水稻耐盐性的遗传和分子育种的研究进展

盐胁迫是造成水稻减产的重要环境因素之一。本文从维护膜系统的完整性、离子的区隔化以及渗透调节等三个方面介绍了水稻耐盐性的机理;简要描述了水稻生物耐盐能力、农艺耐盐能力和离体细胞对盐害反应等三种耐盐鉴定方法。总结了近年来对水稻耐盐种质资源的发掘、耐盐性QTL定位和重要基因的克隆以及耐盐水稻选育所取得的进展。通过长期水稻耐盐性评价、已发掘了一些耐盐的水稻种质;定位了七十多个控制Na+/K+含量、存活天数等耐盐性相关性状的QTL;两个水稻耐盐基因SKC1和DST已被克隆;我们已获得系列不同程度耐盐的转基因植株和SKC1/BADH两类耐盐基因聚合系。本文进一步讨论了水稻耐盐性机制的研究以及在生产实践中应用的前景,试图为深入开展水稻耐盐性研究提供参考。     

植物耐盐基因及小麦耐盐研究进展

随着植物耐盐分子机制研究的不断深入和转基因技术的日趋完善,已有许多与植物耐盐有关的基因被克隆并用于转基因研究中.文章简要论述了植物在盐胁迫下诱导表达的基因及耐盐基因在小麦耐盐研究中的应用现状.     

水稻耐盐相关基因的克隆及转化研究进展

盐分是影响水稻生长发育的重要环境因素之一。伴随工业化进程而出现的土壤盐碱化是当今水稻育种需要突破的一个难点。在此背景下,克隆耐盐基因和培育耐盐转基因水稻则成为水稻育种中的新技术。本文论述了转耐盐基因水稻及克隆耐盐相关新基因的研究现状,并对前景进行了展望。     

向日葵S-腺苷甲硫氨酸合成酶基因克隆与分析

为了研究S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthetase,SAMS)在向日葵(He-lianthus annuus)抗旱和耐盐过程中的作用,先根据计算机辅助克隆结果设计引物,抽提盐胁迫向日葵叶片的总RNA,然后采用RT-PCR扩增技术克隆了向日葵的1个SAMS基因(命名为HaSAMS1),HaSAMS1基因的编码序列长1173bp,编码390个氨基酸残基。HaSAMS1没有跨膜结构域,没有信号肽,含有SAMS蛋白的特征序列。HaSAMS1与其他物种的SAMS具有较高的序列相似性,与茼蒿(Chrysanthemum coronarium)和拟南芥(Arabidopsis thaliana)等高等植物SAMS的氨基酸序列一致性介于86.8%~96.9%。HaSAMS1基因的克隆为进一步研究向日葵抗旱和耐盐机理奠定了基础。     

加工番茄耐盐突变体耐盐相关基因的转录组分析

为筛选鉴定番茄叶片响应盐胁迫应答基因,本研究通过甲基磺酸乙酯(methyl ethyl sulfonate, EMS)诱变处理加工番茄‘JW9’获得的16株耐盐突变植株。经单独收种后,利用(RNA sequencing, RNA-Sep)技术对获得的耐盐突变体加工番茄幼苗叶片进行转录组测序分析。提取样品总RNA进行转录组cDNA文库制备后进行Illumina HiSeq/MiSeq测序。对转录组测序结果进行分析共获得了102 Gb大约810 974 186个高质量的Clean阅读序列,Cufflinks软件重构转录本后得到38 190个已知转录本,1 647个未知转录本。在测序样本中共筛选出4 367个基因在样品间显著差异表达,其中上调表达基因2 606个,下调表达基因1 761个。经qRT-PCR检测表明,10个随机选择的差异表达基因(differentially expressed genes, DEGs)表达量变化与转录组测序结果一致。通过GO和KEGG通路富集分析,从具有耐盐功能的丝氨酸/苏氨酸蛋白激酶和ATPase酶合成相关的基因中,找到5个显著差异表达基因Solyc08g066270.1和Solyc03g083420.2以及Solyc10g018530.1、Solyc09g082890.1和Solyc02g014190.2可能为耐盐相关基因。本研究从耐盐突变体与原始亲本的差异表达基因中筛选出耐盐候选基因,为进一步鉴定和克隆出番茄的耐盐基因培育耐盐品种番茄提供理论参考。     

