[目的]维生素B1(vitamin B1,VB1)是生物所必需的微量元素,其作为多个酶的辅因子,参与重要的细胞代谢途径,而水稻中维生素B1 合成途径还有待深入研究.本研究旨在解析水稻维生素B1 合成相关基因OsTHIC的生物学功能.[方法]综合运用诱变技术、色素测定、Mutmap+、基因编辑及非靶向代谢组分析等手段克隆目的基因并解析其功能.[结果]从EMS诱变的水稻突变体库中发现一个心叶白化致死突变体wll1(white leaf and lethal 1).wll1 从四叶期开始出现心叶白化表型,白化表型逐渐扩展到其他叶片并导致幼苗死亡.与野生型相比,wll1 的叶绿素含量与类胡萝卜素含量显著降低.利用Mutmap+及基因编辑技术,确定目的基因为维生素B1 合成相关基因OsTHIC.OsTHIC在叶片具有较高表达量,OsTHIC蛋白定位于叶绿体.wll1 及OsTHIC的敲除突变体中维生素B1 含量均显著低于野生型,外施维生素B1 可以恢复wll1 的突变表型.外施维生素B1 及OsTHIC突变均会影响维生素B1 合成相关基因的表达.非靶向代谢组测序分析表明,野生型与wll1差异代谢物在氨基酸的生物合成、辅因子的生物合成、ABC转运器及氨酰基-tRNA的生物合成等方面显著富集.[结论]OsTHIC通过调控维生素 B1 含量,参与氨基酸合成等代谢过程,在水稻生长发育过程中发挥重要作用.

在传统的正向遗传学分析过程中,基因定位需要构建复杂的后代群体,并借助大量分子标记进行遗传连锁分析和区间定位,使得这一过程成本高且耗时长.MutMap是近年来发展的基于高通量第二代测序技术的一种新的正向遗传学分析方法.该方法的优点是遗传定位的周期短且效率高.在此基础上扩展的新方法也不断出现,如基于自交的MutMap+、用于识别基因组缺失区间变异的MutMap-Gap、以及用于定位数量性状基因座的分析思路与MutMap类似的QTL-seq方法等.这些方法不需要建立繁琐的定位群体,甚至不依赖于遗传杂交和任何连锁信息即可进行,加快了对感兴趣表型的变异位点所在基因组区域的识别过程.本文对MutMap及其扩展方法进行了介绍,并对它们未来的应用和发展前景进行了讨论,以期为基于第二代测序技术的正向遗传学基因定位和作物遗传改良研究提供参考.

本研究从贵州地方粳稻(Oryza sativa ssp.japonica)品种‘黎平杂边禾’甲基磺酸乙酯(EMS)突变体库中筛选获得一份能稳定遗传的矮秆小粒突变体,暂命名为dss1 (dwarf and small seed 1).与野生型相比,dss1表现为植株矮化、株型直立、叶色深绿、籽粒变小、第二节间严重缩短、穗长增加等典型油菜素内酯(BR)缺陷突变体的性状.显微观察结果表明dss1叶鞘表皮细胞的长度变短,可能是突变体第二叶鞘长度比野生型短的原因.对突变体的暗形态建成与BR敏感性研究表明,黑暗条件下突变体表现为去黄化表型,对外源BR敏感.遗传特性分析证明dss1突变体由一个隐性单基因位点控制.利用MutMap技术将dss1基因定位于3号染色体上,筛选获得一个候选基因,测序结果表明,dss1候选基因为BR合成途径关键酶基因OsDWARF,dss1是由于该基因第5个外显子上第335位的氨基酸由苏氨酸(ACT)突变为异亮氨酸(ATT)所引起的.定位得到的矮化小粒基因DSS1为一个新的OsDWARF等位基因.

Optimizing leaf shape is a major challenge in efforts to develop an ideal plant type.Cucumber leaf shapes are diverse;however,the molecular regulatory mechanisms underlying leaf shape formation are unknown.In this study,we obtained a round leaf mutant (rl) from an ethyl methanesulfonate-induced mutagenesis population.Genetic analysis revealed that a single recessive gene,rl,is responsible for this mutation.A modified MutMap analysis combined linkage mapping identified a single nucleotide polymorphism within a candidate gene,Csa1M537400,as the mutation underlying the trait.Csa1M537400 encodes a PINOID kinase protein involved in auxin transport.Expression of Csa1M537400 was significantly lower in the rl mutant than in wild type,and it displayed higher levels of IAA (indole-3-acetic acid) in several tissues.Treatment of wild-type plants with an auxin transport inhibitor induced the formation of round leaves,similar to those in the rl mutant.Altered expression patterns of several auxin-related genes in the rl mutant suggest that rl plays a key role in auxin biosynthesis,transport,and response in cucumber.These findings provide insight into the molecular mechanism underlying the regulation of auxin signaling pathways in cucumber,and will be valuable in the development of an ideal plant type.

