植物逆境相关C2H2型锌指蛋白的研究进展

锌指蛋白是转录因子的一种,因其具有指状结构特征且能结合Zn2+而得名。植物C2H2型锌指蛋白是研究较多、较为明确的一种,大多包含特有的QALGGH保守结构。该蛋白广泛参与到植物的生长发育和逆境胁迫信号转导过程中。本研究主要综述了植物C2H2型锌指蛋白的结构、功能、分类及逆境相关的作用机制,概括了C2H2型锌指蛋白在参与植物非生物和生物胁迫耐受性的研究成果,并对其进一步的深入研究进行了展望,为将来利用锌指蛋白基因提高作物抗逆性提供较为全面的资料。     

水稻OsCCCH基因家族的组织表达谱及胁迫诱导表达特征研究

CCCH型锌指蛋白是一类生物体中广泛存在的转录因子,在多种植物生物学过程中起着关键的调控作用。在水稻全基因组中已发现67个CCCH基因,分为8个亚家族,而其中大部分基因的功能尚缺少研究。本研究通过荧光定量RT-PCR技术分析了水稻Os CCCH家族基因的组织表达模式以及盐、PEG、低温等非生物胁迫和脱落酸(ABA)对CCCH基因的诱导表达特征。结果表明,大部分Os CCCH基因在各组织中有不同的表达模式,约30%基因为低丰度表达,50%以上基因表现为中丰度表达,极少Os CCCH基因为高丰度表达,包括Os C3H2、Os C3H35、Os C3H41、Os C3H43、Os C3H58和Os C3H67这6个组织优势表达基因。多数Os CCCH基因都不同程度地受到盐、PEG、低温及ABA的诱导或抑制,表明这些CCCH锌指蛋白可能参与水稻的非生物胁迫响应;在四种胁迫下,有7个基因同时受到诱导,其中Os C3H17基因上调倍数高达17倍以上;有10个基因同时被抑制,其中Os C3H16基因下调倍数高达31倍以上。     

玉米C2H2型锌指蛋白基因ZFP225的鉴定、生物信息学分析与克隆

使用生物信息学方法从玉米中预测并克隆了一个锌指蛋白基因,该基因定位于玉米7号染色体上,编码一个由219个氨基酸残基组成并且具有双锌指结构域的C2H2型锌指蛋白,分子量和等电点分别为22.53kDa和8.812。根据其编码蛋白分子量大小将其命名为ZFP225。ZFP225可能具有一个转录抑制活性的DLN-box,与植物中的C2H2型锌指蛋白具有较低的一致性,只在比较保守的锌指区具有相似性。进化分析表明,ZFP225与水稻锌指蛋白ZFP252亲缘关系最近,亲缘关系较近的锌指蛋白多参与植物的胁迫应答反应,而亲缘关系较远的蛋白多数参与调控植物的生长发育。使用RT-PCR方法,从玉米幼苗中成功克隆出了ZFP225基因。     

棉花C3HC4型RING锌指蛋白基因GhSIZ1克隆及表达分析

植物抗逆分子机制非常复杂,克隆并鉴定新的逆境相关基因,有助于解析植物抗逆机理。C3HC4型锌指蛋白在植物生长发育和抵抗非生物胁迫中发挥重要作用。本研究从陆地棉‘Y1169’中克隆了1个C3HC4型锌指蛋白的基因GhSIZ1 (XM＿041085878.1),并分析GhSIZ1对干旱、盐胁迫和脱落酸的转录响应。序列分析表明该基因cDNA全长3 389 bp,含有1个2 613 bp的开放阅读框,利用生物信息学分析了棉花GhSIZ1基因的理化性质及其结构功能域,结果显示棉花GhSIZ1基因编码870个氨基酸,为亲水性蛋白,预测分子量约为279.39 k D,理论等电点为4.86,脂肪指数为29.06。GhSIZ1中含有1个C3HC4型RING锌指结构域。系统进化树分析表明Gh SIZ1与达尔文棉(Gossypium darwinii) TYG＿87958.1、海岛棉(Gossypium barbadense)KAB＿2050370.1和夏威夷棉(Gossypium tomentosum) TYH＿93561.1等植物的蛋白质相似性较高,与达尔文棉TYG＿87958.1处于同一进化枝上,亲缘...     

