水稻卷叶矮化突变体rld的表型鉴定及基因精细定位

为了研究引起水稻叶片卷曲的分子机理,鉴定出新的水稻卷叶基因.用~(60)Co-γ射线辐射诱变籼稻品种镇恢084,获得一份卷叶矮化突变体材料,命名为rld(rolling leaf and dwarf).通过形态学分析水稻表型,石蜡切片观察叶片细胞组织形态,图位克隆和测序技术进行精细定位和确定目的基因,生物信息学分析蛋白序列结构.结果显示:rld突变体叶片极度内卷,株高降低,穗长变短,结实率降低;rld突变体叶片维管束间的下表皮叶肉细胞面积增大;rld基因精细定位在标记Indel2和Indel5间的32.3 kb的物理区间,测序发现rld是调控卷叶基因RL9的一个新等位基因,由于外显子上精氨酸缺失引起rld基因编码的蛋白空间结构发生改变.推测精氨酸在RL9蛋白的正常功能行使过程中是必要的,对维持水稻叶片表型具有至关重要的作用.     

一个新矮秆水稻突变体的光合特性研究

从T-DNA插入的水稻突变体库发现了一个新的水稻矮秆突变体，平均株高约是野生型的37％，茎节间长度明显低于野生型，其叶片直立生长并呈暗绿色，边缘卷曲的性状。光合特性分析表明，单位重量叶片ChlaxChlb和B-Car色素含量显著高于野生型，Chla／b与野生型没有差异。与野生型相比，突变体最大光化学效率（Fv／Fm）、实际光化学效率（φesu）和光化学淬灭系数（qP）与野生型并没有显著差异，但其非光化学淬灭系数显著低于野生型，说明突变体光合效率优于野生型。电镜分析发现了突变体细胞内部出现衰老症状但叶绿体结构正常。     

水稻盐敏感突变体ssm1的生理指标分析

探索水稻对盐胁迫的耐受机制,培育高产耐盐水稻是提高盐碱土地利用率的重要手段.通过对甲基磺酸乙酯(EMS)诱变的种库进行耐盐筛选获得了盐敏感突变体ssm1,该突变体同时具有叶片早衰表型.进一步研究发现,盐处理后突变体中H_2O_2和MDA含量显著高于野生型,总抗氧化能力显著低于野生型.此外,盐胁迫处理6 h后耐盐相关基因MYB2和HKT1.1表达量下调.推测ssm1突变体的盐敏感表型是由于突变体中H_2O_2和MDA的积累导致的膜脂过氧化或膜系统受损,继而造成大量外源Na~+进入细胞导致细胞死亡.通过对盐敏感突变体ssm1的生理指标分析,探究SSM1参与水稻盐胁迫耐受机制,为培育高产耐盐水稻品种提供重要理论依据.     

ATP参与缺氮诱导水稻主根伸长的研究分析

为揭示缺氮诱导植物主根伸长的机制,采用缺氮处理野生型和突变体Osgatb水稻.结果发现缺氮处理后野生型水稻主根变长,而短根突变体Osgatb主根变短,但地上部长度没有明显差异.进一步研究发现,缺氮后野生型和Osgatb突变体根中ATP含量均下降.这一结果表明,ATP可能参与缺氮诱导植物根伸长.     

一个新的水稻幼苗黄叶突变体的遗传分析及其基因的初步定位

在60Coγ射线辐照的水稻突变体库中,发现了一个以粳稻品种日本晴为遗传背景的幼苗叶色黄化突变体syl11(seedling yellow leaf 11).与野生型相比,突变体幼苗第二和第三叶表现黄色,在其完全展开之前叶片自其顶端开始转绿,长到四叶期其叶色恢复正常;并且该突变体syl11幼苗黄色叶片光合色素含量明显下降.遗传分析表明,该突变体的遗传性状由1对隐性核基因控制.本研究以培矮64S/syl11的F2代突变型植株作为定位群体,应用微卫星(SSR)分子标记以及新发展的InDel分子标记,将基因syl11定位在水稻第11号染色体长臂上的RM26652和处于着丝粒附近的ID11974分子标记之间,其遗传距离分别为0.5 cM和0.7 cM.     

水稻spl5突变体类病变受光诱导的机理分析

为了研究光诱导植物类病变发生的机理,以水稻spl5突变体及野生型对照为材料,进行遮光和滤光处理,观察其类病变的发生情况.透射电镜观察叶绿体亚显微结构,分光光度法测定叶绿素含量,DAB染色法分析光照和遮光条件下叶片中H2O2的积累情况.结果表明:2种光合作用的主要光源(蓝光与红光)及紫外光能诱发spl5突变体产生类病变;在spl5突变体未出现类病斑的叶片中,其叶绿体发育异常、叶绿素含量显著降低; spl5突变体叶片经光照后大量积累H2O2,遮光后则无H2O2积累.推测spl5突变体在光照下叶绿体不能进行正常的光能转化,产生大量的活性氧,从而引发细胞坏死.     

