擬南芥幼苗無明顯的差異,凍害脅迫后轉(zhuǎn)基因型MDA的含量低于野生型和空載體型擬南芥幼苗的,說明其細胞膜受到的損害較輕。(6)轉(zhuǎn)AT1G07890基因型SOD和POD的活性在凍害脅迫處理前與野生型和空載體型擬南芥幼苗無顯著差異,脅迫后轉(zhuǎn)基因型SOD和POD的活性明顯高于野生型與空載體型擬南芥幼苗的,因為抗氧化酶系統(tǒng)可以提高植株抗凍的能力,說明轉(zhuǎn)AT1G07890基因提高了擬南芥幼苗抗凍的能力。(7)轉(zhuǎn)AT1G07890基因型可溶性糖和Pro的含量在凍害脅迫處理前與野生型和空載體型擬南芥幼苗是無顯著差異的,脅迫后轉(zhuǎn)基因型可溶性糖和Pro的含量明顯高于野生型與空載體型擬南芥幼苗的,因為滲透調(diào)節(jié)物質(zhì)可以提高植株抗凍的能力,說明轉(zhuǎn)AT1G07890基因提高了擬南芥幼苗抗凍的能力。(8)玉米自交系在凍害脅迫處理前,GRMZM2G137839基因在KR701、B125、黑8834和昌7-2玉米自交系幼苗中的表達量無明顯差異,KR701較高。凍害脅迫后,GRMZM2G137839在4份玉米自交系幼苗的表達量都升高了,抗凍玉米自交系品種KR701和B125上升幅度大,敏感玉米自交系品種黑8834和昌7-2上升幅度低,可見GRMZM2G13783基因在玉米自交系幼苗受到凍害脅迫時上調(diào)表達。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S513
本文編號：2156996
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S513
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