水稻粉质胚乳突变体ws的表型分析及基因克隆

从甲基亚硝基脲(1-Methyl-1-Nitrosourea,MNU)处理的粳稻品种滇粳优1号突变体库中,筛选到一个稳定遗传的胚乳粉质突变体ws,其籽粒的千粒重、籽粒大小、总淀粉含量、直链淀粉含量等指标均降低,淀粉在尿素溶液中的膨胀能力减弱。对成熟及发育中的胚乳淀粉结构进行观察,发现ws突变体的胚乳中产生大量小而不规则排布的单淀粉颗粒。利用F2群体中分离出的92个隐性极端个体将突变基因连锁在第8染色体近着丝粒位置,随后共用2025个极端个体将目标基因定位于95kb的区间。测序发现ws突变体中编码腺苷二磷酸葡萄糖焦磷酸化酶(Adenosine diphosphate glucose pyrophosphorylase,AGPase)小亚基S2的基因发生点突变,导致编码氨基酸的替换。基因表达分析发现,突变体胚乳中编码AGPase各亚基的相关基因表达量没有发生显著改变,而Western杂交分析显示突变体中AGPS2b的蛋白含量下降。同时,ws突变体的胚乳中AGPase活性下降为野生型的一半。研究结果表明,OsAGPS2的突变导致水稻胚乳中AGPase活性降低,从而影响了淀粉合成。     

水稻粉质皱缩胚乳突变体fse4的表型分析与基因克隆

【目的】水稻种子主要以淀粉形式储藏能量。淀粉合成需要多种酶类和调控因子参与，机制较为复杂。本研究利用水稻胚乳发育缺陷突变体，克隆和鉴定新的调控淀粉合成相关基因，旨在为研究淀粉合成及其调控提供理论依据。【方法】从化学诱变剂甲基亚硝基脲（1-methyl-1-nitroso-urea, MNU）处理的宁粳3号(Ningjing 3, WT)突变体库中筛选到一个能稳定遗传的胚乳粉质皱缩突变体，命名为fse4 (floury and shrunken 4 )。与籼稻品种Dular杂交获得F1种子（F2），通过图位克隆的策略确定FSE4候选基因。利用杂合植株（FSE4fse4）分离出的粉质种子，观察形态学特征，分析其理化性质。使用扫描电镜和半薄切片技术观察胚乳结构。使用qRT-PCR和免疫印迹分析淀粉合成相关基因表达模式和淀粉合成相关酶类的蛋白积累量。利用全自动氨基酸分析仪测定成熟胚乳各氨基酸含量。【结果】突变体fse4籽粒宽度、厚度以及千粒重显著下降，同时胚乳中总淀粉、总蛋白、直链淀粉含量亦显著下降，而脂肪含量显著上升；淀粉黏度、崩解值和消减值显著低于野生型。突变体fse4中多为单粒型淀粉颗粒，且排列分散。FSE4定位于第5染色体长臂约252 kb的区间内，测序发现编码Δ1-吡咯啉-5-羧酸合成酶基因 （Delta 1-pyrroline-5-carboxylate synthetase, P5CS）第1外显子上发生单碱基替换，导致一保守的氨基酸发生变异。突变体fse4中大部分淀粉合成相关基因表达量下调，多种淀粉合成相关蛋白积累量减少。突变体fse4米粉中多种氨基酸含量发生显著变化，游离氨基酸含量是其野生型的3.6倍。此外，外源喷施脯氨酸能部分恢复突变体fse4种子萌发缺陷表型。【结论】FSE4编码脯氨酸合成关键限速酶P5CS，该基因对胚乳中氨基酸的合成及代谢起重要的调控作用，并影响淀粉的合成与积累。     

