生理成熟期高温高湿胁迫对春大豆种子生理特性及种子劣变相关基因GmSbh1表达的影响

以种子田间劣变抗性品种"湘豆3号"和不抗品种"宁镇1号"春大豆为试材,研究了生理成熟期高温高湿胁迫[T:40±2℃/30±2℃、RH 95±5%/70±5%、10 h/14 h(昼/夜),CK:30±2℃/20±2℃、RH 75±5%/70±5%、10 h/14 h(昼/夜)]1、5、10、16、24、48、96和168h对种子萌发、膜脂过氧化、渗透调节物质(脯氨酸和可溶性糖)含量、细胞膜保护酶系(SOD、POD、CAT)活性以及种子劣变相关基因Gm Sbh1表达的影响。结果表明:高温高湿胁迫后,发芽势和发芽率呈下降趋势,短时间胁迫(10 h)下降不明显,胁迫24 h后下降显著,各胁迫时间点,不抗品种下降的幅度大于抗性品种;种子细胞膜透性增加,发生膜脂过氧化作用,胁迫5 h后膜脂过氧化作用加剧;相同胁迫时间下,"湘豆3号"的膜脂过氧化程度显著小于"宁镇1号";种子中的脯氨酸(Pro)和可溶性糖含量明显增加,处理显著高于对照;各胁迫时间点,与对照相比,"湘豆3号"的Pro含量上升的幅度显著高于"宁镇1号",可溶性糖含量上升的幅度两品种间差异不明显;随胁迫时间的延长,处理组细胞膜保护酶系活性先升高后降低,对照组酶活性变化不大,各胁迫时间点处理显著高于对照;与对照相比,胁迫后"湘豆3号"种子的酶活性升高幅度大于"宁镇1号",且"湘豆3号"在胁迫后维持高水平酶活性的时间长于"宁镇1号";"宁镇1号"种子劣变相关基因Gm Sbh1呈上调表达趋势,"湘豆3号"呈下调表达趋势。由此认为,春大豆种子田间劣变抗性的生理与分子机制可能与细胞膜透性增加、膜脂过氧化作用加剧、渗透调节物质含量增加、抗氧化酶活性降低及种子田间劣变抗性相关基因Gm Sbh1的差异表达有关。     

生理成熟期高温高湿胁迫对春大豆种子活力、主要营养成分及种皮结构的影响

采用盆栽方式,研究了宁镇1号和湘豆3号2个春大豆品种在种子生理成熟期受到高温高湿胁迫[(40±2)℃/(30±2)℃、相对湿度(95±5)%/(70±5)%、10 h/14 h(昼/夜)]后种子活力指标、主要营养成分及种皮解剖结构的变化.结果表明:生理成熟期植株受到高温高湿胁迫后,2个春大豆品种种子生活力、发芽势、发芽率以及脱氢酶、酸性磷酸酶活性呈下降趋势,种子细胞膜透性增大,浸种液中的H+、可溶性糖和亮氨酸含量上升,种子中的脂肪和蛋白含量无规律性变化,种皮与种脐解剖结构发生改变;短期的高温高湿胁迫(5 h)对春大豆种子活力的影响不明显,胁迫时间超过48 h,种子活力相关指标急剧降低;湘豆3号较耐高温高湿胁迫,表现为发芽势和相关酶活性下降及浸种液外渗物含量增加的幅度小,种皮致密、厚实,种脐结构完整等特征.     

