辣椒高赖氨酸基因（CFLR）导入小麦的研究
摘要
小麦是重要的粮食作物之一,利用基因工程进行遗传改良的研究一直倍受人们关注。小麦的遗传转化还处于不断发展和完善阶段。以Alondra为材料,用直径0.6μm、1μm的金粉作为包裹介质,以直径小于0.5mm、1mm、大于1.5mm的幼胚作受体材料,1-7mg/L浓度的Basta筛选培养,以此建立起良好的再生休系和基因枪转化体系。
赖氨酸为人体必须氨基酸,小麦中赖氨酸含量较匮乏,因此降低了小麦的营养品质。而其他禾谷类作物赖氨酸含量也较低,通过远源杂交的方法获得富含赖氨酸的小麦潜力不大,因此可通过基因工程的方法导入相关基因,如富含赖氨酸的蛋白基因,赖氨酸合成关键酶基因,赖氨酸分解酶基因干扰序列,以提高结合态或游离态赖氨酸含量。本研究把从辣椒中克隆了富含赖氨酸的蛋白基因cflr,(Genbank基因登录号:EU367999),以PAHC-25为基本载体,构建PAHC-cflr载体,通过基因枪法将其转入小麦。Cflr基因总氨基酸数223个,赖氨酸数40个,赖氨酸含量为21.2%(w/w),苏氨酸数24个,苏氨酸含量10.3%(w/w)。
主要的结果:在质粒包裹过程中,采用直径为0.6μm的金粉作为质粒包裹的介质时,不易结块,分散较好,转化效率较高,而直径为1μm时,颗粒较大,容易沉淀,在载体膜上不易分散。用1mm左右的幼胚作为受体时,形成再生苗较好,较小或较大不易形成再生植株。用Basta筛选时,培养基中Basta含量3-4mg/L时筛选效果较好。通过Basta筛选,共获得T0代筛选植株144棵,其中成活并结实的阳性植株5株。T1代共获得33株,通过RNA表达水平PCR检测,有28株阳性,PCR产物测序证明了阳性植株中扩出的片段即为cflr基因。赖氨酸含量检测阳性植株中赖氨酸的含量比对照提高了7.4%。
Being one of important food crops, the application of genetic engineering on wheat improvement has been attracted great attention recently. In contrast to rice, maize and other crops of agronomic importance, genetic transformation of wheat is still in the exploratory stage. In the study, biolistics-mediated transformation was employed. The transformation, development and regeneration systems are as the following: immature embryos of common wheat cv. Alondra was used as the receptor with the diameter less than 0.5mm, 1mm or greater than 1.5mm, coated by 0.6 and/or 1μm gold particles; screening and selection were performed on 1-7mg/L“Basta”culture medium.
As one of the essential amino acids on human nutrient, lysine content is relatively low in wheat which reduces its nutritional quality. Since the situation is the same to other cereal crops, it seems insignificant to perform traditional crossing with the distal wheat varieties. Biolistics-mediated transformation can make it possible through the introgression of genes encoding lysine-rich proteins, a key enzyme in lysine biosynthesis and the proteins and/or enzymes inhibiting or decreasing lysine biolysis process.
cflr(accession number: EU367999), one gene coding high-lysine protein isolated from pepper, was constructed into the vector pAHC25 and then transformed into wheat immature embryos by gold particle bombardment. CFLR consists of 223 amino acids, of them the number of lysine 40, 21.2% (w / w), and the number of threonine 24, 10.3% (w / w), respectively.
Main results: In the process of“bullet”preparation, 0.6μm gold powder can make good results with hard caking, good dispersion, high transformation efficiency, while 1μm or larger particles can precipitate easily and not be dispersed easily in the carrier membrane. Plantlets can be regenerated easily using 1mm immature embryos while smaller or larger ones make bad results. A Basta concentration of 3-4mg/L was more favorable for screening. In total, 144 T0 generation positive plants were obtained. Of them, 5 had produced offsprings. 28 of 33 lines in total were positive in T1 generation using PCR assay at RNA level. Sequencing confirmed the occurrence of cflr transcripts. The content of lysine increased by 7.4% compared to the control.
