Putrescine Modulates Cadmium Fixation Ability of Cell Wall to Decrease Cadmium Accumulation in Rice,a Process Might Depend on Nitric Oxide

Although putrescine(PUT)has been linked to plants'responses to cadmium(Cd)stress,the exact mechanism is still elusive.In the current investigation,the endogenous PUT concentration in rice roots was enhanced by Cd stress.Exogenous PUT increased root cell wall hemicellulose levels,which in turn increased its Cd binding capacity,concurrently decreasing the transcription level of genes such as OsNRAMP1 and OsCd1 that are responsible for root Cd absorption.As a result。

水稻斑马叶突变体zl7的鉴定与基因的精细定位

【目的】斑马叶突变体作为水稻叶色突变体的重要种质资源,是研究植物光合作用机制和高光效育种的理想材料,对于解析光合作用机理和提高水稻产量具有重要意义。【方法】用甲基磺酸乙酯(EMS)诱变粳稻品种春江06建立突变体库,从突变体库中筛选到1份苗期为斑马叶的突变体,该突变体被命名为zl7(zebra leaf7)。在常规大田种植条件下分别比较突变体与野生型在苗期、抽穗期和成熟期叶色表型和产量性状差异,通过透射电镜实验分析叶片叶绿体发育情况,利用图位克隆方法克隆候选基因,利用荧光定量PCR方法分析参与叶绿素合成和叶绿体发育相关基因的表达水平。【结果】从苗期开始,突变体zl7表现出典型的斑马叶,叶绿素含量降低,直到抽穗期,斑马叶表型消失,叶片逐渐复绿,叶绿素含量无明显差异。光合速率测定和电镜观察结果显示,突变体zl7的光合速率、气孔导度下降,叶绿体发育异常。与野生型相比,突变体的株高、分蘖、穗长、一次枝梗、二次枝梗和每穗粒数均显著降低,而粒长、粒宽和千粒重均略有增加。荧光定量PCR结果表明突变体中参与叶绿素降解相关基因的表达量显著升高,而参与叶绿素合成和叶绿体发育相关基因的表达量显著降低。遗传分析表明,该突变体受一对隐性核基因调控。通过图位克隆将该基因定位在第7染色体,测序发现突变体的目标基因ZL7编码区发生单碱基替换,导致一个氨基酸由丝氨酸变为天冬酰胺。【结论】ZL7(Zebra Leaf 7)突变导致叶绿体发育异常,水稻叶片出现斑马叶表型,该基因在叶绿素合成及叶绿体发育中起重要作用。

水稻窄叶突变体nal12的鉴定与基因精细定位

【目的】水稻叶片是理想株型的主要组成部分。筛选和鉴定新的叶形突变材料,可为研究叶发育调控机制和塑造叶片理想形态打下基础。【方法】由粳稻品种武运粳31经EMS(ethyl methane sulphonate)诱变获得窄叶突变体nal12(narrow leaf 12);通过表型测量分析剑叶至倒4叶的叶片长度、宽度、大脉数和小脉数,并进行组织切片和显微观察;将突变体nal12与籼稻品种台中本地1号(TN1)杂交,在F_2分离群体中选取了1709个具窄叶突变表型的单株,通过已有的SSR、STS和新开发的分子标记进行精细定位。【结果】nal12在幼苗期就表现出了窄叶性状,在成熟期植株较野生型矮小,分蘖增多,茎秆变细。经组织切片观察分析,nal12叶片变窄是由于大脉和小脉数量减少造成。遗传分析表明该窄叶表型受单一隐性核基因调控;最终将该基因定位在第10染色体上LC2-RF37与LC4R-RF39标记之间约64.7 kb的范围内。该区间内共有10个开放阅读框,其中并无已报道的窄叶相关基因。qRT-PCR结果表明,NAL12基因的突变会影响生长素合成与运输相关基因的表达。【结论】NAL12为一个新的叶形调控基因。这为进一步研究水稻叶片发育,丰富其分子调控网络打下了基础。