基于CRISPR/Cas9的毛果杨PtrHBI1基因功能解析

目的 利用CRISPR/Cas9系统创制毛果杨PtrHBI1功能缺失突变体,初步解析该转录因子在木材形成过程中的功能,为利用基因工程手段培育制浆造纸优良性状的林木新品种提供新思路。方法 以毛果杨(Populus trichocarpa)为研究材料,根据激光显微切割技术捕获的野生型毛果杨不同细胞类型(形成层、木质部和韧皮部)RNA-seq数据,筛选得到1个bHLH家族转录因子PtrHBI1,利用毛果杨木质部原生质体系统对该基因进行亚细胞定位分析,采用原位杂交技术分析PtrHBI1组织特异性表达,采用CRISPR/Cas9技术创制毛果杨ptrhbi1突变体。对ptrhbi1突变体进行生长表型分析,利用石蜡切片对茎段横切面的各细胞类型进行形态分析,利用Klason酸水解法检测突变体木质素含量,使用高效液相色谱测定突变体纤维素和半纤维素含量。结果 亚细胞定位显示PtrHBI1基因定位于细胞核,原位杂交结果表明PtrHBI1主要在形成层和木质部表达。通过CRISPR/Cas9创制的毛果杨ptrhbi1突变体株高显著增加,茎节数和地径在一定时期显著大于野生型。此外,突变体的导管孔径显著增大,纤维细胞数量显著减少。导管细胞的数量和形成层细胞层数与野生型无差异。木材组分分析表明,与野生型植株相比,ptrhbi1植株的纤维素含量增加,木质素总量无显著变化,紫丁香基木质素与愈创木基木质素之质量比(S/G)显著降低。结论 PtrHBI1参与调控毛果杨的生长及次生木质部发育,可能在木材形成过程起着较为重要的调控作用。     

禾谷丝核菌(Rhizoctonia cerealis vander hoeven)生物学的研究——禾谷丝核菌的分离、形态及培养条件

禾谷丝核菌细胞为双核,立枯丝核菌(Rhizoctonia solani kühn)细胞为多核,这是在形态上的主要区别之一.禾谷丝核菌菌核表层细胞与内部细胞大小、颜色、细胞壁的厚薄都不相同.该菌可以小麦(麦粒、根颈、茎杆、穗轴),谷子(茎杆、叶片),玉米(茎杆、叶片),高粱(茎杆、叶片),水稻(茎杆、叶片),稗子(茎杆、叶片),大豆(茎杆、叶片),绿豆(茎杆、叶片),花生(茎杆、叶片),紫花苜蓿(茎杆、叶片),白花草木栖(茎杆、叶片),芝麻(茎杆、叶片),狗牙根(茎杆)等植物作为生长基质,不能以燕麦茎杆和叶片作为营养基质.病菌生长的良好条件是需要糖、硝态氮、磷、钾、硫等营养物质,PH5—9的酸碱度,15—28℃的温度.温度37℃以上仃止生长.     

Fibrillins突变体对拟南芥开花时间的影响

本研究所使用突变体为fbn8和fbn1a为研究对象,以杂交的方法获得fbn1a/fbn8双重突变体.对Fibrillins突变体在不同的环境下FBN1a、FBN8对开花时间调节的可能机制进行初步探究.研究发现,Fibrillins突变体在长日照、短日照条件下均为早花,并且fbn1a/fbn8双重突变体早花现象更为明显.说明FBN1a、FBN8有可能共同在开花途径中起到作用.通过连续对突变体开花基因的检测发现,和单突变体相比,fbn1a/fbn8双重突变体中的FT、SOC1、CO的基因表达量都大幅度增加,其中FT增加的更为明显,FLC减少也更为明显.而与野生型相比较,这种现象单突变体fbn1a又要比fbn8更为明显.最后,通过检测了植株体内H2O2含量的变化发现,与野生型比较fbn8、fbn1a和fbn1a/fbn8体内H2O2含量升高程度为20%、30%、50%.由实验结果推测Fibrillins可能通过影响体内H2O2含量变化来参与开花时间的改变.     

