【作者】 陈红敏；

【导师】 魏安智；

【作者基本信息】 西北农林科技大学 ， 林木遗传育种， 2010， 硕士

【摘要】 前期工作已克隆与抗逆、抗病相关的基因GmAREB(ABA应答元件结合蛋白基因),EdVP1(焦磷酸化酶基因),GmERF46(乙烯应答元件结合蛋白基因)。基因功能分析证明GmAREB可以提高转基因植物的抗旱性及耐盐性,EdVP1可以促进转基因植物对磷、钾等营养元素的吸收,GmERF46可以提高转基因植物的抗病性。为了提高我国小麦品种的抗逆性及抗病性,本研究利用以上基因分别构建载体转化我国主栽小麦品种,转基因小麦的分子鉴定及抗性鉴定结果如下:利用玉米的Ubiqutin启动子控制GmAREB基因表达,构建了用于小麦转化的载体pSK-GmAREB。采用基因枪共转化法转化受体小麦品种郑147和济麦22。通过PCR检测获得T0代的阳性植株70株,转化率为1.37%。其中,郑147阳性植株共31株,转化率为2.14%,济麦22阳性植株39株,转化率为1.08%。通过PCR跟踪鉴定获得T1代转基因株系18个,其中,以郑147为受体的株系4个;以济麦22为受体的株系14个。对部分株系进行Southern blotting分析,证实GmAREB基因已经整合到小麦基因组中。抗逆性鉴定证明在低温胁迫条件下,3个以济麦22为受体的转基因株系的脯氨酸含量与受体小麦相比有显著增加,证明在小麦中过表达GmAREB基因,可以促进渗透调节物质脯氨酸的积累,脯氨酸的积累可能有助于转基因小麦抗逆性的提高。利用从披碱草中克隆的焦磷酸化酶基因(EdVP1基因)构建载体转化小麦受体郑147、济麦20和杨12。通过PCR检测共获得T0代的阳性植株75株,转化率为2.12%。其中,以郑147为转化受体的阳性植株64株,转化率为3.28%;以济麦20为转化受体的阳性植株3株,转化率为0.29%;以杨12为转化受体的阳性转基因植株11株,转化率为2.02%。通过T1代转基因株系PCR鉴定,获得13个转基因株系。其中,来自郑州147的转基因株系8个,来自杨12的转基因株系5个。T2代转基因小麦阳性植株抗性鉴定证明:在高盐条件下,转基因小麦和受体根系随NaCl浓度的增大,根的总长、总表面积、总体积以及总根尖数下降,在同浓度下转基因小麦根系比受体根系发达,根的总长、总表面积、总体积以及总根尖数比受体明显增加。受体及转基因小麦根的平均直径随NaCl浓度的增加而增大,受体根的平均直径比转基因小麦增加明显;在低钾条件下,转基因小麦根系K+浓度高于受体根系。在低钾和正常钾处理条件下,转基因小麦相对于受体根系总长度、根系总表面积显著增加,而根系平均直径、根系总体积增加不明显,转基因小麦相对于受体在细根和粗根的根长、根系总表面积及体积比方面增加显著;在转EdVP1基因小麦抗寒性鉴定中,分别检测郑147(抗寒性差)、杨12两个受体的转基因材料,在低温条件下,受体为郑147的转基因小麦脯氨酸含量比对照提高58.2%,受体为杨12转基因小麦脯氨酸含量提高9.9%,证明相同基因在不同受体品种中脯氨酸提高量不同,抗寒性差的品种脯氨酸含量提高幅度较大;在干旱条件下,转基因小麦比受体的成活率高。同时,RT-PCR检测证明EdVP1基因在转基因小麦中已表达。利用玉米的Ubiqutin启动子控制GmERF46基因表达构建载体,采用基因枪共转化法将载体转化到小麦品种郑147、杨12和杨15。经PCR鉴定获得T0代阳性植株18株,转化率为0.76%。其中,以郑147为转化受体的阳性植株6株,转化率为0.44%;以杨12为转化受体的阳性植株6株,转化率为1.13%;以杨15为转化受体的阳性植株6株,转化率为1.12%。对T1代转基因株系进行检测,获得4个PCR阳性株系。其中,来自郑147的转基因株系1个,来自杨12的转基因株系3个。白粉病抗性鉴定证明转基因小麦对白粉病具有一定抗性。综上所述,本研究通过基因枪法成功将抗逆、抗病相关的3个基因转化我国主栽小麦品种,通过PCR及Southern blot鉴定证明这些基因已经整合到小麦基因组中,并且可以稳定遗传到下一代。抗逆性鉴定证明转基因小麦株系的抗逆性或抗病性相对于受体对照得到不同程度提高。本研究为进一步筛选遗传稳定的抗逆、抗病转基因小麦新材料奠定了基础。更多还原