籼稻C84和粳稻春江16B重组自交系遗传图谱构建及籽粒性状QTL定位与验证

【目的】本研究旨在挖掘水稻粒型新基因、探索其分子机理,解析籽粒发育调控遗传网络奠定基础,并为通过分子标记聚合有利基因开展超级稻分子设计育种提供理论依据。【方法】以植株和籽粒形态差异较大的晚粳稻品种春江16B(CJ16B)和广亲和中籼稻背景恢复系C84为亲本构建含有188个家系的重组自交系为作图群体,利用158对在双亲中存在多态性差异的分子标记,构建了遗传连锁图谱,总遗传距离为1428.40 cM,平均标记间距为9.04 cM。在构建遗传图谱的基础上,完成RIL188个株系籽粒的粒长、粒宽、粒厚、长宽比和千粒重等5个性状考查并进行QTL定位。【结果】在海南陵水和浙江杭州两地共检测到籽粒相关主效QTL 30个,包括籽粒QTL新座位18个,解释遗传变异3.51%~17.25%。其中粒长、粒宽、粒厚和长宽比QTL位点分别为9个、5个、5个和6个,千粒重QTL位点5个。经基因座位比对,发现有5个QTL区间与已克隆的调控籽粒形态相关基因座位相近,我们通过对双亲目标基因的测序并根据差异位点设计d CAPs分子标记进行验证。【结论】该RIL群体及其遗传图谱可用于水稻重要农艺性状主效QTL基因的定位和克隆,新定位的18个粒型QTL可以为水稻籽粒发育调控网络提供补充和资料积累。

Rapid Creation of New Photoperiod-/Thermo-Sensitive Genic Male-Sterile Rice Materials by CRISPR/Cas9 System

Rice is an important food crop in China, and the development of hybrid rice is a crucial way to increase grain yield. The creation of dual-purpose nuclear-sterile lines for two-line hybrid breeding has become vital for commercial rice breeding. We constructed the pC1300-2 x35 S::Cas9-sgRNA^(PTGMS2-1) expression vector for editing the male fertility gene PTGMS2-1 in two widely compatible rice varieties, 93-11 and Huazhan, by using the CRISPR/Cas9 system. We obtained the marker-free photoperiod-/thermo-sensitive genic male-sterile(P/TGMS) lines in T_1 generation. According to the experiments in phytotron with four temperature and photoperiod treatments, we found the temperature is the main factor for restoring the pollen fertility of ptgms2-1 mutants in 93-11 and Huazhan, and the photoperiod also has some effects on pollen fertility in two different rice backgrounds. The application of cultivating new male-sterile lines by genome editing system will significantly accelerate the rice breeding process.

Functional Analysis of Three Rice Chloroplast Transit Peptides

Chloroplast transit peptides(CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice(Oryza sativa L.) chloroplast-localized proteins and found that their CTPs could be used to transport non-chloroplast-localized proteins into the chloroplasts. Fusion proteins lacking the CTP remained located in the cytoplasm. Furthermore, we constructed green fluorescent protein fusion vectors with the three CTPs and three non-chloroplast-localized proteins, Ghd10, MULTI-FLORET SPIKELET1(MFS1), and SHORTENED UPPERMOST INTERNODE 1(SUI1). After transforming these constructs into rice protoplasts, the fusion proteins all localized in the chloroplasts. Collectively, our results showed that these CTPs can transport non-chloroplast-localized proteins into the chloroplasts, and more importantly, these CTPs can be applied to engineer chloroplast metabolism.

水稻粒宽突变体gw4的鉴定与基因定位

【目的】水稻粒形是影响水稻产量和决定稻米外观品质的主要性状之一。筛选和鉴定新的粒形突变材料,可为研究水稻籽粒发育的调控机制奠定基础。【方法】粳稻品种中花11经1%的EMS处理,在诱变群体中获得一份窄粒突变体gw4(grain width on chromosome 4);分析粒形和其他主要农艺性状,在扫描电镜下观察颖壳细胞变化;利用突变体与籼稻品种台中本地1号配组的F2分离群体,选择隐性个体完成基因的精细定位;开展生物信息和测序分析,确定定位区间的候选基因;采用RT-PCR分析该基因在根、茎、叶、鞘、穗等组织中的表达模式及其他粒形相关基因的表达水平。【结果】与野生型相比,除了表现窄粒外,gw4的粒长、千粒重、每穗粒数、一次枝梗数和二次枝梗数等显著下降;扫描电镜发现gw4的颖壳内外表皮细胞均小于野生型;遗传分析表明该窄粒表型受一对单隐性核基因控制;通过开发的新标记最终将该基因定位在第4染色体BS6与EX49两个标记之间约31.74 kb的范围内;测序结果发现在LOC_Os04g01590基因编码区发生了一个由G至A的单碱基突变,导致原来编码的甘氨酸变成了天冬氨酸;qRT-PCR结果表明,LOC_Os04g01590主要在幼穗中表达,且在突变体中表达显著下降。【结论】GW4主要调控水稻粒宽的发育,预测LOC_Os04g01590为其候选基因。这为进一步丰富粒形的遗传调控网络打下了基础。

QTL analysis of the rice seedling cold tolerance in a double haploid population derived from anther culture of a hybrid between indica and japonica rice

