Molecular adaptation of barley to cold and drought conditions

Summary Molecular adaptation to cold and drought involves a series of biochemical and molecular changes leading plants to improve their winter hardiness or drought resistance.We are interested to study the molecular basis of cold acclimation and drought response of barley to survive under stress. Several genes regulated by low temperatures and sometimes by drought have been isolated from the barley genome. In this review the most significant results of our recent work will be presented and discussed.The protein encoded by cDNA clone pt59 and induced in barley by cold was over-expressed in E. coli to produce the matching antibody, which in vivo recognizes a cold-induced protein of 14 kDa (COR14). The COR14 is stored in amounts only slightly greater in the cold resistant Onice than in the susceptible Gitane, although the former has a higher induction-temperature threshold of COR14 than the latter. This fact is an evolutionary advantage enabling the resistant varieties in the field to prepare for the cold well ahead of the susceptible ones.Two other cDNA clones, paf93 and cdr29, are regulated by low temperature and drought stress but not by exogenous ABA treatment. Indeed during the early stage of dehydration, the mRNAs are expressed before the induction of known ABA regulated genes such as dehydrins and when only a small increase occurs in ABA content. The sequence analysis revealed that paf93 encodes for a protein homologous to the cold-regulated protein COR47 of Arabidopsis, whereas cdr29 represents a plant gene homologous to yeast and mammalian sequences coding for acyl-Coenzyme A oxidase.Abbreviations ABA abscisic acid - COR cold regulated     

棉花亲环素基因(GhCYP1)克隆及在干旱胁迫下的表达分析

本研究从已构建的棉花(Gossypium hirsutum)干旱胁迫抑制性差减杂交(suppressive subtractive hybridization,SSH)cDNA文库分离得到一个含有植物亲环素保守结构域的EST片段,测序并结合cDNA末端快速扩增(5'-RACE)技术获得了1个779bp的cDNA序列。序列分析表明,该cDNA5'非翻译区为70bp,3'非翻译区为187bp,并含有一个编码174个氨基酸蛋白的开放阅读框。Blast分析表明,该基因的编码产物为一个亲环素蛋白,将该基因命名为为GhCYP1,序列提交到GenBank,登录号为GQ292530。半定量RT-PCR分析表明,干旱胁迫处理后,该基因在叶片中的表达量迅速提高,并在胁迫2h达到最高,这一研究暗示该基因的表达与棉花抗旱胁迫相关。     

甘蔗ABA信号转导关键酶SoSnRK2.1基因的克隆与表达分析

蔗糖非酵解型蛋白激酶(SnRK)是植物体内ABA信号转导途径的关键调控酶, 在植物的抗逆境生长过程中发挥着重要的作用。本研究通过RT-PCR和RACE-PCR技术克隆出编码甘蔗SnRK2蛋白的基因SoSnRK2.1。该基因cDNA序列全长为1385 bp, 包含一个1002 bp的开放阅读框(ORF)。根据氨基酸序列预测SoSnRK2.1基因编码333个氨基酸残基, 与其他高等植物中相关蛋白的氨基酸具有高度的相似性, 尤其是与禾本科的玉米和水稻。构建该基因的原核表达载体pET-SoSnRK2.1, 在IPTG诱导下可得到38 kD 左右的蛋白, 与理论值一致。实时荧光定量PCR分析表明, SoSnRK2.1基因在ABA、干旱(PEG)+ABA、干旱(PEG)、NaCl、低温(4℃)和H₂O₂外源胁迫下均呈诱导表达的趋势。推测该基因参与调控干旱、高盐和低温等胁迫过程, 在甘蔗抗逆境胁迫中起重要作用。     

The drought response of landraces of wheat from the northern Negev Desert in Israel

