水稻突变体与功能基因组学

突变体是功能基因组学研究的重要材料。本文综述了水稻突变体的创制方法、变异的机制、水稻突变体/基因分类和数量、克隆的水稻重要基因的研究进展, 包括1 698个水稻突变体/基因的分类和43个克隆的水稻突变体基因, 并提出了利用水稻突变体的展望。     

水稻长日照开花因子Lfm1的图位克隆

开花期是水稻最重要的农艺性状之一,水稻的花期决定着水稻的地区适应性和最终产量。人工选择使水稻从短日照向长日照、低纬度向高纬度扩张,因此水稻已逐渐进化出适应长日照条件下的开花调控机制。目前,虽然鉴定了一些影响水稻长日照的开花基因如SDG724、RFT1、EHD4、DTH2,但是挖掘水稻长日照开花基因还十分有限。本研究通过筛选水稻突变体库,获得一批在长日照下花期有显著差异的突变体材料,其中一份突变体lfm1(late-flowering mutant1),在长日照条件下开花延迟,在短日照条件下开花时间正常。通过图位克隆,将Lfm1基因初定位至第8染色体端粒附近。进一步的精细定位将Lfm1基因定位于分子标记8-0.269和与8-0.283之间,范围为12 kb,该区域包括3个候选基因。经测序分析发现,在突变体lfm1中,LOC_Os08g01420基因的第六外显子2800处缺失9个碱基,突变体lfm1等位于已报道的突变体ehd3。在适度(中日照条件下,~12 h/12 h)的光照条件下,突变体lfm1表现为穗粒数增多,生育期略延长,具有应用于生产的潜力。Lfm1基因的克隆为培育适应不同生态区域的水稻材料提供了重要的基因资源。     

水稻类病斑突变体研究进展

植物类病变突变体是一类在没有病原物侵染情况下就能自发产生坏死斑的突变体。这类突变往往导致植株的抗病增强和防御相关基因的组成性表达。水稻中已报道了将近200个来源不同的类病变突变体,截至2014年12月73个水稻类病变突变体已被鉴定和命名,其中11个控制类病变性状的基因被克隆,它们分别编码不同的蛋白,包括热激蛋白转录因子、E3泛素连接酶、质膜蛋白激酶、锌指蛋白、酰基转移酶。尽管这些蛋白不是直接与植物抗病途径相关,但是在已鉴定的水稻类病变突变体中,绝大多数提高了对白叶枯病或稻瘟病的抗性,表明这些类病变基因的突变激活了植株的防御系统,并且不同的类病变基因可能参与了不同的抗病信号传导途径。深入研究水稻类病变突变体对作物抗病的分子机理研究和栽培品种的遗传改良都具有重要的意义。     

水稻花器官突变体apl (abnormal palea and lodicules) 的表型分析与基因初定位

水稻(Oryza sativa)是重要的粮食作物, 其花器官的正常起始及形态建成直接影响水稻的产量。为了深入分析水稻小花发育的调控机理, 从已构建的水稻EMS诱变突变体库中筛选获得了一个花器官异常发育的突变体apl (abnormal palea and lodicules)。与野生型相比, apl突变体小花的内稃膨大, 浆片伸长或转换成稃状结构, 雄蕊数目减少, 表明APL基因可能参与调控水稻内稃、浆片和雄蕊等多轮花器官属性的建成。遗传学分析表明, 该突变体性状受1个隐性单基因控制。通过图位克隆, 将APL基因初步定位于1号染色体上。该工作为深入研究APL基因在水稻花器官形态建成中的作用机制奠定了基础。     

水稻花器官突变体apl（abnormal palea and Iodicules）的表型分析与基因初定位

水稻（Oryzasafiva）是重要的粮食作物,其花器官的正常起始及形态建成直接影响水稻的产量。为了深入分析水稻小花发育的调控机理,从已构建的水稻EMS诱变突变体库中筛选获得了一个花器官异常发育的突变体apl（abnormal palea and Iodicules）。与野生型相比,apl变体小花的内稃膨大,浆片伸长或转换成稃状结构,雄蕊数目减少,表明APL基因可能参与调控水稻内稃、浆片和雄蕊等多轮花器官属性的建成。遗传学分析表明,该突变体性状受1个隐性单基因控制。通过图位克隆,将APL基因初步定位于1号染色体上。该工作为深入研究APL基因在水稻花器官形态建成中的作用机制奠定了基础。     