A doubled haploid population, derived from anther culture of F hybrid between a typical indica cv. and a japonica cv. has been used to investigate the seedling cold tolerance (SCT) in growth cabinet. By dynamically analyzing every day’s survival percentages of the parents and DH lines under 7-d cold plus 9-d normal temperature condition, the guantitative trait loci (QTLs) for SCT have been mapped based on a molecular linkage map constructed from this population. The results show that two parents had significant differences in SCT and the segregation of SCT in DH lines was basically a continuous distribution with most serious injury on the 6th d of the cold treatment. A total of 4 QTLs for SCT have been identified on chromosomes 1, 2, 3 and 4 respectively. The additive effects of qSCT-1, qSCT-2 and qSCT-3 have been contributed by the japonica ev JX17, but that of qSCT-4 has been contributed by the indica cv ZYQ8. The mechanism of SCT seems complicated since the above 4 QTLs detected at different stages

QTL analysis of rice low temperature germinability

A double haploid population, derived from anther culture of F, hybrid between a typical indica and a japonica (ZYQ8/JX17), has been used to investigate the low temperature germinability (LTG) at 15℃. The low temperature germinability of two parents was significantly different. In 6-11 d, the germination percentage of ZYQ8 was higher than that of JX17. In 12-16 d, the germination percentage of JX17 was higher than that of ZYQ8. The quantitative trait loci (QTLs) of every day for low temperature germinabilityhave been mapped based on a molecular linkage map constructed from this population. In 8-11 d, qLTG-9 was identified in C397B-RZ617B on chromosome 9, the additive effect was positive, showing that, the allele from JX17 could increase low temperature germinability. In 12-16 d, qLTG-4 was mapped between RG908 and CT563 on chromosome 4, the additive effect was negative, showing that the allele from ZYQ8 could increase low temperature germinability. These two QTLs were detected at different stages,

Isolation and genetic charac-terization of a fragile plant mutant in rice (Oryza sativa L.)

The fragile rice mutant was isolated from an M2 population of indica variety Shuang Ke Zao (SKZ) treated with γ-rays, and designated as fp1 {fragile plant 1) because of its fragile leaves and culms. To map FP1 locus, an F2 mapping population was derived from a cross between the fp1 and C-bao, a polymorphic japonic variety. The primary mapping result places the FP1 locus in an interval between two molecular markers, microsatellite marker RM16 (3.1 cM proximal to FP1) and STS marker G144a (9.1 cM distal to FP1) in the centromere region of chromosome 3. A CAPS marker C524a was further developed between RM16 and G144a, with 0.4 cM genetic distances to the FP1 locus, providing a practical starting point for constructing a BAC con-tig spanning the FP1 locus and cloning the fp1 gene. Allelism test demonstrated that fpl is allelic to be 1, a fragile rice mutant reported previously.

Characterization and cloning of a brittle culm mutant (bc88) in rice (Oryza sativa L.)

This study characterizes a brittle culm (bc88) mutant of rice (Oryza sativa L.) obtained by ethylene methylsulfonate (EMS)-induced mutagenesis of Wuyunjing 7. The bc88 mutant exhibits a diversity of pleiotropic phenotypes, including brittle culm at the whole-plant growth stages, withered leaf tips at the seedling stage, and 18-d delay in heading date at the mature stage. Genetic analysis indicates that the bc88 mutant is controlled by a single recessive nuclear gene. The mutated bc88 gene isolated by map-based cloning contains only one point mutation in the 5th exon relative to its wild-type BC88 (LOC_Os09g25490 and Os09g0422500), leading to an amino acid change from P to L in bc88 plants. Alignment of the putative protein sequence with its homologs indicates that the mutation is located in the conserved region of the sequence. Detection of the transcription level of BC88 in rice plants shows that the expression level of BC88 is higher in spikes and culms than in leaves, roots, and leaf sheaths. These contribute to understanding of the molecular mechanism of cellulose synthesis. The target gene BC88 can be a useful tool in molecular marker-assisted selection for rice culm trait breeding.

Characterization and fine mapping of an early senescence mutant (es-t) in Oryza sativa L.

An es-t (early senescence-temporary) mutant, produced by ethylene methylsulfonate treatment of strain Nipponbare, was identified in rice. The leaves of es-t appeared yellow at the seedling stage, and had decreased chlorophyll content. Rust spots were found during growth in es-t, especially at the leaf margin and tip. The plants showed a typical early-senescence phenotype at the milky stage. The leaf surface of es-t appeared smoother than wild-type leaves under a scanning electron microscope, because the leaves lack siliceous protuberances around the stoma. Chloroplasts grow abnormally and are filled with many starch grains in es-t. Paraffin section analysis showed that the development of the sclerenchyma cells and vascular bundles were also abnormal in es-t. Genetic analysis indicated that es-t was controlled by a recessive gene, which was finely mapped to a 42-kb interval on chromosome 5. These results will facilitate the positional cloning and functional studies of the gene.