水稻资源GXM-001-2对稻瘿蚊的抗性鉴定与遗传分析

稻瘿蚊对南方水稻的危害日趋严重,育种上急需新的抗源。利用广西地方品种GXM-001-2作父本,分别与感虫品种TN1和已知抗性基因载体品系W1236(Gm1)、IET2911(Gm2)、BG404-1(gm3)、OB677(Gm4)、ARC5984(Gm5)、多抗1号(Gm6)杂交、自交和回交,获得F1、F2、BC1F1群体,对亲本和各杂交后代进行稻瘿蚊的抗性评价及遗传分析。结果表明,抗源GXM-001-2高抗稻瘿蚊中国Ⅱ型,抗中国Ⅳ型,且抗性均由1对显性基因控制;等位性测定表明抗源中的抗性基因与已知抗性基因Gm1、Gm2、gm3、Gm4、Gm5、Gm6不等位,推测该基因可能是1个新的抗稻瘿蚊基因。     

大豆GmGolS2基因启动子的克隆及功能分析

本研究利用PCR技术克隆了大豆Gm Gol S2基因的启动子序列。系统进化树分析表明,Gm Gol S2与沙冬青的Gols亲缘关系最近。利用Plant CARE分析启动子元件发现,在Gm Gol S2基因启动子序列中含有多个与逆境胁迫、激素等反应相关的元件。在The Bio-Analytic Resource for Plant Biology数据库中分析了Gm Gol S2的同源基因At Gol S2在不同逆境胁迫处理时的基因表达情况,结果表明该基因具有组织表达特异性,并参与盐胁迫、渗透胁迫及干旱胁迫等反应。RT-PCR结果表明Gm Gol S2基因受干旱诱导上调表达。     

阜豆GmABC基因的克隆及表达分析

利用RACE技术从大豆品种‘阜豆11号’中克隆到1个ABC转运蛋白基因,该基因cDNA全长为4 693bp,其中开放读码框4 341bp,编码1 447个氨基酸,分子量162.5kD,具有高度保守的ATP结合位点,命名为GmABC。蛋白序列比对结果显示,该基因编码蛋白属于PDR(pleiotropic drug resistance)多向耐药性蛋白家族成员。系统进化树分析显示,GmABC与苜蓿PDR亲缘关系最近,相似性达84%。基因表达分析显示,GmABC受镉诱导表达,当Cd2+浓度达到100mg.L-1时,其表达量最高。研究表明,GmABC可能对阜豆镉胁迫抗性发挥重要作用。     

菜用大豆蔗糖转运蛋白基因家族的克隆及表达分析

蔗糖是高等植物体内碳水化合物长距离运输的主要甚至唯一的形式,蔗糖转运蛋白在蔗糖转运过程中起着极为重要的作用。该研究从菜用大豆闽豆6号中克隆到9个蔗糖转运蛋白基因,分别命名为Gm SUT-1~GmSUT-9,并对其进行生物信息学、系统发育和组织表达分析。结果表明,GmSUTs属于MFS超家族,根据SUT系统进化分析法将GmSUTs划分为SUT1、SUT2和SUT4三个亚族,其中GmSUT-1、GmSUT-2、GmSUT-4、GmSUT-5、GmSUT-6、GmSUT-7和GmSUT-8属于SUT1亚族, GmSUT-9属于SUT2亚族, GmSUT-3属于SUT4亚族。SUTs各亚族成员氨基酸序列的差异性可能导致它们在蔗糖的跨膜转运中扮演不同的角色,其表达模式和生理功能也不同。大豆SUT1亚族中7个基因总的表达趋势相似,花中表达量相对来说最高,而籽粒、叶片、茎和根等组织中表达量则相对较低; SUT2亚族中的GmSUT-9和SUT4亚族中的GmSUT-3在不同组织中的表达量相对来说都较低。该研究初步阐明了菜用大豆蔗糖转运蛋白家族基因的结构及组织表达特点,为进一步开展蔗糖转运蛋白基因调控菜用大豆籽粒发育中蔗糖的积累和分配的研究奠定了基础。     