细胞周期抑制蛋白KRP1和转录因子GRF5回补拟南芥 Hippo/sik1突变体的表型分析

器官大小调控在多细胞生物的个体发育中非常重要.模式植物拟南芥的叶片大小受到精密的调控:首 先,叶原基通过有丝分裂实现细胞增殖,之后多数细胞迅速退出分裂,进入细胞延展阶段,达到最终的叶片大小. 本课题组前期发现拟南芥丝/苏氨酸蛋白激酶SIK1是限制动物器官大小的Hippo通路核心组分——蛋白激酶 Hippo/MST的同功能蛋白.对第一对真叶发育过程进行观察,发现促进细胞增殖的GRF5转录因子与代表内复 制启动的细胞周期抑制蛋白KRP1的转录水平均显著低于野生型.在此基础上,构建了KRP1和GRF5的过量表 达载体,分别转化野生型与sik1突变体,筛选鉴定获得了T3纯合株系.表型分析显示,KRP1在野生型背景下过 表达,加速了叶片延展;GRF5在野生型背景下过表达,叶片面积稍有增大,叶片呈深绿色.然而KRP1和GRF5在 sik1中的过表达并未改变其叶片较小、植物发育迟缓的表型.研究结果表明sik1的器官变小表型并非由KRP1和 GRF5功能缺失造成,SIK1真正的下游通路还有待探索.     

Genetic analysis and gene fine mapping of a rolling leaf mutant (<Emphasis Type="Italic">rl</Emphasis><Subscript><Emphasis Type="Italic">11(t)</Emphasis></Subscript>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The exploration of new genes controlling rice leaf shape is an important foundation for rice functional genomics and plant archi-tecture improvement. In the present study, we identified a rolling leaf mutant from indica variety Yuefeng B, named rl_11(t), which exhibited reduced plant height, rolling and narrow leaves. Leaves in rl_11(t) mutant showed abnormal number and morphology of veins compared with those in wild type plants. In addition, rl_11(t) mutant was less sensitive to the inhibitory effect of auxin than the wild type. Genetic analysis suggested that the mutant was controlled by a single recessive gene. Gene Rl_11(t) was initially mapped between SSR markers RM6089 and RM124 on chromosome 4. Thirty-two new STS markers around the Rl_11(t) region were developed for fine mapping. A physical map encompassing the Rl_11(t) locus was constructed and the target gene was finally delimited to a 31.6 kb window between STS4-25 and STS4-26 on BAC AL606645. This provides useful information for cloning of Rl_11(t) gene.     

一个水稻白化致死突变体abl25鉴定及其基因定位

经Co60辐照的粳稻嘉花1号得到一个新的致死白化突变体albino lethal 25(abl25),该突变体从发芽至4叶期表现为白化苗,之后逐渐死亡.与野生型嘉花1号相比,abl25突变体的叶绿素含量和类胡萝卜素的含量大大降低,叶绿体结构不正常,说明其叶绿体发育受到严重阻碍,导致植物死亡.遗传分析表明:该突变体受一对隐性核基因(abl25)控制,进一步利用abl25与广占63S杂交的F2分离群体,将该突变体基因(abl25)定位于第2染色体上SSR标记RM424与Indel分子标记ID7330之间,随后利用新开发的分子标记和扩大群体将其定位在Indel分子标记ID9111和ID9261之间的150 kb内,发现abl25是一个新的水稻苗期白化致死基因.     

Regulation of eight avr genes by hrpG and hrpX in Xanthomonas campestris pv. campestris and their role in pathogenicity

Eight putative avirulence genes in Xanthomonas campestris pv. campestris (Xcc) strain 8004 were characterized by Tn5gusA5 mutagenesis and gene expression analysis. The virulence test of mutants on Chinese radish showed that all mutants in individual avr genes except avrBs2 mutant were not significantly different from the wild type in virulence. The avrBs2 mutant showed reduced virulence and bacterial growth in planta. Gene expression analysis using β-glucuronidase as reporter indicated that avrBs1.1，avrBs1，avrXccB，avrXccC，avrXccE1 were regulated by hrpG, whereas avrXccA1, avrXccA2 and avrBs2 were not. RT-PCR analysis showed that all hrpG-regulated genes except avrBs1 were also regulated by hrpX. In addition, it was demonstrated that avrBs1　 was responsible for elicitation of a type III dependent hypersensitive reaction (HR) on nonhost plant pepper ECW-10R, and wild type Xcc 8004 was unable to cause HR on pepper ECW-20R.     

Mutational effect of the “- 35” element of sorghumpsbA gene promoter

Mutations of the first position T and the third position G in TTGACA, the " - 35" element of sorghum psbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants were produced: ATTACA, GTGACA, and ATGACA. Then the protein binding affinity of the mutants and the wild type sorghum psbA gene promoter was tested in a spinach chloroplast protein extract system. Gel retardation assay of the wild type showed a strong protein-binding band. On the other hand, the protein-binding band of the mutant resulting from single base mutation, ATGACA or GTGACA, showed reduced intensity, while that of the mutant resulting from double base mutation, ATTACA, showed increased intensity. It is thus shown that the " - 35" element plays an important role in controlling the binding between psbA gene promoter and the specific chloroplast proteins; mutation of a single base may exert a substantial influence on the binding affinity.     

