【作者】 陈梅；

【导师】 付凤玲；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 干旱是玉米生产的主要限制因素之一。玉米自身的水分生理状况也决定了它比其他禾本科作物更易受干旱危害。尽管玉米不同基因型间存在一定的耐旱性差异,但耐旱性受微效多基因控制,遗传机制复杂,传统的遗传改良难度较大。因此,借助转基因方法,将外源耐旱基因转入玉米,可能是有效提高玉米耐旱能力的快捷方法。近年来耐旱基因研究取得了较快进展,相继克隆了一些重要的耐旱基因,试图提高其耐旱性,但因这些基因耐旱能力不够强,耐旱机制与玉米生理代谢不协调等原因,转基因后代的耐旱性不能达到玉米生产要求(宋松泉等,2000;王关林等,2002;宋凤斌等,2005)。因此需要探索利用耐旱能力更高,适宜玉米生理代谢机制与生产要求的耐旱基因,海藻糖合成酶基因TPS1就是其中之一。TPS1合成的海藻糖在干旱条件下通过氢键与氨基酸相连,可以代替水起到防止蛋白质变性和细胞融合的作用(Crowe等,1992)。如果利用海藻糖的抗逆耐旱特性,采用基因工程将其相关酶的基因转入玉米等作物,进而培育耐旱抗逆新品种,这对于植物遗传改良具有重要意义。常用的玉米转基因方法多以胚性愈伤组织作为外源基因受体,而胚性愈伤组织的诱导和分化再生,严重受到基因型的限制,只有极少数材料能稳定高效诱导胚性愈伤组织,加之胚性愈伤组织培养时间长,操作繁琐,分化再生率不高等原因,严重制约了玉米转基因研究的进展。因此,有必要开展其他转基因方法研究,以提高转化效率。为此,本试验开展了以玉米幼胚为受体、用农杆菌介导法将酿酒酵母海藻糖合成酶基因TPS1转化玉米的研究,结果如下:1)以幼胚为受体转化外源基因,可缩短转化过程。以幼胚为转化受体,与含外源基因的农杆菌共培养后,不经过愈伤组织诱导和筛选,直接再生植株,减少基因型对愈伤组织为受体的转基因限制,避开组织培养的繁锁过程,缩短转化程序。2)以幼胚为受体转化外源基因的优化体系。由于幼胚对农杆菌浸染更敏感,尤其是褐化率比愈伤组织更高,造成转基因失败,为此试验通过对幼胚大小和抗褐化剂半胱氨酸(Cys)的浓度筛选,发现长度在1.5～2.0 mm的幼胚更适合农杆菌介导的幼胚转化,并且在农杆菌与幼胚的共培养阶段添加200 mg/L的半胱氨酸,能够有效降低幼胚在农杆菌浸染过程中的褐化率(29.3%),比对照褐化率(53.7%)减少约一倍。3)通过PCR检测,初步发现外源基因已转入受体。将转基因再生植株移栽后,生长至5叶期,每株取一小块叶片,按5株组成一个混合样,提取DNA,进行PCR特异扩增,有目的条带的再分单株检测,最后得到1株阳性转基因植株。按混样检测法可以提高阳性植株检测效率和准确性。更多还原

【Abstract】 Drought is the major limiting factor in the production of maize, and maize is much easier suffering from drought than other gramineous plants by the physiology of itself. The drought tolerance which is controlled by microeffect polygene are not the same in different gene type. It is bottlenecks in maize improvement. Therefore, transgenic operation is an useful technology to overcome reproductive isolation among species and utilize drought tolerance exotic genes.In recent years, a large number of drought tolerance genes are transferred into maize. But the transgenic plants can not fit the damand of maize production, because the anti-drought ability of the gene is not enough and it is not consistant with the metabolization of maize itself (Song et al., 2000; Wang et al., 2002; Song et al., 2005 ) . It is very important to make use of hight anti-drought ability of the gene which is more consistant with maize.Trehalos-6-phosphate synthase (TPS) is one of them for trait of adversity tolerance. Trehalose has been shown to stabilise proteins and membranes under stress conditions, especially during desiccation. By replacing water through hydrogen bonding to polar residues, trehalose prevents the denaturation of proteins and the fusion of membranes (Crowe J H et al., 1992) . So it’s very significant to transfer the related gene which has the strong ability of drought tolerance into maize.Abundant study on the genetic transformation of maize take embryonic callus induced from immature embryo as recipient. There were only few materials can highly and stable induce embryonic callus due to genetype. The research of mazie gene transform were restrict in long-playing cultureing, cockamamie manipulateing, low seedling rate.It is necessary to advance gene trasform rate by other recipient.Therefore, maize immature embryos were taken as recipient in this study ,and transformed by Agrobacterium tumefaciens EHA105 with TPS1 gene from saccharomyces cerevisiae.The resulta as following:1) The transform process is much shorter for takeing immature embryos as recipient. The immature embryos were concultured with Agrobacterium tumefaciens which contained exotic gene. The process of induceing immature embryos into embyronic calli and selected with antibiotic were cut down,bring immature embryos into seedling directly.That make study much shorter and easier than taking embyronic calli as recipient.2) Optimize the system which takes immature embryos as recipient. Immature embryos is sensitive to agrobacterium tumefaciens,and that make highly brown ratio and fail transform.This study optimize the system by selecting length of immature embryos and concentration of Cys, the results indicated that the 1.5-2.0 mm of immature embryos were better than other length, and adding Cys 200mg/L in coculture can reduce brown ratio (29.3%) effectively, almost half of antitheses (53.7%).3) Result of specific PCR test showed the exotic gene had been transformed in maize. Taking 5 plants as 1 mix-sample to extract DNA, after transplants grow to 5 leaves period .Then test DNA by specific PCR and repeat test if there was positive. Finally one plant was detected to be positive. It is more efficiently and accurately to test mix-sample.更多还原