水稻黄单胞菌hrp基因簇中致病相关基因hpaB的鉴定

由水稻黄单胞菌引起的水稻白叶枯病是水稻最严重的细菌性病害。通过筛选18000个XooTn5转座子插入突变体,得到其中一个致病力缺失的突变体XOG11。TAIL-PCR方法分离该突变体中插入转座子的侧翼序列,发现转座子插入到位于hrp基因簇的hpaB基因中。对该基因进一步的分析表明该基因编码一个含有156个氨基酸,等电点为4.28,亮氨酸含量为14.4%的蛋白HpaB。Southern blot和PCR验证表明Tn5在该突变体中为单拷贝插入且未发生转座子携带侧翼序列的转移。将hpaB克隆到具有广泛寄主的质粒pHM1中,转化重组质粒进入突变体后,突变体恢复了在其寄主水稻IR24上的致病力,而转化空质粒pHM1后的突变体仍然表现为致病力缺失。证实了水稻黄单胞菌中hpaB基因与该细菌的致病力相关,在侵染水稻的过程中起着不可缺失的作用。     

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

水稻(Oryzasativa)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。     

水稻黄单胞菌hrp基因簇中致病相关基因hpaB的鉴定水稻黄单胞菌 TAIL-PCR hrp基因簇 hpaB基因

由水稻黄单胞菌引起的水稻白叶枯病是水稻最严重的细菌性病害.通过筛选18000个Xoo Tn5转座子插入突变体,得到其中一个致病力缺失的突变体XOG11.TAIL-PCR方法分离该突变体中插入转座子的侧翼序列,发现转座子插入到位于hrp基因簇的hpaB基因中.对该基因进一步的分析表明该基因编码一个含有156个氨基酸,等电点为4.28,亮氨酸含量为14.4%的蛋白HpaB.Southern blot和PCR验证表明Tn5在该突变体中为单拷贝插入且未发生转座子携带侧翼序列的转移.将hpaB克隆到具有广泛寄主的质粒pHM1中,转化重组质粒进入突变体后,突变体恢复了在其寄主水稻IR24上的致病力,而转化空质粒pHM1后的突变体仍然表现为致病力缺失.证实了水稻黄单胞菌中hpaB基因与该细菌的致病力相关,在侵染水稻的过程中起着不可缺失的作用.     

提高水稻插入突变体库利用效率的尝试

T-DNA标签法是一种以农杆菌介导的遗传转化为基础来创造插入突变体库, 从而高通量地分离和克隆植物功能基因的方法。但由于种种原因, 水稻插入突变体库的利用效率较低。为了提高水稻插入突变体库的利用效率, 结合水稻一个双拷贝T-DNA插入突变体的发现和鉴定研究, 通过特异PCR检测、侧翼序列与目标性状的共分离分析, 在1个双插入位点均为杂合的植株的后代株系中分拆了2个插入事件, 分离出目标性状存在遗传分离且只带有1个插入事件的后代株系, 为后续的共分离检测和基因克隆研究打下了重要的基础。由此产生了对插入突变体库中的非串联多拷贝插入标签系进行研究的一些思路和方法, 提出来与同行商榷。     

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

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。     

Light-dependent regulation of the jasmonate pathway

Jasmonates (JAs) are plant hormones which are crucial for the response of plants to several biotic and abiotic stresses. Beside this important function, they are involved in several developmental processes throughout plant life. In this short review, we would like to summarize the recent findings about the function of JAs in photomorphogenesis with a main focus on the model plant rice. Early plant development is determined to a large extent by light. Depending on whether seedlings are raised in darkness or in light, they show a completely different appearance which led to the terms skoto- and photomorphogenesis, respectively. The different appearance depending on the light conditions has been used to screen for mutants in photoperception and signalling. By this approach, mutants for several photoreceptors and in the downstream signalling pathways could be isolated. In rice, we and others isolated mutants with a very intriguing phenotype. The mutated genes have been cloned by map-based cloning, and all of them encode for JA biosynthesis genes. The most bioactive form of JAs identified so far is the amino acid conjugate jasmonoyl-isoleucin (JA-Ile). In order to conjugate JA to Ile, an enzyme of the GH3 family, JASMONATE RESISTANT 1, is required. We characterized mutants of OsJAR1 on a physiological and biochemical level and found evidence for redundantly active enzymes in rice.     