施氏假单胞菌Na+/H+逆向转运蛋白基因nhaA的克隆与表达分析

Na+/H+逆向转运蛋白是生物耐盐的关键因子,能够维持高盐胁迫下生物体的正常生长代谢。利 用PCR技术从施氏假单胞菌（Pseudomonas stutzeri）中克隆质膜Na+/H+逆向转运蛋白基因nhaA。序列分 析表明,该基因全长1167 bp,编码388 个氨基酸,含有12 个跨膜结构域和保守的功能氨基酸残基位点： Asp-133、Asp-163、Asp-164、His-225、Gly-338。与大肠杆菌nhaA 基因核苷酸和编码氨基酸序列的同源 性分别为99%和99.2%。将该基因与pET-28a 构建成原核表达载体pET-nhaA,转化E.coli BL21,经IPTG 诱导后获得相对分子量约为41 kD的蛋白。平板法和生长曲线法检测表明,重组菌在800 mmol/L NaCl 胁迫下仍能正常生长,耐盐能力显著提高。以上结果证实克隆到的基因是施氏假单胞菌nhaA 基因家族 的成员,具有盐胁迫下将Na+逆向转运至胞外的功能,为进一步利用该基因进行作物耐盐改良奠定了基 础。该基因的GenBank登录号为EU545468。     

烟草NtNHX1-1基因特征分析

烟草是中国重要的经济作物之一。盐胁迫对烟草生产造成巨大危害,克隆盐胁迫响应基因,为从分子水平抵御盐胁迫提供基因资源。本研究克隆了Na+/H+反向转运蛋白基因。该基因编码的蛋白为膜蛋白,具有12个跨膜区。进化分析表明,Nt NHX1-1与番茄的Sl-NHX1遗传距离最近,相似性为95%,其次为甜辣椒的Ca-NHX2,相似性为94%。组织特性表达分析表明,NtNHX1-1基因在茎中表达最高、其次为叶、花和根。NaCl处理后NtNHX1-1基因的表达明显上调,表明该基因可能在烟草抵抗盐胁迫中发挥重要功能。通过调控NtNHX1-1基因的表达有望获得耐盐胁迫烟草新品系。     

大豆耐盐相关基因STL的克隆与分析

利用RACE技术从大豆中克隆出一条耐盐相关基因STL(GenBank登录号为DQ234265),该基因全长为1286bp,其中编码区为717bp,编码一个由238个氨基酸组成的蛋白质。STL与拟南芥的耐盐蛋白STO(salttoleranceprotein,GenBank登录号NP_849598)具有63.6%的同源性。RT-PCR分析发现,当用1mol/LNaCl处理大豆叶片不同时间时,大豆STL的表达量并未发生显著变化,与拟南芥STO的表达模式相似,推测STL在大豆中也具有类似的耐盐功能。     