SUPERMAN类单锌指蛋白的研究进展

植物SUPERMAN类锌指蛋白属于C2H2型锌指蛋白中的单锌指蛋白,具有N端QALGGH和C端富含亮氨酸L(I)DLXLR(K)L的保守结构域,主要在植物花发育、胚发育、种子发育、侧根和分枝发育过程中起重要作用。本文阐述了SUPERMAN类单锌指蛋白在结构、作用机制、功能等方面的研究进展。随着不同作物中SUPERMAN家族的基因克隆,其生理功能和对植物生长发育的调控机制将更清晰,为进一步的基因遗传转化奠定了基础。     

海岛棉GbWRKY40基因的克隆及特征分析

【目的】WRKY转录因子调控多种生物学进程,包括植物生长发育和应答多种环境胁迫。本研究旨在分析WRKY转录因子在海岛棉纤维发育中的功能。【方法】从海岛棉中克隆了1个WRKY转录因子基因GbWRKY40,进行同源性分析、多序列比对,利用荧光定量聚合酶链式反应分析其表达模式,通过构建酵母表达载体并转化酵母菌株AH109研究其转录激活活性。【结果】该基因c DNA全长1713 bp,5'非编码区长261 bp,3'非编码区长510 bp;开放阅读框长942 bp,编码313个氨基酸,预测相对分子质量约为34.138×103,等电点为8.46,包含5个外显子和4个内含子;其编码蛋白含有1个WRKY保守区(WRKYGQK)和1个锌指基序(C-X5-C-X23-H-X1-H),属于WRKY家族第Ⅱ类a组,包含3个核定位信号区,与陆地棉GhWRKY40同源性最高。GbWRKY40在根和开花后25 d纤维中表达量高,而且不具有转录激活活性。【结论】GbWRKY40可能参与调控棉纤维次生壁发育。     

海岛棉GbWRKY32基因的克隆及特性分析

利用RT-PCR技术从海岛棉品种新海21中克隆了1个WRKY基因,在GenBank数据库中比对发现其与陆地棉GhWRKY32的序列高度同源,因此命名为GbWRKY32。该基因ORF为1 077 bp,编码358氨基酸,预测分子量为39.288 k D,等电点为5.02。序列分析表明该基因含有一个WRKY保守结构域,锌指结构为:C-X5-C-X23-H-X1-H,属于WRKY转录因子家族Ⅱ类D组成员。GbWRKY32蛋白为疏水性蛋白,不具有跨膜区和信号肽结构。GbWRKY32转录因子不具有转录自激活活性。本研究成功构建GbWRKY32基因的植物表达载体并转入根癌农杆菌EHA105菌株中,这有助于该基因功能的后续研究。     

拟南芥中一个锌指蛋白AT 5 g 47610的基因克隆及表达分析

逆境胁迫是植物正常生长发育与作物产量提高的重要限制因素,具有锌指结构的E 3泛素连接酶作为一个重要的蛋白家族,在植物逆境胁迫应激中起着重要的调控作用。本文从一个含有锌指结构的蛋白AT 5 g 47610出发,通过PCR对其基因进行克隆,然后连接到PET 32 a原核表达载体,37℃通过0.2 mM IPTG诱导出了目标蛋白,为后续的蛋白功能鉴定研究奠定了基础。     