水稻(Oryza sativa)籽粒大小是影响其产量的关键农艺性状,克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义.为深入探究水稻籽粒大小的调控机制,通过EMS诱变品种宽叶粳(KYJ),分离了一系列水稻籽粒大小改变的突变体,其中smg12表现为籽粒变小,株高变矮,一级枝梗数和二级枝梗数减少.遗传分析表明,该小粒突变体受隐性单基因控制.细胞学分析显示,该突变体颖壳纵向细胞长度显著变短,表明SMG12主要影响细胞扩展.利用Mutmap方法对候选基因进行克隆,筛选出SMG12的候选基因OsBRI1,该基因编码油菜素内酯受体激酶.OsBRI1外显子上的第2 074个碱基发生了由C到T的置换,产生非同义突变,使得该位置编码的脯氨酸变为丝氨酸,从而影响OsBRI1的功能.综上,该研究鉴定了OsBRI1基因的1个新等位变异,揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础.

Bulked segregant analysis (BSA) is an efficient and low-cost strategy that is widely used to identify causal genes in segregating populations.BSA-based methods, such as BSA sequencing (Wenger et al., 2010), bulked segregant RNA sequencing (BSR-seq) (del Viso et al., 2012), and MutMap (Abe et al., 2012), are powerful tools that can be used for rapidly discovering genetic markers and gene mapping.Although BSA is increasingly being used in wheat (Triticum aestivum) gene mapping efforts, few user-friendly BSA tools have been developed for researchers lacking a strong bioinformatics background.Here, we developed the web-based BSA platform WheatGmap (https://www.wheatgmap.org), which integrates multiple BSA mapping models and large amounts of public data to accelerate gene cloning and functional research and facilitate resource sharing.

叶片颜色通常与叶绿体数量、结构、光合能力等相关,以C4模式植物谷子的叶色突变体为材料,克隆突变基因并研究其功能,对于解析C4植物叶绿体发生发育及光合作用调控机制具有重要作用.本研究从谷子品种豫谷1号EMS突变体库中分离鉴定到一个条纹叶突变体t122.该突变体生长发育迟缓,且叶片呈现不规则白色条纹.农艺性状测定结果显示,t122的株高、叶长、叶宽、主穗粗、主穗重、结实率等性状均显著降低,而单株穗数相比野生型显著增加,同时光合性能受到影响.叶片解剖结构观察发现t122维管束间距离、维管束间细胞层数、叶片横截细胞面积均无明显改变,而叶片细胞长度显著增加.叶绿体超微结构观察表明t122一部分叶片细胞叶绿体缺失,而另一部分叶片细胞含有发育正常的叶绿体.遗传分析结果显示,t122突变表型由一对隐性核基因控制.利用MutMap法,将候选基因初步定位于3号染色体24.0～30.0 Mb区间内,研究结果为谷子条纹叶基因的克隆及功能研究奠定了基础.

Grain size is a key agronomic trait that determines the yield in plants. Regulation of grain size by brassinosteroids (BRs) in rice has been widely reported. However, the relationship between the BR signaling pathway and grain size still requires further study. Here, we isolated a rice mutant, named small grain2 (sg2), which displayed smaller grain and a semi-dwarf phenotype. The decreased grain size was caused by repressed cell ex-pansion in spikelet hulls of the sg2 mutant. Using map-based cloning combined with a MutMap approach, we cloned SG2, which encodes a plant-specific protein with a ribonuclease H-like domain. SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2 (GSK2) in re-sponse to BR signaling, and its mutation causes insensitivity to exogenous BR treatment. Genet-ical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2. We further found that BRs enhance the accumulation of SG2 in the nucleus, and subcellular distribution of SG2 is regulated by GSK2 kinase activity. In addition, Oryza sativa OVATE family protein 19 (OsOFP19), a negative regulator of grain shape, interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size. Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regu-lates grain size by interacting with OsOFP19.

Ghd7 is an important gene involved in the photo-period flowering pathway in rice. A Ghd7-involved transcriptional regulatory network has been estab-lished, but its translational regulatory pathway is poorly understood. The mutant suppressor of overexpression of Ghd7 (sog7) was identified from EMS-induced mutagenesis on the background of ZH11 overexpressing Ghd7. MutMap analysis re-vealed that SOG7 is allelic to Ghd8 and delayed flowering under long-day (LD) conditions. Biochemical assays showed that Ghd8 interacts with OsHAP5C and Ghd7 both in vivo and in vitro. Surprisingly, a point mutation E96K in theα2 helix of the Ghd8 histone fold domain (HFD) destroyed its ability to interact with Ghd7. The prediction of the structure shows that mutated amino acid is located in the interaction region of CCT/NF-YB/YC com-plexes, which alter the structure ofα4 of Ghd8. This structural difference prevents the formation of complex NF-YB/YC. The triple complex of Ghd8-OsHAP5C-Ghd7 directly bound to the promotor of Hd3a and downregulated the expression of Ehd1, Hd3a and RFT1, and finally resulted in a delayed heading. These findings are helpful in deeply un-derstanding the Ghd7-involved photoperiod flow-ering pathway and promote the elucidation of rice heading.