Summary Diverse landraces of wheat, collected from the semi-arid (150 to 250 mm of total annual rainfall) Northern Negev desert in Israel were considered as a potential genetic resource of drought resistance for wheat breeding. These materials were therefore evaluated for their reponses to drought stress in agronomical and physiological terms. Up to 68 landraces, comprising of Triticum durum, T. aestivum, and T. compactum were tested in two field drought environments, in one favourable field environment, under post-anthesis chemical plant desiccation which revealed the capacity for grain filling from mobilized stem reserves, under a controlled drought stress in a rainout shelter and in the growth chamber under polyethylene glycol (PEG)-induced water stress. Biomass, grain yield and its components, harvest index, plant phenology, canopy temperatures, kernel weight loss by chemical plant desiccation, growth reduction by PEG-induced drought stress and osmotic adjustment were evaluated in the various experiments.Landraces varied significantly for all parameters of drought response as measured in the different experiments, which was in accordance to their documented large morphological diversity. Variation in grain yield among landraces under an increasing drought stress after tillering was largely affected by spike number per unit area. Kernel weight contributed very little to yield variation among landraces under stress, probably because these tall (average of 131 cm) landraces generally excelled in their capacity to support kernel growth by stem reserve mobilization under stress. Yield under stress was reduced with a longer growth duration of landraces only under early planting but not under late planting. Landraces were generally late flowering but they were still considered well adapted phenologically to their native region where they were always planted late.Landraces differed significantly in canopy temperature under drought stress. Canopy temperature under stress in the rainout shelter was negatively correlated across landraces with grain yield (r=0.67^**) and biomass (r=0.64^**) under stress. Canopy temperature under stress in the rainout shelter was also positively correlated across landraces (r=0.50^**) with canopy temperature in one stress field environment. Osmotic adjustment in PEG-stressed plants was negatively correlated (r=–0.60^**) with percent growth reduction by PEG-induced water stress. It was not correlated with yield under stress in any of the experiments. In terms of yield under stress, canopy temperatures and stem reserve utilization for grain filling, the most drought resistant landrace was the Juljuli population of T.durum.     

一个新的棉花MYB类基因(GhTF1)的克隆及染色体定位分析

MYB类转录因子是指含有MYB结构域的一类转录因子, 广泛参与植物发育和代谢调节。含2个MYB结构域的R2R3类MYB转录因子在植物体内主要参与次生代谢的调节和控制细胞的形态发生。从优质材料7235不同发育时期的棉纤维混合cDNA文库中克隆了一个棉花MYB转录因子基因GhTF1(GenBank登录号: EF651783)。该cDNA序列长1 115 bp, 其开放读码框长度为771 bp, 编码256个氨基酸。表达特征分析表明, 该基因在陆地棉7235不同组织中均表达, 但表达量不同, 特别在开花前1 d, 开花后8 d和11 d的纤维细胞中优势表达。该基因在二倍体棉种非洲棉和雷蒙德氏棉中开放读码框区的序列较保守, 但在非编码区差异较大, 在内含子区存在大片段插失和碱基替换现象。Southern杂交结果表明该基因在陆地棉基因组中存在2个拷贝, 推测A、D亚组中各有1个拷贝。利用海7124和TM-1两亲本配置的BC₁作图群体, 将GhTF1定位在染色体10上。     

水稻(Oryza sativa)新型液泡Na+/H+逆向转运蛋白基因的特征分析和表达

以拟南芥AtNHX1 cDNA 片段作为探针，筛查水稻盐胁迫植株叶片cDNA 文库，获得与AtNHX1同源的水稻新型液泡Na⁺/H⁺逆向转运蛋白基因（OsANT1）。序列分析表明，OsANT1 全长cDNA为2 178 bp，包括一个长度为1 608 bp的完整开放阅读框，编码535个氨基酸残基。在DNA水平上，OsANT1基因含有15个外显子和14个内含子，长度为4 835 bp。OsANT1含有12个跨膜域，系统进化树分析结果表明，与来自拟南芥、水稻、小麦、玉米、大麦、马蔺和芦苇等的Na⁺/H⁺逆向转运蛋白高度同源。盐胁迫条件下，OsANT1的表达具有盐分诱导特征，且随着胁迫的增大而增加。表明该基因可能在水稻抵御盐分胁迫的过程中具有一定作用。     

花生AhSOS2基因的克隆及功能初探

SOS2 (Salt Overly Sensitive 2)作为植物中一类重要的耐盐相关基因, 在调控细胞内离子平衡及参与植物对盐害的响应及适应过程中具有重要作用。本研究通过RACE的方法, 从花生叶片中分离到一长1462 bp、包含1341 bp开放阅读框(ORF)的cDNA片段, 生物信息学分析显示, 该基因属SOS2类基因, 被命名为AhSOS2(GenBank登录号为HG797656), 编码446个氨基酸, 为丝氨酸/苏氨酸蛋白激酶。对基因表达特性的荧光定量PCR分析显示, AhSOS2在花生中为组成型表达; 且受盐胁迫及干旱诱导。经250 mmol L^-1 NaCl处理后, 该基因在花生幼苗茎中被诱导表达, 表达量约是对照茎中的30倍; 而在30% PEG-6000模拟干旱处理下, 该基因在花生幼苗叶中表达量也明显升高。综合以上结果, 显示AhSOS2可能参与并调控花生对逆境的抗性及耐受性。目前, 已成功构建了AhSOS2的植物双元表达载体pCAMBIA1301P-AhSOS2并获得转基因植株, 初步功能分析显示, 过表达AhSOS2基因的转基因水稻对盐胁迫的耐受性提高。预期这方面的工作对于解析花生对盐害、干旱等逆境的适应及防御机制, 进而指导花生抗性育种及品质改良具有重要意义。     