植物C_2H_2型锌指蛋白研究进展

C_2H_2型锌指蛋白是锌指家族中最经典也是目前研究最为深入的一类锌指蛋白。植物中该类锌指蛋白大多具有高度保守的基序QALGGH,并且该序列除了Q其他任何一个氨基酸的突变都会导致该蛋白完全失去结合DNA的功能,而Q突变则会大大降低结合DNA的能力。本综述综述了C_2H_2型锌指蛋白的结构特征、分类方法以及近年来的功能研究成果,系统介绍了植物中该类蛋白在生长发育、非生物胁迫如干旱胁迫、盐胁迫以及冷胁迫等方面的作用,为今后C_2H_2型锌指蛋白功能研究的深入奠定基础。     

大豆胁迫相关蛋白GmSAP3基因的克隆和表达分析

胁迫是影响植物生长发育和农作物产量的重要因素.胁迫相关蛋白(stress associated protein,SAP)是一类锌指蛋白,广泛参与植物发育和胁迫响应.为了研究大豆SAP基因的功能,本研究以'商豆1201'为材料克隆了GmSAP3基因,并进行了生物信息学分析,采用RT-PCR分析该基因在大豆不同组织和温度胁迫下的表达模式.结果 表明,GmSAP3基因CDS序列全长为513 bp,编码170个氨基酸,GmSAP3蛋白等电点pI为6.79,分子量为18.30568kD;序列分析发现,GmSAP3含有2个保守结构域(zf-A20和ZnF-AN1),序列比对和进化分析发现GmSAP3与GmSA P26亲缘关系最为接近.RT-PCR结果表明,GmSAP3在大豆不同组织中均有表达,其中根和叶片表达较高;GmSAP3受高温胁迫诱导表达,推测该基因在大豆胁迫响应中具有重要作用.本研究为GmSAP3基因功能研究提供了一定的理论支持.     

In-vitro Fertilization of Ovules of Some Species of Brassicaceae*

The method of in-vitro fertilization of ovules can be successfully applied to various species of Brassincaceae. Mature embryos and plants were obtained after in-vitro pollinating the ovules of Arabis caucasica, Brassica napus, B. oleracea var. sabellica (kale), B. oleracea var. italica (broccoli), Diplotaxis tenuifolia, Moricandia arvensis and Sisymbrium Loeselli. In the case of Sinapis alba fertilization and embryo development did not occur. The same method has been successfully used for obtaining hybrid immature embryos at different stages of development from crosses between B. napus X D. tenuifolia, B. napus X M. arvensis, B. oleracea var. italica X D. tenuifolia, D. tenuifolia x B. napus, D. tenuifolia. X M. arvensis and D. tenuifolia X S. Loeselli. The present findings show that in-vitro pollination of ovules of various species of Brassicaceae makes it possible to (a) perform the whole process of fertilization and embryogenesis; and (b) obtain intergeneric hybrid embryos.     

The potential role of PR-8 gene of apple fruit in the mode of action of the yeast antagonist,Candida oleophila,in postharvest biocontrol of Botrytis cinerea

Several pathogenesis-related (PR) genes of apple have been cloned and their response to different pathogens has been studied. Different PR genes, however, may have a variable response depending on the specific organ or tissue as well as microbe. The objective of the current study was to characterize the expression of specific apple PR genes in fruit tissues in response to the antagonistic yeast, Candida oleophila (a common postharvest biocontrol agent), and the fungal pathogen, Botrytis cinerea (a major postharvest pathogen). Apple PR-5 and PR-8 gene expression was characterized in fruit in response to C. oleophila and B. cinerea. Results indicated that PR-8 expression was significantly elevated in response to both fungi. In contrast, neither C. oleophila nor B. cinerea treatment markedly affected PR-5 expression. The PR-8 gene was then synthesized and cloned into a Pichia pastoris expression system to study the antifungal activity of the PR-8 protein against B. cinerea both in vitro and in vivo. Collectively, the PR-8 gene of apple is associated with the response to B. cinerea infection, and may play a role in the mechanism by which C. oleophila effectively inhibits B. cinerea disease in apple fruit, namely by the induction of this specific host PR gene.     