【Abstract】 Previously, we had isolated three stress-related or pathogen-resistent genes, namely: GmAREB (ABA responsive element binding protein), EdVP1 (H + pyrophosphorylase) and GmERF46 (ethylene responsive element binding protein). Functional analysis indicated that overexpression of GmAREB can enhance tolerance of transgenic plants to drought or high salt streeses, and expression of EdVP1 can improve uptake of phosphorus or passium mineral in transgenic plants, expression of GmERF46 improves pathogen resistant in transgenic plants. In order to improve biotic or abiotic stresses resistant of Chinese wheat varieties, we plan to transform those three genes into wheat in this research. The moleculer and resistant analysis results were following:In this study, GmAREB was inserted in downstream of maize Ubiqutin promoter to construct vector, pSK-GmAREB, which was transformed into wheat varieties, Zheng 147 and Jimai 22 using biolistic. After transformation, 70 T0 transgenic plants were identified using PCR and the transformational efficiency was 1.37%. Among transgenic plants, 31 plants came from host Zheng 147 and the transformational efficiency was 2.14%, and 39 plants came from host Jimai 22 and the transformational efficiency was 1.08%. So far, 18 of T1 transgenic lines were identificated. Among those transgenic plants, 4 plants came from host Zheng 147 and 14 plants came from host Jimai 22. The southern blot proved that GmAREB was inserted into wheat genome. The functional analysis showed that under low temperature stress condition, proline accumulation in three transgenic plants hosted by Jimai 22 were higher than that in wild type plants, which suggested that overexpression of GmAREB increased accumulation of proline in transgenic wheat, which might be contributed for stress-tolerance of transgenic wheat plants.Similarly, the EdVP1 gene was inserted in downstream of maize Ubiqutin promoter to construct vector and was transformed into wheat varieties, Zheng 147, Jimai 20 and Yang 12 using biolistic. 75 transgenic plants were identificated by PCR in T0 generation plants and totally transformational efficiency was 2.12%. Among those transgenic plants, 64 plants came from host Zheng 147 and the transformational efficiency was 3.28%, and 3 plants came from host Jimai 20 and the transformational efficiency was 0.29%, and 11 plants came from host Yang 12 and the transformational efficiency was 2.02%. In T1 transgenic plants, totally 13 plants were obtained. Among those T1 transgenic plants, 8 transgenic plants came from Zheng 147 and 5 plants came from host Yang 12. Founctional analysis for T2 transgenic plants proven that under high salt stress codition, the total length and face area of root system, and total root tip number in both transgenic and wild type wheat plants increased following with increasing of salt concentration. Under same concentration salt treatment, root of transgenic wheat grew more healthly than that in wild type, and total length, total face area, total volume of root system and total root tip number increased remarkly comparing with wild type. Average diameter of roots increased fowllowing with increasing of salt concentration in both transgenic plant and wild type, whereas average diameter of wild type increased more remarkly than that in transgenic plants. Under starvation of passium condition, K+ content in transgenic plants root were higher than that in wild type. Under starvation or nomal of passium condition, total length and total face area increased more remarkly comparing with wild type, whereas average diameter of roots and total volume of root system were same between transgenic plants and wild type. Root length, total root erea and ratio of root volume in transgenic plants increased more remarkly than that in wild type. Two type of EdVP1 transgenic wheat plants hosted by Zheng 147 (sensitive to low temperature stress) and Yang 12, separately were used to test low temperature resistance. Results indicated that under cold stress, accumulated proline content in transgenic plants hosed by Zheng 147 was 58.2% higher than that in wild type, and accumulated proline content in Yang 12 transgenic plants was 9.9% higher than that in wild type, which suggesting that different host wheat varieties transformed with same gene accumulated different content of proline and cold sensitive host wheat variety accumulated higher level of proline comparing with cold un-sensitive host wheat variety. Under drought condition, survive rate of transgenic plants was higher than that of wild type. Morever, results of RT-PCR proven that EdVP1 can be transcripted in transgenic plants.In this study, GmERF46 under control of maize ubiqutin promoter was used to constructe vector and were transformed into wheat varieties Zheng 147, Yang 12 and Yang 15. 18 T0 transgenic plants were identificated using PCR and the transformational efficiency was 0.76%. Among those transgenic plants, 6 plants came from host Zheng 147 and the transformational efficiency was 0.44%; 6 plants came from Young 12 and the transformational efficiency was 1.13%; 6 plants came from Young 15 and the transformational efficiency was 1.12%. Total 4 T1 transgenic plants were identificated using PCR. Among those transgenic plants, 1 plant came from host Zheng 147 and 3 plants came from the Yang 12. Pathogen resistant analysis indicated that GmERF46 transgenic wheat showed resistant to wheat powdery mildew.In short, three stress-resistent and pathogen-resistant genes were transformed in Chinese wheat varieties and those genes were identificated to insert wheat genome and inherited to next generation using PCR or Southern blot analysis. Founctional analysis indicated that overexpression of those genes in host wheat imropved stress-tolerance or pathogen-resistant of transgenic wheat plants. This study was nice beginning of further screening of the steady and reliable abiotic or biotic resistant transgenic wheat lines in the further.更多还原