轉(zhuǎn)EuCHIT1基因提高小麥對條銹病的抗性
發(fā)布時間：2018-08-02 16:44
【摘要】：小麥條銹病是一種嚴(yán)重的真菌病害,導(dǎo)致小麥(Triticum aestivum)產(chǎn)量和品質(zhì)的下降,探索利用外源基因提高條銹病抗性對小麥育種具有重要意義。本研究利用農(nóng)桿菌(Agrobacterium tumefaciens)介導(dǎo)的遺傳轉(zhuǎn)化法將杜仲幾丁質(zhì)酶基因(eucommia ulmoides chitodextrinase gene,EuCHIT1)遺傳轉(zhuǎn)化小麥品種"貴紫3",通過β葡萄糖醛酸酶(β-glucuronidase,GUS)組織化學(xué)染色和PCR鑒定獲得T1代轉(zhuǎn)基因小麥植株,對轉(zhuǎn)基因和野生型小麥的幾丁質(zhì)酶活性、保護性酶活性、條銹病抗性以及病程相關(guān)蛋白基因相對表達量比較分析。結(jié)果表明,轉(zhuǎn)基因小麥幾丁質(zhì)酶活性平均為3 328.63 U/g FW,比野生型高42.21%。接種條銹菌(Puccinia striiformis f.sp.tritici)小種CYR32后7 d,轉(zhuǎn)基因小麥過氧化氫酶(catalase,CAT)、超氧化物歧化酶(super-oxide dismutase,SOD)和過氧化物酶(peroxidase,POD)平均活性分別為211.91、448.37和81.30U/g FW,野生型植株CAT、SOD和POD分別為159.95、294.38和37.87 U/g FW,轉(zhuǎn)基因植株比野生型分別高32.48%、49.76%和114.68%;轉(zhuǎn)基因小麥丙二醛(malondialdehyde,MDA)含量平均為8.69 nmol/g FW,比野生型低29.23%(12.28 nmol/g FW)。對野生型和轉(zhuǎn)基因小麥接菌后,轉(zhuǎn)基因小麥葉片發(fā)病時間比野生型推遲了9 d,接菌14 d后對小麥葉片條銹病進行抗性鑒定,結(jié)果表明,轉(zhuǎn)基因小麥表現(xiàn)為高抗,野生型小麥表現(xiàn)為中感。小麥旗葉病變長度統(tǒng)計結(jié)果顯示,轉(zhuǎn)基因植株旗片病變長度極顯著低于野生型。病程相關(guān)蛋白基因相對表達量分析表明,接菌前轉(zhuǎn)基因小麥中病程相關(guān)蛋白1(pathogenesis-related protein,PR-1)、病程相關(guān)蛋白2(pathogenesis-related protein,PR-2)和病程相關(guān)蛋白5(pathogenesis-related protein,PR-5)基因表達量平均為野生型1.14、6.61和3.87倍,接菌后轉(zhuǎn)基因小麥表達量平均為野生型的2.14、3.41和7.55倍。綜上所述,轉(zhuǎn)基因小麥提高了對條銹病抗性,可能與保護性酶活性的提高以及病程相關(guān)蛋白基因表達上調(diào)有關(guān)。本研究為創(chuàng)制抗條銹病轉(zhuǎn)基因小麥材料提供了基礎(chǔ),同時為進一步研究EuCHIT1基因功能機制提供理論依據(jù)。
[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.
【作者單位】： 貴州大學(xué)農(nóng)業(yè)生物工程研究院/生命科學(xué)學(xué)院/山地植物資源保護與種質(zhì)創(chuàng)新省部共建教育部重點實驗室;貴州省農(nóng)業(yè)科學(xué)院;
【基金】：國家高技術(shù)研究發(fā)展計劃(863)項目(No.2013AA102605-05) 國家自然科學(xué)基金項目(No.31360272) 國家轉(zhuǎn)基因生物新品種培育科技重大專項(No.2016ZX08010003-009)
【分類號】：S435.121.42
，
本文編號：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.
【作者單位】： 貴州大學(xué)農(nóng)業(yè)生物工程研究院/生命科學(xué)學(xué)院/山地植物資源保護與種質(zhì)創(chuàng)新省部共建教育部重點實驗室;貴州省農(nóng)業(yè)科學(xué)院;
【基金】：國家高技術(shù)研究發(fā)展計劃(863)項目(No.2013AA102605-05) 國家自然科學(xué)基金項目(No.31360272) 國家轉(zhuǎn)基因生物新品種培育科技重大專項(No.2016ZX08010003-009)
【分類號】：S435.121.42
，
本文編號：2160052
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