人工老化处理对结球甘蓝种子生理生化特性的影响

以结球甘蓝品种‘冬升’种子为材料,研究高温(40℃)高湿(相对湿度100%)人工老化处理过程中种子的萌发特性、种子浸出液相对电导率和丙二醛、可溶性蛋白、可溶性糖含量以及抗氧化酶活性的变化,以揭示种子劣变的机理。结果表明:(1)人工老化处理甘蓝种子的含水量和不正常苗率均随着老化时间的延长逐渐增加,而种子发芽势、发芽率、发芽指数和活力指数的增加均逐渐降低。(2)随着处理天数的延长,老化处理甘蓝种子的浸出液电导率显著增大,浸出液可溶性糖含量逐渐升高,可溶性蛋白含量表现出显著下降趋势,而种子MDA含量呈先升高后逐渐下降的趋势。(3)在结球甘蓝种子老化进程中,其种子中SOD、POD、CAT活性变化的趋势相似,均随老化程度的加深而逐渐降低;而APX活性在老化处理的最初2d显著增加,第6天显著降低。研究发现,在结球甘蓝种子老化进程中,种子活力和萌发率显著降低,其种子浸出液电导率、可溶性蛋白含量、可溶性糖含量、保护酶活性变化与种子老化及劣变程度密切相关,膜脂过氧化作用可能是引起或加剧种子老化劣变的重要原因之一。     

大豆类钙调磷酸酶B亚基GmCBL1互作候选蛋白的筛选

Ca2+是非生物胁迫信号转导途径中的重要信号分子,植物类钙调磷酸酶B亚基蛋白(CBL,calcineurin B-like proteins)是一类重要的钙信号受体蛋白,主要通过与其他蛋白的特异结合传递信号,使植物形成对非生物胁迫的响应。本实验室已经获得大豆Gm CBL1基因,功能鉴定显示Gm CBL1增强了转基因拟南芥对非生物胁迫的耐性。为了进一步研究Gm CBL1的作用机理,本研究构建诱饵载体p GBKT7::Gm CBL1,利用酵母双杂交技术筛选大豆Gm CBL1的互作蛋白。通过对筛选获得的106个蛋白基因测序和Blast比对分析,并根据其可能的生理功能对这些候选蛋白归类,整理得到4类蛋白:能量代谢相关蛋白、修饰蛋白、防御蛋白、钙信号转导相关蛋白。筛选得到候选蛋白的功能预测初步表明,大豆Gm CBL1参与多条信号途径,为进一步研究探索大豆CBL介导的抗逆信号转导途径奠定了基础。     

菜用大豆CIPK基因对逆境胁迫及激素的响应特征

CIPK(calcineurin B-like-interacting protein kinase)是一类丝氨酸/苏氨酸蛋白激酶,在植物响应逆境胁迫和激素信号转导中发挥重要作用。本研究利用大豆基因组数据库,在全基因组水平鉴定获得52个CIPK蛋白激酶。蛋白比对分析发现所有Gm CIPK含有高度保守特征性的N端激酶区、连接区和C端调控区。系统进化树分析发现大豆Gm CIPK与拟南芥、水稻CIPK分类一致,分为4个亚家族,且每个亚家族含有3个不同物种的成员,表明Gm CIPK基因的分化早于物种的分化。启动子分析表明,多数Gm CIPK基因的启动子区含有逆境和激素应答元件。组织表达分析发现,Gm CIPK基因呈现多样化的组织表达特性。进一步选取组织表达量相对较高的14个Gm CIPK进行荧光定量PCR分析,结果表明这些菜用大豆CIPK基因在不同程度上均受高温、干旱、高盐胁迫以及ABA、ACC、SA、Me JA激素的诱导表达。采用蛋白同源比对和蛋白互作在线数据库对拟南芥及大豆同源CIPK蛋白激酶与其他蛋白的互作关系进行了预测分析,发现17对同源CIPK与其他蛋白(激酶、磷酸酶、转录因子等)存在互作。本研究为菜用大豆CIPK基因的功能研究与利用奠定了基础。     

