转EuCHIT1基因提高小麦对条锈病的抗性
发布时间：2018-08-02 16:44
【摘要】：小麦条锈病是一种严重的真菌病害,导致小麦(Triticum aestivum)产量和品质的下降,探索利用外源基因提高条锈病抗性对小麦育种具有重要意义。本研究利用农杆菌(Agrobacterium tumefaciens)介导的遗传转化法将杜仲几丁质酶基因(eucommia ulmoides chitodextrinase gene,EuCHIT1)遗传转化小麦品种"贵紫3",通过β葡萄糖醛酸酶(β-glucuronidase,GUS)组织化学染色和PCR鉴定获得T1代转基因小麦植株,对转基因和野生型小麦的几丁质酶活性、保护性酶活性、条锈病抗性以及病程相关蛋白基因相对表达量比较分析。结果表明,转基因小麦几丁质酶活性平均为3 328.63 U/g FW,比野生型高42.21%。接种条锈菌(Puccinia striiformis f.sp.tritici)小种CYR32后7 d,转基因小麦过氧化氢酶(catalase,CAT)、超氧化物歧化酶(super-oxide dismutase,SOD)和过氧化物酶(peroxidase,POD)平均活性分别为211.91、448.37和81.30U/g FW,野生型植株CAT、SOD和POD分别为159.95、294.38和37.87 U/g FW,转基因植株比野生型分别高32.48%、49.76%和114.68%;转基因小麦丙二醛(malondialdehyde,MDA)含量平均为8.69 nmol/g FW,比野生型低29.23%(12.28 nmol/g FW)。对野生型和转基因小麦接菌后,转基因小麦叶片发病时间比野生型推迟了9 d,接菌14 d后对小麦叶片条锈病进行抗性鉴定,结果表明,转基因小麦表现为高抗,野生型小麦表现为中感。小麦旗叶病变长度统计结果显示,转基因植株旗片病变长度极显著低于野生型。病程相关蛋白基因相对表达量分析表明,接菌前转基因小麦中病程相关蛋白1(pathogenesis-related protein,PR-1)、病程相关蛋白2(pathogenesis-related protein,PR-2)和病程相关蛋白5(pathogenesis-related protein,PR-5)基因表达量平均为野生型1.14、6.61和3.87倍,接菌后转基因小麦表达量平均为野生型的2.14、3.41和7.55倍。综上所述,转基因小麦提高了对条锈病抗性,可能与保护性酶活性的提高以及病程相关蛋白基因表达上调有关。本研究为创制抗条锈病转基因小麦材料提供了基础,同时为进一步研究EuCHIT1基因功能机制提供理论依据。
[Abstract]:Wheat stripe rust is a serious fungal disease, which leads to the decrease of yield and quality of wheat (Triticum aestivum). It is important for wheat breeding to explore the use of exogenous genes to improve the resistance to stripe rust. In this study, the genetic transformation of eucommia chitinase gene (eucommia ulmoides chitodextrinase gene Eugene EuCHIT1 was carried out by Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediated genetic transformation into wheat variety "Guizin3". The transgenic plants of T1 generation were obtained by histochemical staining and PCR identification of 尾 -glucuronidase Gus. Chitinase activity, protective enzyme activity, stripe rust resistance and relative expression of pathogenesis-related protein genes in transgenic and wild-type wheat were compared and analyzed. The results showed that the average chitinase activity of transgenic wheat was 3 328.63 Ur / g FW, which was 42.21 higher than that of wild type. The average activities of catalase (cat), superoxide dismutase (super-oxide dismutase) and peroxidase (peroxidase) in transgenic wheat were 211.91448.37 and 81.30U/g FWrespectively at 7 days after inoculation with CYR32, and CATH-SOD and POD of wild-type plants were 159.95294.38 and 37.87 UgFWrespectively. The content of malondialdehyde (MDA) in transgenic wheat was 8.69 nmol/g FW, which was 29.23% (12.28 nmol/g FW).) lower than that of wild type. After inoculation of wild type and transgenic wheat, the onset time of transgenic wheat leaf was delayed by 9 days than that of wild type, and the resistance to stripe rust of wheat leaf was identified 14 days after inoculation. The results showed that transgenic wheat showed high resistance to stripe rust. Wild type wheat showed middle sense. The results showed that the lesion length of flag leaf of transgenic plants was significantly lower than that of wild type. The relative expression of pathogenesis-related protein gene in transgenic wheat before inoculation was 1.146.61 and 3.87 times higher than that in wild-type wheat. The average expression of transgenic wheat after inoculation was 2.14 ~ 3.41 and 7.55 times of that of wild type. In conclusion, transgenic wheat increased resistance to stripe rust, which may be related to the increase of protective enzyme activity and the up-regulation of pathogenesis-related protein gene expression. This study provides a basis for the creation of transgenic wheat materials resistant to stripe rust, and provides a theoretical basis for further study on the functional mechanism of EuCHIT1 gene.