Light-dependent regulation of the jasmonate pathway

Jasmonates (JAs) are plant hormones which are crucial for the response of plants to several biotic and abiotic stresses. Beside this important function, they are involved in several developmental processes throughout plant life. In this short review, we would like to summarize the recent findings about the function of JAs in photomorphogenesis with a main focus on the model plant rice. Early plant development is determined to a large extent by light. Depending on whether seedlings are raised in darkness or in light, they show a completely different appearance which led to the terms skoto- and photomorphogenesis, respectively. The different appearance depending on the light conditions has been used to screen for mutants in photoperception and signalling. By this approach, mutants for several photoreceptors and in the downstream signalling pathways could be isolated. In rice, we and others isolated mutants with a very intriguing phenotype. The mutated genes have been cloned by map-based cloning, and all of them encode for JA biosynthesis genes. The most bioactive form of JAs identified so far is the amino acid conjugate jasmonoyl-isoleucin (JA-Ile). In order to conjugate JA to Ile, an enzyme of the GH3 family, JASMONATE RESISTANT 1, is required. We characterized mutants of OsJAR1 on a physiological and biochemical level and found evidence for redundantly active enzymes in rice.     

Rice Lesion Mimic Gene Cloning and Association Analysis for Disease Resistance

Lesion mimic mutants refer to a class of mutants that naturally form necrotic lesions similar to allergic reactions on leaves in the absence of significant stress or damage and without being harmed by pathogens. Mutations in most lesion mimic genes, such as OsACL-A2 and OsSCYL2, can enhance mutants’ resistance to pathogens. Lesion mimic mutants are ideal materials for studying programmed cell death (PCD) and plant defense mechanisms. Studying the genes responsible for the rice disease-like phenotype is of great significance for understanding the disease resistance mechanism of rice. In this paper, the nomenclature, occurrence mechanism, genetic characteristics, regulatory pathways, and the research progress on the cloning and disease resistance of rice lesion mimic mutant genes were reviewed, in order to further analyze the various lesion mimic mutants of rice. The mechanism lays a theoretical foundation and provides a reference for rice breeding.     

Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice

Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the beta (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links. A novel rice (Oryza sativa) XTH-related gene, OsXTH8, was cloned and characterized after being identified by cDNA microarray analysis of gibberellin-induced changes in gene expression in rice seedlings. OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids. Phylogenetic analysis revealed that OsXTH8 falls outside of the three established subfamilies of XTH-related genes. OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. In situ hybridization and OsXTH8 promoter GUS fusion analysis revealed that OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes.     

水稻广谱抗稻瘟病基因研究进展

稻瘟病是水稻生产中的最严重病害之一,由于稻瘟菌小种的高度变异性,垂直抗性基因难以持续控制稻瘟病的危害,因此,克隆和利用广谱持久抗瘟基因被认为是解决稻瘟病问题最经济有效的策略。本文从广谱抗源的筛选与利用,广谱抗瘟基因的定位、克隆与应用等方面对水稻广谱抗稻瘟病基因研究取得的进展进行了概述,并介绍了广谱抗性分子机理的最新研究进展。基于国内外稻瘟病抗性基因研究的现状及趋势,以及我国丰富的抗瘟水稻种质资源,克隆越来越多的广谱抗瘟基因具有重要的理论与应用价值。     

水稻突变体des2和des5的研究和定位

小穗发育是决定水稻产量的主要农艺性状,鉴定控制小穗发育的关键基因对研究和分析调控农艺性状的分子机理是至关重要的.本文中,我们鉴定了一组小穗数目明显减少的突变体,命名为decteased spikelets(des),这里详细研究des2和des5两个突变体.结果显示des2是由单基因隐性位点控制,图位克隆将此位点定位到6号染色体的长臂上,并最终克隆了此基因,发现des2是moc1的一个新的等位突变体.定位克隆和序列分析显示在des5中,LAX基因的编码HLH(螺旋一环一螺旋)结构域的区域发生了一个点突变,暗示des5是lax的一个新的等位突变体.我们的结果暗示小穗和水稻叶腋分枝的发育受相同的遗传途径调控.     

Recent Advances in Cloning and Characterization of Disease Resistance Genes in Rice

Rice diseases caused by fungi, bacteria and viruses are one of the major constraints for sustainable rice （Oryza sativa L.） production worldwide. The use of resistant cultivars is considered the most economical and effective method to control rice diseases. In the last decade, a dozen resistance genes against the fungal pathogen Magnaporthe grisea and the bacterial pathogen Xanthomonas oryzae pv. oryzae have been cloned. Approximately half of them encode nuclear binding site （NBS） and leucine rich repeat （LRR）-containing proteins, the most common type of cloned plant resistance genes. Interestingly, four of them encode novel proteins which have not been identified in other plant species, suggesting that unique mechanisms might be involved in rice defense responses. This review summarizes the recent advances in cloning and characterization of disease resistance genes in rice and presents future perspectives for in-depth molecular analysis of the function and evolution of rice resistance genes and their interaction with avirulence genes in pathogens.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.