灰分对煤自燃特性影响的实验研究

为了研究灰分对煤自燃能力的影响作用,利用绝热氧化实验装置对不同灰分含量煤样进行升温氧化实验,采用R₇₀、T_CPT、B 3种指标表征灰分含量对煤样自发氧化过程的影响。结果表明：1）灰分含量越大,煤样低温氧化阶段温升速率越小,温升加速点温度越高,煤样的自发氧化过程越慢,煤越不易自燃;灰分含量大于40%后,煤自燃倾向性快速减弱。温升加速点是反应微观信息的零活化能温度的宏观累计结果,具有直观且滞后的特点。灰分越大,滞后越明显,温差越大。2）R₇₀、T_CPT、B 3种指标与灰分关系表现为二次函数。R₇₀和T_CPT两种指标显示灰分越大,自燃倾向性越弱,与实践经验相符。受水分权重影响,B指标显示煤样在灰分小于40%时,灰分越大,煤样自燃倾向性越强,这与实践经验相悖。因此,B在判定灰分对煤样自燃倾向性的影响时具有一定的局限性。     

Yield Performance of Wheat Isolines with Different Dosages of the Short Arm of Rye Chromosome 1

Translocations of the short arm of rye (Secale cereale L.) chromosome 1 (1RS) in wheat (Triticum aestivum L. cv. Pavon 76) are known to increase root biomass. Such an increase enhances water and nutrient uptake and may improve grain yield. Two greenhouse experiments and a field experiment were carried out at the University of California, Riverside, in 2012 and 2013 under well‐watered and terminal drought treatments to evaluate phenotypic characters associated with varying dosages of 1RS, including grain yield. The genotypes used were cultivar Pavon 76 (R₀), Pavon 76/Pavon1RS.1AL (F₁ hybrid) with a single dosage of 1RS (R₁A), Pavon 1RS.1AL with two dosages of 1RS (R₂A), Pavon 1RS.1DL (R₂D) also with two dosages of 1RS and Pavon 1RS.1AL‐1RS.1DL (R₄AD) with four dosages of 1RS. There was a significant positive correlation between number of dosages of 1RS and root biomass. However, no correlation was found between root biomass and grain yield per plant. Drought in the field experiment reduced grain yield significantly. Under well‐watered field conditions, grain yield of R₂A (215.9 g plant^?1) was significantly greater than those of R₂D (191.8 g plant^?1) and R₄AD (161.7 g plant^?1). Also, grain yield of R₄AD was significantly less than those of F₁, Pavon 76 and R₂D under well‐watered conditions. Under drought field conditions, no significant differences were found among the genotypes for grain yield was found between F₁ (14.7 g plant^?1) and R₄AD (12.4 g plant^?1). Harvest index was significantly greater in well‐watered (44.2 %) than in drought (34.6 %) field conditions. On average, genotypes F₁ (42.3 %) and R₂A (40.6 %) had higher harvest index than R₂D (38.3 %) and R₄AD (35.5 %) in the field. Also, Pavon 76 (40.2) and R₂D (38.3) had higher harvest index than R₄AD. Drought tolerance was lowest for R₄AD due to its relatively lower grain yield potential. In general, Pavon 1RS.1AL carrying two dosages of 1RS showed higher grain yield under wet treatments. Pavon 1RS.1AL‐1RS.1DL carrying four dosages of 1RS produced the largest shoot and root biomasses, but the least grain yield.     

Evolutionary studies of the soybean: the frequency and distribution of alleles among collections of Glycine Max and G. Soja of various origin

Summary Allelic frequencies at 10 loci common throughout the genus Glycine subgenus Soja were determined for 27 geographical area-species samples. The samples included 366 accessions of Glycine soja, the wild soybean, and 193 accessions of G. max, the domesticated soybean, from the USDA Soybean Germplasm Collection. The data indicate that the alleles for grey pubescence (t ₁), low seed coat peroxidase level (ep), and blunt pubescence tip (pb) probably arose as mutations during the domestication of G. max. The remaining seven loci studied(Fr, Pa ₁, Pa ₂, Fg ₁, Fg ₂, Fg ₃and Fg ₄)are polymorphic throughout the subgenus Soja; differences among collections of G. max and G. soja seem to be the result of differing selection pressures. A cluster analysis of allelic frequencies reveals two distinct groups within the subgenus corresponding to G. soja and G. max, Semi-wild accessions of G. max, while morphologically more similar to cultivated plants, clustered with samples of G. soja. The semi-wild accessions examined are thought to have arisen via hybridization between G. soja and G. max.Joint contribution: North Central Region, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, and Journal Paper No. J-9868 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011; Project 2107.     