碱蓬PEAMT基因的克隆及表达分析

研究旨在克隆碱蓬的耐逆相关基因——磷酸乙醇胺甲基转移酶(phosphoethanolamine N-methyltransferase,PEAMT)的编码基因,分析PEAMT基因调控机制,为进一步研究PEAMT基因的功能和碱蓬的耐盐机制奠定分子基础。依据GenBank数据库发表的其他物种的PEAMT序列设计简并引物,通过降落PCR法获得碱蓬PEAMT基因全长cDNA,命名为SgPEAMT,生物信息学软件分析其序列特点,荧光定量PCR检测其表达特性。序列分析表明SgPEAMT基因开放阅读框为1485bp,编码494个氨基酸,推测其为亲水性蛋白。保守结构域分析表明,SgPEAMT含有2个独立的S-腺苷甲硫氨酸依赖性甲基转移酶的保守结构域,每个结构域含有4个基序。系统进化树分析确认SgPEAMT与同属的碱蓬属植物亲缘关系最近。实时荧光定量PCR分析显示,盐胁迫或ABA胁迫下碱蓬根、茎、叶中SgPEAMT基因的表达上调,特别是叶中表达量最高。研究结果表明,SgPEAMT基因受NaCl和ABA诱导表达,预示SgPEAMT基因可能在碱蓬对盐胁迫的反应中起重要作用,是一种参与碱蓬耐盐反应的有效耐逆基因,为植物基因工程提供有效的耐逆基因。     

载有抗逆性基因的多枝赖草染色体连锁群的特定TAC克隆的鉴定

分别以耐黄矮病耐蚜虫小赖麦附加系Line24、耐盐耐旱小赖麦附加系Line15、耐盐小赖麦易位附加系Line14为材料,以它们各自所附加的含有相应抗逆基因的多枝赖草染色体(简称为抗逆染色体)的6个特定SSR分子标记为探针,通过两轮菌落杂交法筛选多枝赖草基因组可转化人工染色体(TAC)文库,最终得到经过验证的阳性克隆77个,它们分别对应于3个(易位)附加系中所附加抗逆染色体(臂)连锁群的6个特定SSR标记,每一标记所对应的TAC克隆数目变化在5～19个,平均约13个.进一步从来自Line24的抗逆染色体连锁群的同一个探针筛选到的阳性克隆中抽查2个TAC克隆15J18和09I02,通过双色荧光原位杂交技术(Double-color FISH)进行初步定位,直观地证明了文库筛选结果的准确性.为各条抗逆染色体的识别鉴定和相关抗逆基因的精细定位奠定了基础.     

In vitro selection of strawberry (Fragaria × ananassa Duch.) clones tolerant to salt stress

The method of in vitro selection for increased salt tolerance at the seed germination and early growth phase of strawberry seedlings is proposed. Clone Pau/27 was selected on medium containing 200 mM of NaCl from population obtained by free pollination of cv. ‘Paula2019;. Subsequently, on the same medium 18 salt tolerant clones were attained from the population of seeds collected from self pollinated Pau/27 plants. In the next step we examined the influence of a mild salt stress (75 mM of NaCl) on vegetative growth parameters of selected clones and two cvs.; ‘Paula2019; and ‘Senga Sengana’. All materials in the study on the basis of calculated indexes were divided into four groups differing in reaction to salt. First group (clustered together cv. ‘Paula’, Pau/27 and three second generation clones: Pau/27/11, Pau/27/24, Pau/27/30) represents sensitive genotypes. Second group, including ‘Senga Sengana’, Pau/27/06, Pau/27/10, Pau/27/12, Pau/27/13, Pau/27/15, Pau/27/18, Pau/27/20, Pau/27/21, Pau/27/26, Pau/27/27, Pau/27/31 and Pau/27/32 was designated tolerant. Third group contains only one highly tolerant clone Pau/27/08. The last group comprises two highly sensitive clones (Pau/27/01 and Pau/27/03). This revised version was published online in August 2006 with corrections to the Cover Date.     

