表达播娘蒿突变基因DsALS-108的抗苯磺隆甘蓝型油菜植株构建

发布时间：2018-04-10 03:03

  本文选题：乙酰乳酸合成酶突变基因(ALS)　切入点：播娘蒿　出处：《农业生物技术学报》2016年04期

【摘要】：利用化学杀雄剂如苯磺隆(tribenuron-methyl,TBM)除草剂诱导雄性不育,是甘蓝型油菜(Brassica napus)杂种优势应用的主要途径之一。选育具有TBM除草剂抗性的甘蓝型油菜作为父本,有利于简化制种程序,降低制种成本,对提高甘蓝型油菜的产量和品质具有重要意义。乙酰乳酸合成酶(acetolactate synthase,ALS)是合成支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)的关键酶。ALS抑制类除草剂以乙酰乳酸合成酶为靶标,通过抑制其活性阻碍支链氨基酸的合成,导致植株死亡。TBM属于一种磺酰脲类(sulfonylureas,SU)ALS抑制类除草剂。为了改良甘蓝型油菜抗除草剂的遗传特性,本研究首先从一个抗TBM除草剂的播娘蒿(Descurainia sophia)天然突变体(#108)中克隆了ALS基因(Gen Bank登录号:EU520490),命名为Ds ALS-108。和拟南芥(Arabidopsis thaliana)乙酰乳酸合成酶At ALS蛋白序列的比对结果显示,保守氨基酸位点脯氨酸197在突变型蛋白Ds ALS-108中替换为苏氨酸(即P197T)。为了验证含有P197T突变的Ds ALS-108基因是否使植株产生TBM除草剂抗性的原因,构建表达载体PBI121-Ds ALS-108,并通过农杆菌(Agrobacterium tumefaciens)介导法将该载体转化拟南芥,共获得12个阳性转基因株系。喷洒浓度大于等于1.0×10-4g/L TBM后,野生型植株停止生长,趋于死亡,而拟南芥转化植株均能正常生长,说明表达突变基因Ds ALS-108致使拟南芥植株抗TBM除草剂。在甘蓝型油菜转化中,以下胚轴为外植体,通过农杆菌介导法将载体p CAMBIA3301-Ds ALS-108导入甘蓝型油菜中,共获得23个阳性转基因株系。结果发现,Ds ALS-108的表达使甘蓝型油菜对苯磺隆的抗性提高至野生型致死浓度的3倍,即7.5×10-3g/L。而且,当用化学杀雄使用剂量(0.05 mg/L TBM)处理甘蓝型油菜转化植株后,其花粉育性正常,说明该转化植株可作为父本应用于化学诱导的雄性不育系统,进行杂交制种。本研究为利用化学诱导的雄性不育系统实现杂种优势提供新材料。
[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.
【作者单位】： 华中农业大学作物遗传改良国家重点实验室;
【基金】：湖北省自然科学基金(No.2014CFB945)
【分类号】：S565.4
，

本文编号：1729356