蓖麻种子油脂累积与可溶性糖变化的关系

通过高效液相色谱、RNA-seq测序和放射性碳同位素示踪等技术研究了蓖麻种子发育过程中可溶性糖代谢与油脂累积过程的关系。结果表明,蓖麻种子可溶性糖主要由葡萄糖、果糖和蔗糖构成。随着种子发育和油脂累积,可溶性糖含量呈现明显的下降趋势。其中蔗糖含量变化与油脂累积存在极显著的负相关性(r=0.980)。在种子发育早期,己糖/蔗糖比值较高,糖代谢相关基因大量表达,其中蔗糖合成酶在蔗糖代谢过程中起关键性作用;而在发育中后期,随着种子油脂快速累积,己糖/蔗糖比值和糖代谢水平下降,参与糖代谢的相关基因表达量下调,而脂肪酸合成与油脂累积相关基因表达量明显升高。通过14C蔗糖同位素示踪实验证实:降低种子蔗糖摄入量可以显著抑制糖类向油脂转化,限制蓖麻种子油脂累积。因此,可溶性糖代谢(主要是蔗糖)在蓖麻种子油脂累积过程中具有重要作用。     

Changes in germination, growth and soluble sugar contents of Sorghum bicolor (L.) Moench seeds under various abiotic stresses

The effect of various abiotic stresses on germination rate, growth and soluble sugar content in Sorghum bicolor (L.) Moench cv. CSH 6 seed embryos and endosperm during early germination was investigated. Under stress conditions germination, water potential and tissue water content decreased markedly. Subsequently, this reduction resulted in marked decreases in fresh weight both in embryos and endosperm. Conversely, a substantial increase in dry weight was observed. Furthermore, a considerable increase in the sugar contents in both embryo and endosperm was detected. The fructose level was always higher than glucose and sucrose in response to various stresses. However, as compared to the control the level of glucose and sucrose was higher in embryos and endosperm after stress treatments. Based upon these results a possible physiological role of sugars in the germination of sorghum seeds is discussed.     

The relationship between carbohydrate composition of some tree seeds and their longevity

Fresh mature tree seeds of 16 species plus soybean were usedfor the analysis of soluble neutral sugar content, as well asfor the determination of longevity in terms of the time requiredfor the seeds to decrease to 50% of the original germinationunder 5C and 80–91% relative humidity. Oligosaccharideswere sometimes found in species of recalcitrant; although themass ratio of oligosaccharide/total sugar or mole ratio of stachyoseand raffinose/sucrose was less than 0.05 or 0.044, respectively.The time required for seeds to decrease to 50% germination variedfrom a few days to 8.3 months for the seeds of desiccation-sensitivespecies. The low ratio of oligosaccharide to sucrose is, however,most unlikely to be a cause of short life-span in recalcitrantseeds. It is suggested that 0.05 mole of oligosaccharide needs to beassociated with one mole sucrose to confer the seeds with desiccationtolerance. Orthodox seeds which have a high ratio of oligosaccharide/totalsugars happened to have a low ratio of disaccharide/total sugarindicating active biosynthetic activity of oligosaccharide.There was a positive correlation between the longevity and themass ratio of oligosaccharide/total sugar or oligo-/disaccharidefor those desiccation-tolerant seeds tested. These results supportthe conclusion that the ratio of oligo-/disaccharide plays arole in the desiccation tolerance and, consequently, the longevityof orthodox seeds. Key words: Tree seed, storage behaviour, recalcitrant, soluble carbohydrate, oligosaccharide, seed longevity     

Mapping of the genomic regions controlling seed storability in soybean (Glycine max L.)