Development of two new molecular markers specific to cytoplasmic male sterility in tuber mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tumida</Emphasis> Tsen et Lee)

A novel and stable cytoplasmic male sterility CMS line of tuber mustard has been bred by subsequent backcrosses for 10 years. Two specific markers atpA and orf220 were cloned and partially characterized in our previous study (Zhang et al. 2003). In this study, two new molecular markers, orf256 and orf305/orf324, have been isolated and identified. The orf256 gene size was found to be 825 bp in CMS line and a 1,357 bp in its maintainer line. Sequence analysis indicated that the orf256 gene was an entire coding sequence and downstream of the cox1 gene. Interestingly, the 906 bp fragment, which contains part of the sequence of orf222, nad5 and orf139 genes, was found to be inserted from the 451st bp of 5′-flank of the 1,357 bp fragment. In the same way, the orf324 gene was isolated from CMS line and orf305 gene from its maintainer line. Both of them are entire coding sequences, upstream from nad3 and rps12 gene, and co-transcribed with the nad3 and rps12 genes. In addition, two molecular markers, orf256 and orf324/orf305, have been successfully converted into the SCAR markers. Subsequently, ORF256, ORF324, ORF305 protein and ORF256-M-431 fragment are predicated to contain signal peptide sequences, and ORF220 was predicated to contain signal anchor sequence. RFLP analysis results revealed that all of the molecular markers exhibited polymorphisms. Northern blot analysis indicated that the expression level of these genes in CMS line is higher than that of the maintainer line. In the mass, all of these genes are expressed lower in the leaf than that of floral organs between the CMS line and its maintainer line. The difference in expression pattern of different mitochondrial specific marker genes suggests that the abundance of mitochondrial proteins is differentially regulated in the organ/tissue development in tuber mustard. Results of this study also provide some novel and useful clues to explore the biological function of these specific marker genes in the tuber mustard.     

香蕉乙醇脱氢酶基因的克隆及其逆境胁迫表达

香蕉乙醇脱氢酶基因的克隆及在逆境胁迫下的表达分析,将为进一步研究ADH在香蕉中的功能奠定基础。本研究从香蕉果实抑制差减文库中获得一条香蕉乙醇脱氢酶基因片段,采用RACE技术获得其全长,命名为MaADH。MaADH的cDNA全长1140 bp,编码379个氨基酸。生物信息学分析该基因编码的蛋白分子量为40.7 kDa,等电点为7.09。保守结构域分析发现,该基因具有MDR保守结构域,包含22个NAD结合位点、11个底物结合位点和4个锌结合位点。系统进化树分析表明,MaADH与拟南芥和甘蓝亲缘关系较近。MaADH在盐胁迫和低温胁迫处理的香蕉苗中上调表达;在干旱胁迫下该基因是先上调表达,随后下调表达;在伤害胁迫下是先下调表达后上调表达,但变化不明显。研究说明,香蕉中的乙醇脱氢酶基因在香蕉适应盐、低温、干旱和伤害等逆境中发挥重要的作用。     

The role of Lr34 in imparting durable resistance to wheat leaf rust through gene interaction

Summary The leaf rust responses of wheat lines carrying the complementary genes Lr27 and Lr31 and the same genes in a Chinese Spring background which contains Lr34, indicate that Lr34 interacts with the complementary genes to give enhanced levels of field resistance to leaf rust. Lr34, particularly in combination with other genes, is considered to be an important gene for imparting a high degree of durable resistance to leaf rust. Its similarity to Sr2, an adult plant gene for resistance to stem rust and its association with adult plant resistances to stem and stripe rusts are discussed.     

棉花腺苷高半胱氨酸水解酶cDNA的克隆、表达及染色体定位

腺苷高半胱氨酸水解酶是调节细胞内甲基反应的一个关键酶。通过对高品质纤维陆地棉品系7235的棉纤维混合cDNA文库随机测序, 得到一个棉花腺苷高半胱氨酸水解酶(编号: g073a03a,GhSAHH)的cDNA序列。该cDNA序列长1 598 bp, 利用5′RACE技术得到上游318 bp的片段,序列拼接获得全长为1 916 bp的cDNA序列, ORF为1 458 bp, 编码485个氨基酸, 其理论上的等电点pI＝5.69, 分子量MW＝53.2 kD。该基因在不同组织、器官中均表达, 在根、下胚轴和纤维发育早期优势表达。根据Southern杂交结果推测GhSAHH基因在陆地棉基因组中为单拷贝。利用本实验室陆地棉遗传标准系TM-1和海岛棉海7124培育的含140个单株的BC1作图群体, 将GhSAHH基因定位在第20号染色体上。     

