摘要
转录后基因沉默(post-transcriptional gene silencing,PTGS)是一种序列特异性的RNA降解过程,主要作用于同源性较高的转录产物,也称为RNA沉默(RNAsilencing)。PTGS只会特异性地降解同源性mRNA不会影响其他基因的表达,是生物体在基因调控水平上的一种自我保护机制。对基因沉默进行深入研究,可进一步揭示生物体基因遗传表达调控的本质,在基因工程中克服基因沉默现象,使外源基因能更好地按照人们的需要进行有效表达。植物PTGS的稳定表达为研究植物基因表达调控及鉴定基因功能及作物改良提供了新的途径。
番茄红素β-环化酶(Lyc—b)是类胡萝卜素代谢途径中催化番茄红素转变成为β-胡萝卜素的关键酶,它决定β-胡萝卜素的生成量,也决定了生成维生素A的量。利用RT-PCR技术从番茄叶片中克隆出Lyc-b基因,将PCR产物与pGEM-T载体连接。并以它为模板,PCR获得了约350bp左右的目的片段,将目的片段直接插入到基因沉默载体pJawoh18中,构建出高效沉默的ihpRNA结构载体pJawoh18-2b。在沉默载体中选bar基因作为选择标记基因,使转基因再生植株能抗除草剂Basta,可广泛适用于大田。将PCR和酶切鉴定为阳性的克隆转化农杆菌LBA1100,利用根癌农杆菌Ti质粒介导的叶盘转化法转化番茄获得了抗性愈伤及17株再生苗,其中PCR、RT-PCR检测结果都为阳性的植株有4棵。通过PCR、RT-PCR检测证明了pJawoh18-2b沉默载体中沉默Lyc-b的ihpRNA结构目的片段已成功的插入了番茄基因组中。番茄转化阳性植株对200 mg/L浓度的Basta有明显抗性。番茄转化阳性植株的果实成熟过程中,在黄色背景下逐渐出现红色斑纹,表明果实中番茄红素β—环化酶基因可能被沉默,使上游番茄红素在果实中积累。本实验为培育出果实中高番茄红素含量的优良番茄品种,并进一步了解番茄红素β-环化酶(Lyc—b)基因的功能,以及Lyc—b在类胡萝卜素代谢过程中的重要作用奠定了基础。
Post-transcriptional gene silencing (PTGS) is a nucleotide sequence-specific process of RNA degradation. It leads to the degradation of homologous RNA from among endogenous genes, transgenes or RNA virus. RNA silencing represents an evolutionarily conserved defense mechanism that plays a key antiviral role in protecting plants against virus infection. Post-transcriptional gene silencing (PTGS) can be used to control the gene expression and study the gene function in transgenic plants.
Lycopeneβ-Cyclase is a key enzyme in carotenoid biosythetic pathway and catalyzes the formation ofβ-carotene from lycoene. Tomato lycoeneβ-Cyclase gene was amplified by RT-PCR from tomato leaf mRNA and cloned in pGEM-T vector. A 350 bp lycoeneβ-Cyclase gene fragment was subcloned in pDon221 and pJawoh18 from pGEM-T-Lyc-b. After verification by PCR and restriction analysis, ihpRNA vector pJawohl8-2b containing lycoeneβ-Cyclase gene fragment was constructed successfully by Gateway technology and was transfered into tomato plants by Agtobacterium-mediated transformation. We constructed a efficient gene silencing vector to silence the Lycopeneβ-Cyclase Gene, then investigate the function of Lycopeneβ-Cyclase gene in caroteniod biosynthesis pathway. Transfected tomato got restriction callus and 17 regeneration plants, four of which are positive by PCR and RT-PCR detection. Positive plants are restrictive to 200 mg/L Basta. During ripening process, the fruits of the positive tomatoes become red gradually. That means Lycopeneβ-Cyclase gene may be silent. The bar gene was used as the selective marker gene in transgenic tomato. The transgenic tomato plants were produced in a herbicide tolerence system, so that the transgenic plants can be used in the fields.
引文
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