Seed storability is especially important in the tropics due to high temperature and relative humidity of storage environment that cause rapid deterioration of seeds in storage. The objective of this study was to use SSR markers to identify genomic regions associated with quantitative trait loci (QTLs) controlling seed storability based on relative germination rate in the F_2:3 population derived from a cross between vegetable soybean line (MJ0004-6) with poor longevity and landrace cultivar from Myanmar (R18500) with good longevity. The F_2:4 seeds harvested in 2011 and 2012 were used to investigate seed storability. The F₂ population was genotyped with 148 markers and the genetic map consisted of 128 SSR loci which converged into 38 linkage groups covering 1664.3 cM of soybean genome. Single marker analysis revealed that 13 markers from six linkage groups (C1, D2, E, F, J and L) were associated with seed storability. Composite interval mapping identified a total of three QTLs on linkage groups C1, F and L with phenotypic variance explained ranging from 8.79 to 13.43%. The R18500 alleles increased seed storability at all of the detected QTLs. No common QTLs were found for storability of seeds harvested in 2011 and 2012. This study agreed with previous reports in other crops that genotype by environment interaction plays an important role in expression of seed storability.     

Translationally controlled tumor protein GmTCTP interacts with GmCDPKSK5 in response to high temperature and humidity stress during soybean seed development

The developing seed of soybean is susceptible to high temperature and humidity (HTH) stress, resulting in pre-harvest seed deterioration in the field. Many genes are found to respond to the stress. Based on our previous proteomics study, an HTH-responsive gene, GmCDPKSK5, was isolated from soybean seed. GmCDPKSK5 encodes a cytoplasm- and membrane-associated protein, which belongs to Group I of the CDPK family. By yeast two-hybrid (Y2H) from soybean seed cDNA library, GmTCTP was screened as a GmCDPKSK5-interacting protein. The interaction between GmCDPKSK5 and GmTCTP was further verified using bimolecular fluorescence complementation and GST pull down assays. Expression levels of both GmCDPKSK5 and GmTCTP were induced by HTH stress in soybean seed. Our results indicated that GmCDPKSK5 and GmTCTP interact with each other and may function in responses to HTH stress in soybean developing seed.     

模拟四季温度层积对南方红豆杉种子生理性状的影响

为了解濒危物种南方红豆杉(Taxus chinensis var. mairei)种子内含物含量受温度和湿度层积的影响,设置4个季节、2种湿度(16%和24%)基质层积处理,对种子的可溶性糖、淀粉、可溶性蛋白和脂肪等内含物质的变化进行研究。结果表明,不同层积处理下种子贮藏物质的含量有显著变化,春季层积9个月后,可溶性蛋白含量达到最高值;可溶性糖含量呈现降低-升高-降低的变化趋势;淀粉和脂肪含量均随层积逐渐减少。秋季层积9个月后,淀粉含量降至最低。相比于24%湿度,16%湿度的春季、秋季、冬季层积9个月后,脂肪含量均减少较多,说明16%湿度下种子代谢活动更强。春季和秋季的暖温更能促进种子代谢,促进种子形态后熟。夏季温度过高,导致种子生活力下降,夏季层积处理3个月后,种子已经发霉和腐烂。层积过程中,种子内含物在相关酶的作用下,降解为可溶性蛋白、可溶性糖等,为种子萌发提供物质与能量。种子层积时间、温度和湿度及交互作用可作为种子内含物的调控因子。     

Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein

The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K(d)) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K(d)=2.79+/-0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.     

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the pre-storage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.     

基于cDNA-AFLP技术的玉米种子劣变相关基因分析

以人工控制劣变处理（温度45℃,相对湿度100%）3 d的玉米杂交种‘郑单958’种子为材料,通过cDNA-AFLP技术,共获得约5 400个转录本。对差异片段进行测序分析,最终获得122个种子劣变相关基因序列（DRS）,其中,上调表达74个,下调表达48个,功能涉及初生代谢、次生代谢、蛋白代谢、转录与调控、胁迫响应、信号转导和运输。采用qRT-PCR技术分析了11个上调表达差异基因的表达模式,结果显示不同基因的表达模式存在较大差异,说明不同基因在种子劣变中可能扮演不同角色。