Phenotypic Analysis and Gene Cloning of Rice Floury Endosperm Mutant fse4

【Objective】Starch is the main energy reserve of rice endosperm. The biosynthesis of starch is complex, requiring a large number of synthetic enzymes and regulators. Screening rice endosperm defective mutants and cloning the underlying genes will lay theoretical basis for starch biosynthesis and its regulation. 【Method】 A stable genetic floury and shrunken endosperm mutant termed as fse4 (floury and shrunken4) were obtained from the mutant library of Ningjing 3 (WT), which was induced by N-methyl-N-nitrosourea (MNU). An F2 mapping population was generated by crossing the fse4 mutant with Dular (an indica rice variety) and the gene was finally isolated. The floury seeds segregated from the fse4 heterozygous plants were used to observe the morphological features, and the physicochemical properties of the brown rice flour were analyzed. The endosperm structure was observed with a scanning electron microscopy by the semi-thin section technology. The expression of starch synthesis related genes during grain filling was determined by qRT-PCR; Immunoblotting was used to detect the accumulation of proteins related to starch synthesis. The amino acids contents of each mature endosperm were determined with the fully automatic amino acid analyzer.【Result】The 1000-grain weight and grain size were significantly reduced in fse4. Compared with WT, the contents of total starch, amylose and total protein were signi?cantly lower in fse4, while the lipid content was signi?cantly higher. The starch viscosity, breakdown viscosity and setback viscosity of the fse4 mutant were lower than WT. The endosperm of the mutant had many single dispersed starch granules with large spaces between each other. Using 1568 recessive individuals, FSE4 was narrowed down to a 252 kb region. Sequencing revealed a single base substitution in the first exon of the delta 1-pyrroline-5-carboxylate synthetase (P5CS), resulting in a conserved amino acid variation. Most of the genes related to starch synthesis were downregulated in fse4 and the protein accumulation related to starch synthase were reduced. The contents of various amino acids in fse4 rice flour were increased or decreased, the total free amino acids contents in fse4 seeds was 2.6 times higher than those in WT. Exogenous proline was applied during the germination of fse4 seeds, and the embryonic lethal phenotype was partially recovered.【Conclusion】FSE4 encode the key rate-limiting enzyme P5CS of proline synthesis, which plays an important role in the biosynthesis and metabolism of amino acids in endosperm and affects the accumulation of starch.     

水稻白条纹突变体st13的表型分析及基因定位

【目的】叶色突变相关基因鉴定和克隆有助于研究光合作用,补充并完善叶绿体发育机理和色素合成代谢途径,为开展水稻的高光效育种提供理论依据。【方法】从粳稻品种Dongjin的组培后代中分离出一个白条纹突变体st13,成熟期测定野生型和st13的主要农艺性状,苗期测定色素含量并观察叶绿体的超微结构;将st13和Dongjin进行正反交,观察F_1植株表型,并对F_2表型分离进行卡方检验,对st13进行遗传分析;利用st13×南京11(籼稻品种)的F_2和F_(2:3)群体,对st13突变基因定位;采用qPCR分析叶绿体发育和叶绿素合成相关基因在st13与野生型相对表达量。【结果】与野生型Dongjin相比,该突变体的株高、单株有效穗数、穗长、结实率和千粒重等主要农艺性状显著下降。苗期的色素含量降低,分蘖期无差异。突变体的叶绿体中既有含丰富的类囊体膜结构的正常叶绿体,也存在无类囊体结构的叶绿体。遗传分析和基因定位结果表明,st13的突变表型受1对隐性核基因控制,突变基因位于第3条染色体长臂InDel(Insertion-Deletion)标记I3-21和I3-22之间。进一步在这两个标记之间设计了6对InDel标记,最终将基因定位在94kb区间内,此区间共有8个候选基因。【结论】这8个候选基因中,有5个假定的蛋白,其他三个都是有功能注释的蛋白,而这三个蛋白在水稻中均未见报道,因此,st13突变是由一个新的叶色基因突变引起的;同时st13中叶绿体发育、叶绿素合成和光合系统相关基因的表达也发生了显著改变,推测ST13可能是调控叶绿体发育的关键基因。     

水稻类病斑突变体lm8015-2的鉴定与基因的精细定位

[目的]为进一步解析水稻抗病分子机制,对类病斑突变体lm8015-2进行了鉴定.[方法]利用EMS诱变籼稻中恢8015,从其后代中鉴定出一个类病斑突变体lm8015-2.对突变体lm8015-2及其野生型的叶片进行超氧化物歧化酶活性、过氧化氢酶活性、过氧化物酶活性、过氧化氢含量、丙二醛含量、可溶性蛋白含量、光合色素含量...     