国槐无性系对NaCl胁迫的生长生理响应及耐盐性综合评价

为揭示国槐无性系对盐渍环境的适应能力,筛选出耐盐速生的优良无性系,以5个国槐无性系盆栽2年生植株为试材,研究不同浓度NaCl(0‰、2‰、4‰、6‰)胁迫对其生长及生理生化指标的影响。采用盆栽方法进行盐胁迫实验,运用主成分分析法对不同处理下各无性系生理指标进行综合评价。结果表明,随NaCl胁迫的增加,5个国槐无性系生长指标（苗高、地径、生物量）均受到了明显抑制,‘华夏’和‘华硕’分别在6‰和4‰处理下表现出相对较强的适应性;叶绿素含量随胁迫的增加而下降,其中下降幅度最小的为‘华夏’和‘华硕’;叶片脯氨酸含量随NaCl胁迫的增加而增加,其中‘华夏’和‘华硕’显著高于其他胁迫条件下的无性系;NaCl胁迫导致叶片细胞膜透性和丙二醛(MDA)含量提高,在4‰NaCl处理下‘华夏’、‘华硕’和卷叶无性系‘嫣红3号’MDA含量低于其他无性系,在6‰NaCl处理下‘华夏’和‘嫣红3号’含量最低;超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)均呈先增大后减小的变化趋势,SOD活性在盐浓度2‰时达到最大值,CAT活性和POD活性在盐浓度4‰时达到最大值,其中‘华夏’的CAT酶活性和POD酶活性在盐浓度6‰时始终高于其他无性系;在6‰NaCl处理下,苗高增长量与生物量、叶绿素、脯氨酸、丙二醛之间,MDA与电导率、SOD、POD之间的相关性达到显著或者极显著相关,表明在对无性系进行耐盐性评价时,综合利用生长指标和生理指标能更好地反映植物受NaCl胁迫的响应。利用主成分分析对5个国槐无性系生长及生理代谢指标进行综合评价,各无性系耐盐性由强到弱为‘华夏’>‘华硕’>‘鲁槐1号’>‘嫣红3号’>无性系564。因此,认为‘华夏’和‘华硕’具有显著耐盐性和速生性,可作为盐碱地造林品种或抗逆育种材料应用。     

盐胁迫下陆地棉耐盐品种根系的抑制消减文库构建

 采用抑制消减(SSH)杂交技术构建了陆地棉耐盐品种中棉所35在盐(0.4%NaCl)胁迫后1 d、3 d、5 d和7 d的混合SSH文库。文库质量检测表明,差减杂交效率较高,质量较好。随机挑选200个阳性克隆进行测序,获得160余条高质量的表达序列标签（EST）。对序列进行BLAST比对及功能注释,其中有62条unigene 未获得同源性匹配, 42条与未知功能的序列同源性较高;其余56条功能已知的unigene中,与抗逆直接相关的基因有14个,占8.8%,参与新陈代谢及信号转导的基因有11条和10条, 分别占6.9%和6.3%,参与转录调控的基因8条,占5.0%。最后,对这些基因在盐胁迫中的作用进行了注释与讨论。     

二倍体马铃薯耐盐材料的离体筛选

马铃薯(Solanum tuberosum L.)是一种对盐中度敏感的作物,土壤盐渍化严重影响马铃薯的生长和发育,给马铃薯的生产带来严重的损失。研究选用四倍体品种‘Bintje’为耐盐对照,‘Mainechip’为感盐对照,在0、40、80、120 mmol/L盐(NaCl)胁迫下,采用单茎节切段离体培养的方法对45个适应长日照的二倍体马铃薯富利亚(S. phureja,PHU)与窄刀薯(S. stenotomum,STN)杂种(PHU-STN)无性系进行耐盐性的评价。通过测量芽长、根长、及其鲜质量和干物质量这6个生长参数,筛选出6个耐盐性显著高于‘Bintje’的二倍体无性系(89-2-1、188-1、267-1、566-1、472-1、463-1),4个耐盐性显著低于‘Mainechip’的二倍体无性系(354-1、138-1、507-1、270-2)。这些二倍体材料是选育耐盐新品种和进行耐盐机制研究的宝贵资源材料。     

