本文選題：乙酰乳酸合成酶突變基因(ALS)　切入點：播娘蒿　出處：《農(nóng)業(yè)生物技術(shù)學報》2016年04期

【摘要】：利用化學殺雄劑如苯磺隆(tribenuron-methyl,TBM)除草劑誘導(dǎo)雄性不育,是甘藍型油菜(Brassica napus)雜種優(yōu)勢應(yīng)用的主要途徑之一。選育具有TBM除草劑抗性的甘藍型油菜作為父本,有利于簡化制種程序,降低制種成本,對提高甘藍型油菜的產(chǎn)量和品質(zhì)具有重要意義。乙酰乳酸合成酶(acetolactate synthase,ALS)是合成支鏈氨基酸(纈氨酸、亮氨酸和異亮氨酸)的關(guān)鍵酶。ALS抑制類除草劑以乙酰乳酸合成酶為靶標,通過抑制其活性阻礙支鏈氨基酸的合成,導(dǎo)致植株死亡。TBM屬于一種磺酰脲類(sulfonylureas,SU)ALS抑制類除草劑。為了改良甘藍型油菜抗除草劑的遺傳特性,本研究首先從一個抗TBM除草劑的播娘蒿(Descurainia sophia)天然突變體(#108)中克隆了ALS基因(Gen Bank登錄號:EU520490),命名為Ds ALS-108。和擬南芥(Arabidopsis thaliana)乙酰乳酸合成酶At ALS蛋白序列的比對結(jié)果顯示,保守氨基酸位點脯氨酸197在突變型蛋白Ds ALS-108中替換為蘇氨酸(即P197T)。為了驗證含有P197T突變的Ds ALS-108基因是否使植株產(chǎn)生TBM除草劑抗性的原因,構(gòu)建表達載體PBI121-Ds ALS-108,并通過農(nóng)桿菌(Agrobacterium tumefaciens)介導(dǎo)法將該載體轉(zhuǎn)化擬南芥,共獲得12個陽性轉(zhuǎn)基因株系。噴灑濃度大于等于1.0×10-4g/L TBM后,野生型植株停止生長,趨于死亡,而擬南芥轉(zhuǎn)化植株均能正常生長,說明表達突變基因Ds ALS-108致使擬南芥植株抗TBM除草劑。在甘藍型油菜轉(zhuǎn)化中,以下胚軸為外植體,通過農(nóng)桿菌介導(dǎo)法將載體p CAMBIA3301-Ds ALS-108導(dǎo)入甘藍型油菜中,共獲得23個陽性轉(zhuǎn)基因株系。結(jié)果發(fā)現(xiàn),Ds ALS-108的表達使甘藍型油菜對苯磺隆的抗性提高至野生型致死濃度的3倍,即7.5×10-3g/L。而且,當用化學殺雄使用劑量(0.05 mg/L TBM)處理甘藍型油菜轉(zhuǎn)化植株后,其花粉育性正常,說明該轉(zhuǎn)化植株可作為父本應(yīng)用于化學誘導(dǎo)的雄性不育系統(tǒng),進行雜交制種。本研究為利用化學誘導(dǎo)的雄性不育系統(tǒng)實現(xiàn)雜種優(yōu)勢提供新材料。
[Abstract]:Male sterility induced by chemical androgenicides such as tribenuron-methyl TBMis is one of the main ways of heterosis application of Brassica napus. in Brassica napus.The breeding of Brassica napus with TBM herbicide resistance as male parent is beneficial to simplify seed production procedure and reduce seed production cost. It is of great significance to improve the yield and quality of Brassica napus.Acetolactate synthase (ALS) is a key enzyme in the synthesis of branched amino acids (valine, leucine and isoleucine). ALS inhibits the synthesis of branched amino acids by inhibiting the activity of acetolactate synthases.The result of plant death. TBM belongs to a sulfonylurea sulfonylurea supragalus inhibitory herbicide.In order to improve the genetic characteristics of herbicide resistance in Brassica napus, a natural mutant of Descurainia sophia, which is resistant to TBM herbicide, was cloned from ALS gene Genin Bank accession number: EU520490, named DS 108.The results of alignment with the ALS protein sequence of Arabidopsis thaliana showed that the conservative amino acid site proline 197 was replaced by threonine in the mutant protein Ds ALS-108.In order to verify whether Ds ALS-108 gene containing P197T mutation could induce TBM herbicide resistance in plants, the expression vector PBI121-Ds ALS-108 was constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens. Twelve positive transgenic lines were obtained.After spraying with concentration of 1.0 脳 10-4g/L TBM, wild-type plants stopped growing and tended to die, while Arabidopsis transformed plants could grow normally, indicating that the transgenic plants were resistant to TBM herbicides due to the expression of mutant gene Ds ALS-108.In the transformation of Brassica napus, the following cotyls were used as explants. The vector p CAMBIA3301-Ds ALS-108 was introduced into Brassica napus by Agrobacterium tumefaciens, and 23 positive transgenic lines were obtained.The results showed that the expression of ALS-108 increased the resistance of Brassica napus to bensulfuron by three times of wild-type lethal concentration, that is 7.5 脳 10 ~ (-3) g 路L ~ (-1) 路L ~ (-1).In addition, the pollen fertility of transformed plants of Brassica napus was normal when treated with chemical androgen (0. 05 mg/L TBM), which indicated that the transformed plants could be used as male parents in the chemically induced male sterile system for hybrid seed production.This study provides a new material for realizing heterosis by chemically induced male sterility system.
【作者單位】： 華中農(nóng)業(yè)大學作物遺傳改良國家重點實驗室;
【基金】：湖北省自然科學基金(No.2014CFB945)
【分類號】：S565.4
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本文編號：1729356