赖氨酸为人体必须氨基酸,小麦中赖氨酸含量较匮乏,因此降低了小麦的营养品质。而其他禾谷类作物赖氨酸含量也较低,通过远源杂交的方法获得富含赖氨酸的小麦潜力不大,因此可通过基因工程的方法导入相关基因,如富含赖氨酸的蛋白基因,赖氨酸合成关键酶基因,赖氨酸分解酶基因干扰序列,以提高结合态或游离态赖氨酸含量。本研究把从辣椒中克隆了富含赖氨酸的蛋白基因cflr,(Genbank基因登录号:EU367999),以PAHC-25为基本载体,构建PAHC-cflr载体,通过基因枪法将其转入小麦。Cflr基因总氨基酸数223个,赖氨酸数40个,赖氨酸含量为21.2%(w/w),苏氨酸数24个,苏氨酸含量10.3%(w/w)。
主要的结果:在质粒包裹过程中,采用直径为0.6μm的金粉作为质粒包裹的介质时,不易结块,分散较好,转化效率较高,而直径为1μm时,颗粒较大,容易沉淀,在载体膜上不易分散。用1mm左右的幼胚作为受体时,形成再生苗较好,较小或较大不易形成再生植株。用Basta筛选时,培养基中Basta含量3-4mg/L时筛选效果较好。通过Basta筛选,共获得T0代筛选植株144棵,其中成活并结实的阳性植株5株。T1代共获得33株,通过RNA表达水平PCR检测,有28株阳性,PCR产物测序证明了阳性植株中扩出的片段即为cflr基因。赖氨酸含量检测阳性植株中赖氨酸的含量比对照提高了7.4%。
Being one of important food crops, the application of genetic engineering on wheat improvement has been attracted great attention recently. In contrast to rice, maize and other crops of agronomic importance, genetic transformation of wheat is still in the exploratory stage. In the study, biolistics-mediated transformation was employed. The transformation, development and regeneration systems are as the following: immature embryos of common wheat cv. Alondra was used as the receptor with the diameter less than 0.5mm, 1mm or greater than 1.5mm, coated by 0.6 and/or 1μm gold particles; screening and selection were performed on 1-7mg/L“Basta”culture medium.
As one of the essential amino acids on human nutrient, lysine content is relatively low in wheat which reduces its nutritional quality. Since the situation is the same to other cereal crops, it seems insignificant to perform traditional crossing with the distal wheat varieties. Biolistics-mediated transformation can make it possible through the introgression of genes encoding lysine-rich proteins, a key enzyme in lysine biosynthesis and the proteins and/or enzymes inhibiting or decreasing lysine biolysis process.
cflr(accession number: EU367999), one gene coding high-lysine protein isolated from pepper, was constructed into the vector pAHC25 and then transformed into wheat immature embryos by gold particle bombardment. CFLR consists of 223 amino acids, of them the number of lysine 40, 21.2% (w / w), and the number of threonine 24, 10.3% (w / w), respectively.
Main results: In the process of“bullet”preparation, 0.6μm gold powder can make good results with hard caking, good dispersion, high transformation efficiency, while 1μm or larger particles can precipitate easily and not be dispersed easily in the carrier membrane. Plantlets can be regenerated easily using 1mm immature embryos while smaller or larger ones make bad results. A Basta concentration of 3-4mg/L was more favorable for screening. In total, 144 T0 generation positive plants were obtained. Of them, 5 had produced offsprings. 28 of 33 lines in total were positive in T1 generation using PCR assay at RNA level. Sequencing confirmed the occurrence of cflr transcripts. The content of lysine increased by 7.4% compared to the control.
引文
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