水稻低淀粉粘滞性突变体的获得与特性分析

利用300 Gy [sup]60[/sup]Co-γ射线辐照早籼稻品系W8-1干种子,诱发产生了低淀粉粘滞性突变体[i]lsv-1[/i]。对其淀粉特性分析表明,突变体的表观直链淀粉含量、胶稠度和碱消值与原亲本相仿,但最高粘度、冷胶粘度、崩解值和消减值明显小于原亲本,达最高粘度时间更短,开始糊化温度更高。突变体糊化后的米粉特性和胚乳内淀粉粒的形态与原亲本显著不同。     

一个水稻叶片白化转绿叶突变体的遗传分析和精细定位

 在水稻品种宜香B中发现了一个白化转绿叶突变体,经过多代自交获得了稳定的白化转绿叶色突变体。该突变体在4叶期前叶色为黄绿色,之后逐渐变绿,从苗龄4周到12周,突变体/野生型叶绿素含量比值从34.5%逐渐升高到99.4%。遗传分析表明该突变受1对隐性核基因控制,暂命名为gra。利用微卫星标记将gra初步定位于第10染色体标记RM596和RM5620之间,进一步利用极端个体定位法把gra精细定位于标记RM25522和RM25535之间。gra基因距RM25522和RM25535标记的遗传距离均为0.05 cM, 其物理距离约为136 kb。     

一个水稻分蘖角度突变体tac2的遗传分析和基因初步定位

  水稻散生突变体tac2是以恢复系缙恢10号为材料经甲基磺酸乙酯（EMS）化学诱变所得。该突变体苗期表型正常,分蘖期株型松散,分蘖角度较野生型显著增大,株高明显降低。外源赤霉素处理可以使该突变体株高恢复,但分蘖角度不受影响。遗传分析表明该突变性状受1对隐性主效基因控制。利用分子标记将该基因初步定位于第9染色体上的RM3320与RM201之间,遗传距离分别为19.2和16.7 cM。     

Genetic Analysis and Mapping of TWH Gene in Rice Twisted Hull Mutant

A mutant with twisted hulls was found in a breeding population of rice (Oryza sativa L.). The mutant shows less grain weight and inferior grain quality in addition to twisted hulls. Genetic analysis indicated that the phenotype of mutant was controlled by a single recessive gene (temporarily designated as TWH). To map the TWH gene, an F2 population was generated by crossing the twh mutant to R725, an indica rice variety with normal hulls. For bulked segregant analysis, the bulk of mutant plants was prepared by mixing equal amount of plant tissue from 10 twisted-hull plants and the bulk of normal plants was obtained by pooling equal amount tissue of 10 normal-hull plants. Two hundred and seven pairs of simple sequence repeat (SSR) primers, which are distributed on 12 rice chromosomes, were used for polymorphism analysis of the parents and the two bulks. The TWH locus was initially mapped close to the SSR marker RM526 on chromosome 2. Therefore, further mapping was performed using 50 pairs of SSR primers around the marker RM526. The TWH was delimited between the SSR markers RM14128 and RM208 on the long arm of chromosome 2 at the genetic distances of 1.4 cM and 2.7 cM, respectively. These results provide the foundation for further fine mapping, cloning and functional analysis of the TWH gene.     