利用CRISPR /Cas9技术快速创制香型“秀水134”水稻

以目前上海市主栽的高产常规水稻"秀水134"为材料,利用CRISPR/Cas9技术成功敲除甜菜碱醛脱氢酶2基因,获得了两种类型纯合突变体植株.采用表达载体特异性结合的引物检测T_1代转基因植株,成功获得6株不携带载体骨架的转基因植株.定量PCR分析显示,突变体植株甜菜碱醛脱氢酶2基因表达量极显著低于野生型对照(p0.01),但突变体植株成熟种子香味物质2-乙酰-1-吡咯啉(2AP)含量极显著高于野生型对照(p0.01).比较野生型对照与突变体植株的主要农艺性状和产量性状,两者间都没有显著差异(p0.05).本研究可为加快高产香型水稻在上海及周边地区的推广应用,以及为今后利用CRISPR/Cas9技术快速培育其他高产香型水稻新品种研究奠定基础.     

Genetic analysis and gene mapping of a narrow leaf mutant in rice (Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (un-der saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. More-over, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than tho...     

Control of tillering in rice

Tillering in rice (Oryza sativa L.) is an important agronomic trait for grain production, and also a model system for the study of branching in monocotyledonous plants. Rice tiller is a specialized grain-bearing branch that is formed on the unelongated basal internode and grows independently of the mother stem (culm) by means of its own adventitious roots. Rice tillering occurs in a two-stage process: the formation of an axillary bud at each leaf axil and its subsequent outgrowth. Although the morphology and histology and some mutants of rice tillering have been well described, the molecular mechanism of rice tillering remains to be elucidated. Here we report the isolation and characterization of MONOCULM 1 (MOC1), a gene that is important in the control of rice tillering. The moc1 mutant plants have only a main culm without any tillers owing to a defect in the formation of tiller buds. MOC1 encodes a putative GRAS family nuclear protein that is expressed mainly in the axillary buds and functions to initiate axillary buds and to promote their outgrowth.     

Identification and gene mapping of a narrow and upper-albino leaf mutant in rice (Oryza sativa L.)

The leaf blade consists of color and shape traits. Studies of leaf-blade development are important for improvement of rice yield and quality because it is an essential organ for photosynthesis. A narrow and upper-albino leaf mutant (nul1) was identified from among progeny of the indica restorer line Jinhui10 raised from seeds treated with ethyl methane sulfonate. Under field conditions, the mutant displayed narrow and upper-albino leaf blades with significantly decreased photosynthetic pigment contents throughout their development. The narrow-leaf trait is caused by a decreased number of small veins. In contrast to the wild type, the growth period was extended by approximately 8 d and agronomic traits, such as effective panicle number, percentage seed set and 1000-grain weight, declined significantly in the nul1 mutant. Genetic analysis suggested that the narrow and upper-albino leaf characteristics showed coseparation and were controlled by one recessive gene. The Nul1 gene was mapped onto chromosome 7 between the Indel marker Ind07-1 and the Simple Sequence Repeat marker RM21637. The physical distance between the markers was 75 kb and eight genes were annotated in this region based on the rice Nipponbare genome sequence. These results provide a foundation for cloning and function analysis of Nul1.     

Genetic analysis and molecular mapping of a presenescing leaf gene psll in rice （Oryza sativa L.）

A rice psl1 （presenescing leaf） mutant was obtained from a japonica variety Zhonghua 11 via radiation of ^60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the biggest length, while the leaves of the wild variety could keep green for 25--35 d. In this study, genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene （psl1） located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nipponbare and 93-11. The psl1 was further mapped between two STS markers, STS2-19 and STS2-26, with genetic distances of 0.43 and 0.11 cM, respectively, while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus, the region being delimited to 48 kb. This result was very useful for cloning of the psl1 gene.     

F-box基因FOF2在拟南芥盐和冷胁迫响应中的功能分析

FOF2为F-box蛋白家族成员,其生物学功能尚不清楚.采用实时荧光定量PCR和生理学实验相结合的方法,对FOF2基因的表达模式及其在拟南芥抗盐和冷胁迫响应中的作用进行了分析.研究发现,FOF2在拟南芥根、茎生叶和果荚中表达较高,并且其表达受盐和冷胁迫诱导.FOF2过表达株系对盐胁迫敏感,与野生型相比种子萌发率低、幼苗主根较短;相反,fof2突变体对盐胁迫的敏感性则减弱.FOF2过表达和缺失突变体种子萌发对冷胁迫无响应,但其主根在冷处理中分别比野生型短或者长.盐处理下,FOF2过表达株系中盐胁迫反应相关基因的表达量显著降低,fof2突变体中则升高;冷处理下,FOF2过表达株系中冷胁迫反应相关基因的表达量显著升高,fof2突变体中则降低.结果表明,FOF2在植物抗盐胁迫响应中起负调控作用,在抗冷胁迫响应中则可能起正调控作用.