Genetic control of crossability of triticale with rye

Limited genetic knowledge is available regarding crossability between hexaploid triticale (2n= 6x= 42, 21″, AABBRR, amphiploid Triticum turgidum L.‐Secale cereale L.) and rye (2n= 14, 7″, RR). Our objectives were to determine (1) the crossability between triticales and rye and (2) the inheritance of crossability between F₂ progeny from intertriticale crosses and rye. First, ‘8F/Corgo’, a hexaploid triticale, was crossed as a female with two landrace ryes, ‘Gimonde’ and, ‘Vila Pouca’ and two derived north European cultivars, ‘Pluto’ and ‘Breno’. These crosses produced 21.7, 20.9, 5.9, and 5.6%, seed‐set or crossability, respectively, showing that the landrace ryes produced higher seed‐set than the cultivars. Second, ‘Gimonde’ rye was crossed as a male with four triticales for 3 years. The control cross, ‘Chinese Spring’ wheat × rye, produced 80‐90% seed‐set. Of the four triticales, ‘Beagle’ produced 35.7‐56.8% seed‐set. The other three triticales produced less than 20% seed‐set, showing that the triticales differ in crossability with ‘Gimonde’ rye. Third, six FiS from intertriticale crosses (‘8F/Corgo’בBeagle’, ‘Beagle’בCachirulo’, ‘Lasko’בBeagle’, ‘8F/Corgo’בCachirulo’, ‘Lasko’בCachirulo’, ‘Lasko’ב8F/Corgo’) were crossed to ‘Gimonde’ rye. Results indicated that lower crossability trait was partially dominant in the two F₁S from crosses involving ‘Beagle’(high crossability) with‘8F/Corgo’ and ‘Cachirulo’(low crossability) and completely dominant in the ‘Beagle’בLasko’ cross, as it happens in wheat. Fourth, segregants in four F₂ populations (‘Lasko’בBeagle’, ‘8F/Corgo’בBeagle’, ‘Lasko’ב8F/Corgo’, and‘8F/Corgo’בCachirulo’) were crossed with rye. Segregation for crossability was observed, although distinct segregation classes were blurred by environmental and perhaps other factors, such as self‐incompatibility alleles in rye. Segregation patterns showed that ‘Beagle’, with high crossability to rye, carries either Kr1 or Kr2. The three triticales with low crossability with rye were most likely homozygous for Kr1 and Kr2. Therefore, it is likely that the Kr loci from A and B genomes acting in wheat also play a role in triticale × rye crosses.     

Characterization of sensitivity of grove and packing house isolates of Penicillium digitatum to pyrimethanil

In most northeast Argentinean citrus packing houses, postharvest fungicide treatments are based on the use of thiabendazole and imazalil. However, these fungicides have been used in a manner highly conducive to the selection and proliferation of resistant biotypes of Penicillium digitatum, the main fruit decay fungus in the area. Recently, a new fungicide, pyrimethanil (PYR), was introduced to control molds. Aims of this study were to determine the baseline sensitivities for PYR against isolates of P. digitatum considering its use in the region is not yet widespread and to evaluate the control of the fungus in vivo. One hundred and nine (109) P. digitatum isolates were collected from diseased fruit within citrus groves (43 isolates) and packing houses (66 isolates). EC₅₀ was determined for each isolate by measuring colony diameters on different agar dilutions of the fungicide. The mean EC₅₀ value of the green mold isolates collected from the groves was 0.14 ± 0.03 mg L⁻¹ while the mean EC₅₀ of those collected from packing houses was 0.13 ± 0.05 mg L⁻¹. No resistant isolates were found in the field where the fungicide is not used, while one isolate originated from a packing house showed an EC₅₀ of 3.40 mg L⁻¹, 26-fold higher than the mean level. This isolate was collected from lemons stored in cool rooms of a packing house where PYR had not been used. Fruit decay by sensitive isolates was reduced approximately 80% by PYR applied at 500–600 mg L⁻¹ by immersion for 60 s at room temperature to inoculated oranges and mandarins. In contrast, the resistant isolate was not controlled by PYR applied at 1000 mg L⁻¹. Thus, the introduction of PYR applied into packing houses should be done carefully and control strategies should be implemented in order to minimize the development of resistant isolates.     