Genetic Analysis and Molecular Mapping of a Novel Chlorophyll-Deficit Mutant Gene in Rice

A rice etiolation mutant 824ys featured with chlorophyll deficiency was identified from a normal green rice variety 824B.It showed whole green-yellow plant from the seedling stage,reduced number of tillers and longer growth duration.The contents of chlorophyll,chlorophyll a,chlorophyll b and net photosynthetic rate in leaves of the mutant obviously decreased,as well as the number of spikelets per panicle,seed setting rate and 1000-grain weight compared with its wild-type parent.Genetic analyses on F1 and F2 generetions of 824ys crossed with three normal green varieties showed that the chlorophyll-deficit mutant character was controlled by a pair of recessive nuclear gene.Genetic mapping of the mutant gene was conducted by using microsatellite markers and F2 mapping population of 495R/824ys,and the mutant gene of 824ys was mapped on the shon arm of rice chromosome 3.The genetic distances from the target gene to the markers RM218,RM282 and RM6959 were 25.6 cM,5.2 cM and 21.8 cM,respectively.It was considered to be a now chlorophyll-deficit mutant gene and tentatively named as chl11(t).     

一个新的水稻叶绿素缺失突变基因的遗传分析和分子标记定位

从正常绿色水稻品种824B中发现1个黄化突变体824ys。该突变体具有叶绿素缺失突变特性,表现为植株黄绿色,分蘖数减少,生育期延长,总叶绿素、叶绿素a、叶绿素b的含量以及净光合速率比野生型亲本824B明显下降,每穗着粒数、结实率、千粒重等降低。对824ys与3个正常绿色品种杂交F1、F2的遗传分析表明,控制824ys的叶绿素缺失突变性状为1对隐性核基因。以495R/824ys F2作为定位群体,应用微卫星标记将824ys的叶绿素缺失突变基因定位于水稻第3染色体短臂,与RM218、RM282和RM6959等标记之间的遗传距离分别为25.6、 5.2和21.8 cM。认为该基因为一个新的水稻叶绿素缺失突变基因,暂命名为chl11(t)。     

Genetic Analysis and Gene Mapping of a Rice Tiller Angle Mutant tac2

Tiller angle, a very essential agronomic trait, is significant in rice breeding, especially in plant type breeding. A tiller angle controlling 2 (tac2) mutant was obtained from a restorer line Jinhui 10 by ethyl methane sulphonate mutagenesis. The tac2 mutant displayed normal phenotype at the seedling stage and the tiller angle significantly increased at the tillering stage. A preliminary physiological research indicated that the mutant was sensitive to GA. Thus, it is speculated that TAC2 and TAC1 might control the tiller angle in the same way. Genetic analysis showed that the mutant trait was controlled by a major recessive gene and was located on chromosome 9 using SSR markers. The genetic distances between TAC2 and its nearest markers RM3320 and RM201 were 19.2 cM and 16.7 cM, respectively.     

水稻多分蘖矮秆突变体d63的遗传分析与基因定位

 多分蘖矮秆突变体d63来源于SARⅢ二倍体与明恢63杂交得到的双胚苗株系的自然突变。与野生型相比,其株高显著下降,分蘖明显增多,剑叶变细变短,结实率和千粒重均大幅下降。遗传分析表明,该突变性状受一对隐性单基因控制。该基因位于水稻第8染色体短臂上距离RM22195约0.4 cM的位置。用水稻基因组注释软件Rice Genome Annotation预测,发现从端粒区至RM22195间共有14 个注释基因,未发现已经报道的与株高、分蘖相关的同源基因。因此,推测D63可能是一个未被报道的新基因。     

水稻类病斑突变体wy3的鉴定和基因定位

在粳稻品种武育粳3号栽培群体中获得一个类病斑突变体wy3。该突变体类病斑出现于苗期,分蘖期扩散至整张叶片,属于扩散型类病斑突变体。相比野生型,突变体wy3的株高明显降低,有效分蘖数减少,穗长、每穗粒数、结实率均显著降低。遮光处理表明,突变体wy3类病斑的产生受自然光诱导。台盼蓝染色结果表明,类病斑部位有大量的死亡细胞。突变体wy3的光合色素含量和净光合速率较野生型显著降低,SOD、POD、CAT活性和MDA含量均显著高于野生型。遗传分析表明突变体表型受单隐性核基因控制,采用BSA将该基因初步定位在第2染色体短臂端粒附近。采用F2群体中1099株类病斑单株将基因定位在标记W2-17和W2-18之间28kb的物理距离内。测序结果表明,突变体wy3中的LOC_Os02g02000编码区(CDS)第375位碱基C缺失,导致翻译提前终止,突变体中该候选基因为OsHPL3的一个新等位基因。     