Evaluation of seedling and adult plant resistance to stem rust in European wheat cultivars

A total of 105 European wheat cultivars were assessed for seedling and adult plant resistance (APR) to stem rust using an array of Australian isolates of Puccinia graminis f. sp. tritici. Twenty-seven cultivars were susceptible at both seedling and adult plant growth stages. Twelve catalogued seedling stem rust resistance genes (Sr7b, Sr8a, Sr8b, Sr9b, Sr9g, Sr11, Sr15, Sr17, Sr29, Sr31, Sr36 and Sr38) were detected in the remaining cultivars, and 13 cultivars carried additional seedling resistance genes that could not be postulated with the isolates used. Low levels of APR to stem rust were found in the cultivars Artaban, Forno, Mec, Mercia, Pandas and Vlada. Although the genetic identity of this APR was not determined, it was clear that the only designated stem rust APR gene Sr2 was not present in any of the cultivars tested based on the absence of the linked traits seedling chlorosis and pseudo black chaff. One of these cultivars, Forno, is believed to carry the leaf rust APR gene Lr34, previously reported to be associated with improved resistance to stem rust. A detailed genetic characterisation of the APRs in these cultivars will be needed to understand their modes of inheritance and relationships with catalogued stem rust resistance genes. Such knowledge may help in developing cultivars with effective gene combinations that confer higher levels of protection.     

Androgenesis in a Lolium multiflorum × Festuca arundinacea hybrid to generate genotypic variation for drought resistance

The pentaploid hybrid of Lolium multiflorum and Festuca arundinacea (2n = 5x = 35) combines the high growth rate of L. multiforum with the drought resistance and freezing-tolerance of F. arundinacea. Unfortunately, it also displays the deleterious traits associated with Festuca, namely those associated with high leaf fibre content giving rise to poor palatability and digestibility. To access different combinations of these characters, anther cultures were initiated and regenerated into single embryo derived plants. The anther culture method was very productive since out of a total of 2349 androgenic plants derived from the same parent plant, 57% were green plantlets, although only 507 (22%) subsequently established into plants following transfer to soil. Chromosome counts of randomly selected lines showed that plants with euploid chromosome numbers (14, 21, and 28) would appear to have selective advantage during regeneration. There was wide variation between mature androgenic plants grown under field conditions in plant height, leaf length, leaf width, tiller number and herbage dry matter. The variation between genotypes in response to drought stress was assessed by placing replicate clones under rain-out shelters or under irrigated control conditions in the field. Herbage dry matter under drought was higher in a number of androgenic lines than either parents, but not higher than the pentaploid hybrid. Androgenesis was shown to be a highly effective procedure to expose diverse phenotypic variation all derived from the same Lolium × Festuca hybrid genotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular markers in some medicinal plants of the Apiaceae family

The internal transcribed spacers ITS1 and ITS2 in the18S-5.8S-26S rDNA repeat units were amplified and cloned from Angelica gigas Nakai, Angelica acutiloba (Siebold & Zucc.) Kitagawa, A. dahurica Maxim, Angelica decursiva (Miq.) Franch. & Savat, Bupleurum falcatum L. and Peucedanum japonicum Thunb. Sequence analyses showed that ITS1 is approx. 215 bp, the 5.8S gene is 162 bp and the ITS2 approx. 221 bp in all six species. The sequences are deposited at the EMBL Nucleotide Database. By including these new sequences in the Apiaceae phylogenetic tree, a third branch consisting of P. japonicum, A. gigas, P. decursivum and A. decursiva is added to theAngelica clade. Peucedanum does not forma distinct branch. The sequence obtained from Angelica dahurica collected in S. Korea is identical to that reported for the same species originating from China. A Bupleurum clade of three species was added to the tree showing closer relationship to theDaucus Laserpitium clade than the Angelica clade. RAPD analysis of all six species showed that the 10-base primer OPC-17 only, out of the20 Kit-C primers from Operon gave polymorphic banding patterns. This revised version was published online in July 2006 with corrections to the Cover Date.     