Analysis of mode of inheritance of Fusarium wilt resistance in castor (Ricinus communis L.)

Castor (Ricinus communis L.) is an important industrial oilseed crop grown worldwide. Wilt caused by Fusarium oxysporum f.sp. ricini is a devastating disease in castor. The inheritance mode of wilt resistance was investigated. The F₁, F₂ and backcross generations of four crosses involving four resistant and three susceptible parents were developed. The role of digenic (R¹ and R²) epistatic interactions on wilt resistance was confirmed. The 15 : 1, 9 : 7 and 13 : 3 ratios indicated duplicate dominant, duplicate recessive and dominant and recessive epistatic interactions, respectively. Castor parents used in the crosses exhibited varied inheritance modes. All generations of a cross exhibited similar inheritance mode when parents were comparable. However, generations varied in inheritance mode when parents were not comparable in inheritance mode. These results would have practical interest when decisions are required regarding the choice of parents and methodology in resistance and hybrid breeding. The results also provided a basis for investigating molecular genetics of wilt resistance mechanisms.     

Distribution of S haplotypes in commercial cultivars of Brassica rapa

Self‐incompatibility in Brassicaceae plants is sporophytically controlled by a single multi‐allelic locus (S locus), which contains at least three highly polymorphic genes expressed in the stigma (SLG and SRK) and in the pollen (SCR/SP11). Using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis with SXG‐specific primer pairs, the S haplotypes of F₁ hybrid and open‐pollinated commercial cultivars of Brassica rapa were identified. The number of S haplotypes detected in the F₁ hybrid cultivars of Chinese cabbage, komatsuna, pak‐choi, turnip, open‐pollinated cultivars of Chinese cabbage and turnip were 9, 9, 4, 11, 13 and 12, respectively. Nine of them had different PCR‐RFLP profiles from those of the S‐tester lines that determined the SLG sequences. Four SLG sequences in the F₁ hybrid cultivars were determined and named S⁵³, S⁵⁴, S⁵⁵ and S⁵⁶, respectively. It is demonstrated that the PCR‐RFLP analysis using specific primer pairs of SLG and SRK is useful for identification of the S haplotypes, in both, S homozygous and S heterozygous plants of B. rapa. The possibility of using this method routinely in breeding programmes, and in the evaluation of F₁ hybrid seed purity, is discussed.     

添加硝酸钙对池塘沉积物中理化因子的影响研究

为减少池塘底泥对养殖水环境的影响,以硝酸钙和养殖池塘底泥沉积物为研究对象,采用模拟试验方法,探讨了添加硝酸钙对池塘沉积物理化因子的影响。结果表明：添加硝酸钙后,表层沉积物间隙水中NH₄⁺-N浓度持续降低,最终降至初始值的14.78%;同时可以显著降低表层和深层沉积物间隙水中的PO₄^3--P的浓度,在表层降低的幅度较大,第7~28天可降低超过85%的PO₄^3--P。试验结果表明,添加硝酸钙可以显著改善池塘底泥沉积物中NH₄⁺-N和PO₄^3--P的含量,研究结果对减少池塘底泥沉积物的修复具有积极的生态学意义。