TILLING技术及其在农作物中的应用现状

TILLING技术是一种全新的反向遗传学研究方法。文章简要介绍了TILLING技术的原理、操作流程、技术要点及其特点,并总结了TILLING技术在麦类、玉米及水稻研究中的应用现状。     

Potential of a mutant-based reverse genetic approach for functional genomics and molecular breeding in soybean

Mutant-based reverse genetics offers a powerful way to create novel mutant alleles at a selected locus. This approach makes it possible to directly identify plants that carry a specific modified gene from the nucleotide sequence data. Soybean [Glycine max (L.) Merr.] has a highly redundant paleopolyploid genome (approx. 1.1 Gb), which was completely sequenced in 2010. Using reverse genetics to support functional genomics studies designed to predict gene function would accelerate post-genomics research in soybean. Furthermore, the novel mutant alleles created by this approach would be useful genetic resources for improving various traits in soybean. A reverse genetic screening platform in soybean has been developed that combines more than 40,000 mutant lines with a high-throughput method, Targeting Local Lesions IN Genome (TILLING). In this review, the mutant-based reverse genetic approach based on this platform is described, and the likely evolution of this approach in the near future.     

EMS诱发小麦京411突变群体构建及Wx-A1基因突变体鉴定

为扩大小麦突变群体类型,提高目的基因点突变挖掘效率,选用北部冬麦区骨干亲本京411为材料,利用甲基磺酸乙酯(ethyl methylsulfonate,EMS)化学诱变剂构建小麦突变群体,以Wx-A1为候选基因,用TILLING技术检测所创建的突变群体。结果表明,0.90%EMS溶液处理8 h和1.50%EMS溶液处理4 h均能获得较好的损伤效果,致死率分别达到38.47%和56.00%;在此基础上创建了包含867个株系的M2突变群体,在该群体中获得Wx-A1基因的7个点突变,突变密度为1/67.0 kb;其中预测有功能变异的4个错义突变系均可以稳定遗传至下一代,其直链淀粉含量降低2.8%~7.4%。本研究所构建的小麦突变群体可以作为其他目的基因的TILLING检测材料,用于小麦目的基因突变挖掘及功能基因组学研究。     

TILLING技术研究进展及其在植物空间诱变上的应用前景

在回顾TILLING/ecoTILLING技术的发展及其应用的基础上,阐述了其在植物空间诱变研究领域的应用前景。     

TILLING技术应用于家蚕化学诱导突变检测的体系优化

TILLING（Targeting Induced Local Lesions In Genomes）即定向诱导基因组局部突变技术,是近年发展起来的一种高通量检测点突变的反向遗传学方法。本研究从PCR模板量及酶切体系中CELI的酶量两个方面对家蚕TILLING技术的检测体系进行了优化。结果表明：本实验在应用TILLING技术筛选家蚕突变体过程中,用于PCR扩增的家蚕基因组DNA模板最佳用量为20ng;CEL I酶切的20μL反应体系中,加入的最适酶量为0．5μL（本实验室从芹菜中自提的CELI酶10倍稀释液）。本研究初步对TILLING技术应用于家蚕化学诱导突变检测体系进行了优化,为TILLING技术在家蚕功能基因组研究中的应用奠定了基础。     

Availability of Micro-Tom mutant library combined with TILLING in molecular breeding of tomato fruit shelf-life

Novel mutant alleles of an ethylene receptor Solanum lycopersicum ETHYLENE RESPONSE1 (SlETR1) gene, Sletr1-1 and Sletr1-2, were isolated from the Micro-Tom mutant library by TILLING in our previous study. They displayed different levels of impaired fruit ripening phenotype, suggesting that these alleles could be a valuable breeding material for improving shelf life of tomato fruit. To conduct practical use of the Sletr1 alleles in tomato breeding, genetic complementation analysis by transformation of genes carrying each allele is required. In this study, we generated and characterized transgenic lines over-expressing Sletr1-1 and Sletr1-2. All transgenic lines displayed ethylene insensitive phenotype and ripening inhibition, indicating that Sletr1-1 and Sletr1-2 associate with the ethylene insensitive phenotype. The level of ethylene sensitivity in the seedling was different between Sletr1-1 and Sletr1-2 transgenic lines, whereas no apparent difference was observed in fruit ripening phenotype. These results suggested that it is difficult to fine-tune the extent of ripening by transgenic approach even if the weaker allele (Sletr1-2) was used. Our present and previous studies indicate that the Micro-Tom mutant library combined with TILLING could be an efficient tool for exploring genetic variations of important agronomic traits in tomato breeding.     

An EMS mutagenesis protocol for sugar beet and isolation of non-bolting mutants

An annual sugar beet line homozygous for the dominant gene for early bolting (B) has been mutagenized with different doses of ethylmeth‐anesulfonate (EMS). Approximately 15 000 M1 seeds were treated with EMS doses between 0.5 and 1% for 4, 6, 8, 12 and 14 h. Among 10 066 M1s, plants with chlorophyll defects and other abnormalities were found. Germination rates ranged between 30 and 100%, whereas the fertility of M1s dropped to 36%. A dose of 1% EMS applied for 8 h was found to yield an acceptable rate of M2 sterility (16%). Exactly 0.5% of the M2 families contained plants with altered bolting behaviour. After selfing these M2 plants, five non‐bolting M3 lines were selected. These plants do not exhibit shoot elongation even after cultivation under long‐day conditions. Thus, they are homozygous for new mutagenized, recessive non‐bolting alleles. Moreover, four M3 lines showed delayed bolting which was clearly different from the early bolting parent. This demonstrates varying activities of the bolting gene due to different mutational events.     

大麦EMS突变群体的创建及功能评价

为开展麦类作物功能基因组学研究,选用综合性状突出的大麦品种Tamalpais,通过化学诱变剂甲基磺酸乙酯(Ethyl methane sulfonate,EMS)处理,创建了含有10 389个M2单株的突变群体。该群体数据分析表明,温室条件下,6.21%的M2幼苗呈现叶片颜色变异;大田实验中,M2群体出现丰富的表型变异,主要包括幼苗匍匐、分蘖、株高、生育期、叶色、叶形、叶条纹、叶斑、穗部特征、育性等,其中幼苗匍匐、分蘖、株高、叶色、叶条纹、叶斑的突变频率分别为0.11%、6.03%、0.13%、2.5%、0.18%、0.17%。抽样调查显示,M2世代的胚坏死率较低,仅有9%左右的单株表现出过半的胚坏死率。运用TILLING技术成功获得大麦COI1同源基因的突变体,筛选结果同时表明,该突变群体的突变频率约为平均每673kb一个点突变。因此,Tamal-pais群体突变表型丰富、TILLING检测可行,可作为麦类作物基因图位克隆与功能验证的重要素材,适用于麦类作物的正向和反向遗传学研究。     

TILLING技术的原理、特点及其在点突变筛选中的应用

TILLING(TargetingInducedLocalLesionsINGenomes),是一种全新的反向遗传学研究方法,它提供了一种高通量、低成本规模化高效筛选化学诱变剂EMS诱发产生点突变的技术。本文简要介绍了TILLING的原理和技术优势,并对其在植物功能基因组学、突变分子育种和预测突变频率中的应用作了初步探讨。