水稻淡褐斑叶突变体lbsl1的遗传分析与基因定位

 通过EMS诱变籼稻品种IR64获得一个稳定遗传的淡褐色斑点叶突变体lbsl1（light brown spotted leaf 1）。在自然条件下,突变体播种后10~14 d,叶片上出现淡褐色斑点,随后逐渐扩散至全叶,第1叶至剑叶上均有淡褐色斑,为全生育期性状。斑点性状的表达对株高、生育期、结实率和千粒重等农艺性状具有显著的影响。遗传分析结果表明,该淡褐色斑点叶性状受一个隐性核基因控制。将突变体lbsl1与正常叶色水稻Morobereken杂交构建F2定位群体,利用SSR标记,最终将该淡褐叶基因lbsl1(t)定位在第6染色体短臂上一个约130 kb的区段上。定位的结果和发展的群体为该基因的进一步精细定位和克隆奠定了基础。     

一个新的水稻半矮化小穗突变体的遗传分析与基因定位

 从粳稻中花 11转基因后代中发现了一个半矮化小穗突变体,表现为植株半矮化、生长势弱、半包茎穗、穗型变小等特点,将其命名为sd sp2(semi dwarf and small panicle 2)。遗传分析显示,该突变体表型受1对隐性核基因控制。以sd sp2突变体为母本与龙特甫B杂交构建F2分离群体,将该基因定位在水稻第6染色体的RH6 32和RH6 40之间的116 kb的物理距离内。通过水稻基因组注释系统在此区域预测到14个开放阅读框,未发现与已报道穗型发育相关基因的同源基因。     

水稻白叶白穗突变体wlwp7的鉴定与基因定位

水稻叶穗色泽突变体为解析不同器官叶绿素生物合成之间的内在联系提供了优良的遗传材料。本研究鉴定了1份白叶白穗突变体wlwp7（white leaf and white panicle 7）,分析了wlwp7的形态、生理和遗传特点。结果表明：wlwp7对低温敏感,当环境温度为20 ℃时苗期叶片白化,但温度升高至30 ℃后叶色正常;大田环境下wlwp7抽穗后颖壳白化,叶绿素含量降低至野生型的40.73%;除结实率较野生型T98B下降6.28%外,其他产量性状不受影响;遗传分析发现,wlwp7与T98B的正反杂交F₂群体中都未出现白叶绿穗和绿叶白穗重组单株,经卡方检测白叶白穗突变单株与绿叶绿穗野生型单株的理论分离比符合1∶3,表明白叶白穗性状受同一隐性核基因控制;利用BSA策略进一步将wlwp7定位在第3染色体上一个280 kb的区域内,该区域未有已报道的白叶白穗基因。本研究发现了wlwp7同时控制叶部和穗部叶绿素合成,精细定位结果为最终克隆wlwp7奠定了基础。     

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-bi and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region (SCAR) marker tightly linked with Stv-bi and a cleaved amplified polymorphic sequence (CAPS) marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, N...     

Genetic Analysis and Gene Mapping of Multi-tiller and Dwarf Mutant d63 in Rice

A spontaneous mutation,tentatively named d63,was derived from the twin-seedling progenies of rice crossed by diploid SARIII and Minghui 63.Compared with wild-type plants,the d63 mutant showed multiple abnormal phenotypes,such as dwarfism,more tillers,smaller flag leaf and reduced seed-setting rate and 1000-grain weight.In this study,two F 2 populations were developed by crossing between d63 and Nipponbare,d63 and 93-11.Genetic analysis indicated that d63 was controlled by a single recessive gene,which was located on the short arm of chromosome 8,within the genetic distance of 0.40 cM from RM22195.Hence,D63 might be a new gene as there are no dwarf genes reported on the short arm of chromosome 8.