巴西橡胶树NAC转录因子HbNAC1基因的克隆及生物信息学分析

为了研究植物特有的NAC(NAM,ATAF1/2和CUC2)转录因子在巴西橡胶树生长发育、胁迫应答和激素调节中的功能,对巴西橡胶树NAC转录因子进行了克隆和分析。根据橡胶树胶乳cDNA文库中筛选到的NAC EST序列,利用RACE-PCR技术克隆了橡胶树HbNAC1的cDNA序列。生物信息学分析显示HbNAC1基因开放阅读框(ORF)为891 bp,编码了一个296个氨基酸组成的中性不稳定水溶性蛋白,该蛋白的N-端具有保守的NAC结构域,C-端高度变异,具备了NAC转录因子的基本特征。HbNAC1定位在细胞核中,具有3个糖基化位点和21个磷酸化位点,三级结构由3个α-螺旋和9个β-折叠组成。序列比对和系统进化分析显示HbNAC1蛋白属于NAC转录因子家族中的ANAC011亚族,推测其可能作为一种氨基酸合成相关的调节酶参与调控植物的免疫反应。     

Cytogenetical studies in wheat XIX. Location and linkage studies on gene Yr27 for resistance to stripe (yellow) rust

The stripe (yellow) rust resistance gene Yr27 was located in wheat (Triticum aestivum L.) chromosome 2B and shown to be closely linked to the leaf (brown) rust resistance genes Lr13 and Lr23 in the proximal region of the short arm. Gene Yr27 was genetically independent of Lr16, which is distally located in the same arm. While Yr27 was often difficult to score in segregating seedling populations, it is apparently quite effective in conferring resistance to avirulent cultures under field conditions. The occurrence of Yr27 in Mexican wheat germplasm and the current over-dependence on Yr27 for crop protection in Asia are discussed.     

Tissue culture-derived variation in crop improvement

Tissue culture generates a wide range of genetic variation in plant species which can be incorporated in plant breeding programmes. By in vitro selection, mutants with useful agronomic traits, e.g. salt or drought tolerance or disease resistance, can be isolated in a short duration. The successful use of somaclonal variation is very much dependent on its genetic stability in the subsequent generations for which molecular markers such as RAPDs, AFLPs, SSRs and others can be helpful. The potential of somaclonal variation has yet to be fully exploited by breeders, even though a few cultivars have been developed in crops such as Brassica juncea, rice and others. This revised version was published online in July 2006 with corrections to the Cover Date.     

Induced mutations – A new paradigm in plant breeding

The use of ionizing radiation, such as X-rays, gamma rays and neutrons and chemical mutagens for inducing variation, is well established. Induced mutations have been used to improve major crops such as wheat, rice, barley,cotton, peanuts, and beans, which are seed propagated. Since the establishment of the Joint FAO/IAEA Division of the Nuclear Techniques in Agriculture, more than 1800 cultivars obtained either as direct mutants or derived from their crosses have been released worldwide in 50 countries. In vegetatively propagated plants, many of mutants were derived from irradiating rooted stem cuttings, detached leaves, and dormant plants. According to the FAO/IAEA database, of the 465 mutants released among the vegetatively propagated plants, most are in the floricultural plants and a few in fruit trees. These include chrysanthemum, Alstroemeria, dahlia, bougainvillea, rose, Achimenes,begonia, carnation, Streptocarpus, and azalea. The irradiation of in vitro cultured date palm, apple, potato, sweet potato and pineapple now provides a means to treat large populations which would not have been possible before. Irradiation of micropropagated plants, axillary and adventitious buds, apical meristems, regenerative callus cultures, anthers and microspores, and somatic embryos provides a miniaturized version of trees and seeds in the Petridish instead of the field. During the last decade, the use of radio-actively labeled probes in recombinant DNA research for cloning and mapping plant genes and transgenesis, particularly for RFLP, micro satellite based DNA fingerprinting, has become a routine procedure. Many homeotic mutants that change floral development have been isolated in Arabidopsis, Petunia, Antirrhinum and Lycopersicon. Mutants of Arabidopsis are being used to analyze genes, which determine response to auxins, cytokinins, gibberellin, abscisic acid and ethylene in plant growth, floral development and senescence, fruit formation and ripening. These mutants are facilitating the isolation, identification and cloning of the genes, which would ultimately help in designing crops with improved yield, increased stress tolerance, longer shelf-life and reduced agronomic inputs. The identification and analysis of mutants by using molecular techniques of DNA fingerprinting and mapping with PCR based markers, such as RAPDs, AFLP and STMS, and mutant tagging shall bring a new dimension in gene technology. Already, mutations can be linked to changes in DNA sequences for some plant traits and to establish molecular maps in structural and functional genomics of crop plants. These in turn would lead to a rapid enhancement of crop yields and quality. This revised version was published online in July 2006 with corrections to the Cover Date.