【作者单位】： 贵州大学农业生物工程研究院/生命科学学院/山地植物资源保护与种质创新省部共建教育部重点实验室;贵州省农业科学院;
【基金】：国家高技术研究发展计划(863)项目(No.2013AA102605-05) 国家自然科学基金项目(No.31360272) 国家转基因生物新品种培育科技重大专项(No.2016ZX08010003-009)
【分类号】：S435.121.42
，
本文编号：2160052
[Abstract]:Wheat stripe rust is a serious fungal disease, which leads to the decrease of yield and quality of wheat (Triticum aestivum). It is important for wheat breeding to explore the use of exogenous genes to improve the resistance to stripe rust. In this study, the genetic transformation of eucommia chitinase gene (eucommia ulmoides chitodextrinase gene Eugene EuCHIT1 was carried out by Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediated genetic transformation into wheat variety "Guizin3". The transgenic plants of T1 generation were obtained by histochemical staining and PCR identification of 尾 -glucuronidase Gus. Chitinase activity, protective enzyme activity, stripe rust resistance and relative expression of pathogenesis-related protein genes in transgenic and wild-type wheat were compared and analyzed. The results showed that the average chitinase activity of transgenic wheat was 3 328.63 Ur / g FW, which was 42.21 higher than that of wild type. The average activities of catalase (cat), superoxide dismutase (super-oxide dismutase) and peroxidase (peroxidase) in transgenic wheat were 211.91448.37 and 81.30U/g FWrespectively at 7 days after inoculation with CYR32, and CATH-SOD and POD of wild-type plants were 159.95294.38 and 37.87 UgFWrespectively. The content of malondialdehyde (MDA) in transgenic wheat was 8.69 nmol/g FW, which was 29.23% (12.28 nmol/g FW).) lower than that of wild type. After inoculation of wild type and transgenic wheat, the onset time of transgenic wheat leaf was delayed by 9 days than that of wild type, and the resistance to stripe rust of wheat leaf was identified 14 days after inoculation. The results showed that transgenic wheat showed high resistance to stripe rust. Wild type wheat showed middle sense. The results showed that the lesion length of flag leaf of transgenic plants was significantly lower than that of wild type. The relative expression of pathogenesis-related protein gene in transgenic wheat before inoculation was 1.146.61 and 3.87 times higher than that in wild-type wheat. The average expression of transgenic wheat after inoculation was 2.14 ~ 3.41 and 7.55 times of that of wild type. In conclusion, transgenic wheat increased resistance to stripe rust, which may be related to the increase of protective enzyme activity and the up-regulation of pathogenesis-related protein gene expression. This study provides a basis for the creation of transgenic wheat materials resistant to stripe rust, and provides a theoretical basis for further study on the functional mechanism of EuCHIT1 gene.
【作者单位】： 贵州大学农业生物工程研究院/生命科学学院/山地植物资源保护与种质创新省部共建教育部重点实验室;贵州省农业科学院;
【基金】：国家高技术研究发展计划(863)项目(No.2013AA102605-05) 国家自然科学基金项目(No.31360272) 国家转基因生物新品种培育科技重大专项(No.2016ZX08010003-009)
【分类号】：S435.121.42
，
本文编号：2160052
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2160052.html
最近更新
教材专著