海棠无性系耐盐碱区试研究

为选育耐盐碱海棠良种,用2年生海棠优良无性系容器苗在分别在滨州、东营和潍坊进行耐盐碱区域试验,通过物候期和形态指标观测、生长量测定等进行抗逆性和适应性评价。结果表明,在3个试验点,7个海棠无性系（‘傲霜’、‘高川’、‘乃馨’、‘拿斯露’、‘奥运红’、‘艾瑞斯’、‘磊丽红’）物候期较为相似,只是随区试点位置不同略有差异,其中滨州经济开发区里则镇的物候期最早,其次是东营市孤岛镇,昌邑市围子镇最晚;开花较早的品系为‘乃馨’、‘拿斯露’、‘艾瑞斯’和西府海棠,除‘高川’和‘奥运红’外,其他海棠品系的花期都较长;除‘傲霜’和西府海棠外,其他海棠品系果实宿存。7个海棠无性系在生长量、生长适应性、抗逆性和抗病虫害方面明显优于对照（‘绚丽’海棠和西府海棠）,其中‘艾瑞斯’生长量最大,其次是‘高川’。参加区域性试验的7个海棠无性系,具有观赏效果好、耐盐碱能力强、生长快等优点,适宜在山东滨海盐碱地及条件相似区域推广应用。     

Oxidative stress indicators as selection tools for salt tolerance

Oxidative stress indicators, malondialdehyde (MDA) accumulation and chlorophyll retention, were tested as tools for salt‐tolerance screening in Chloris gayana (Kunth), a forage grass exhibiting inter‐ and intra‐cultivar variability in the response to salt stress. Three types of experimental system were compared, salt shock, gradual salt treatment and leaf segments floated on control and saline solutions. Results followed the same trend in the three systems, but leaf segments or gradually salinized plants are the most convenient. Lower mean MDA and higher mean chlorophyll content were found under salinity in cv. ‘Katambora’, which is considered, from previous field trials, to be more salt tolerant than cv. ‘Boma’. Nevertheless, chlorophyll content did not give consistent results in other tests and it is not recommended for selection purposes. Within cv. ‘Boma’, clones rating higher in a salt tolerance evaluation under greenhouse conditions had lower MDA content under salinity and lower mean MDA ratios between salt‐treated and control samples. The survival of 80‘Boma’ plants under increasing NaCl concentration was assessed and MDA was measured in the leaves of very plant before the final NaCl level was reached, to assess whether it was predictive of survival capability. MDA values were significantly lower in the group surviving longer, thus, a selection based on low MDA values would have enriched the original population with more tolerant individuals.     

Identification of some Populus deltoides Marsh. x P. nigra L. clones,developed in North America,with the aid of allozymes

Summary Horizontal starch gel electrophoresis was done to assay 10 enzyme systems in root tips of 14 Populus deltoides x P. nigra clones developed by controlled hybridization and selected in North America, and some clones sharing one or both parents. The genotypes of the clones were determined for 31 loci coding for 10 enzyme systems. The interclonal allozyme variability was controlled by 12 loci. Each of the 14 clones had unique 12-locus genotypes, thus could be distinguished from each other. The clones differed from each other on an average of 4.2 loci. The first two Principal Components from Principal Component Analysis of the clonal allozyme genotype data accounted for 48% of the total variation in the 12 variant loci. 6-Pgd-3, Per-2, 6-Pgd-1, Aco-1 and Per-1 were found to be the most discriminating loci for the clones. The ordination of the clones on Principal Component axes 1 and 2 was in general agreement with the origin of the clones.     

Identification of salt tolerance‐improving quantitative trait loci alleles from a salt‐susceptible rice breeding line by introgression breeding

To improve salt tolerance of two elite rice varieties, Ce258 and Zhongguangxiang1 (ZGX1), two sets of introgression lines (ILs) each comprising 200 BC₁F₁₀ lines derived from a common donor, IR75862, and two recipient parents, Ce258 and ZGX1, were used for mapping of QTLs for four salt tolerance‐related traits at the seedling stage. Although the three parents were susceptible to salt, the two IL populations showed transgressive segregations for salt tolerance with 12 and 8 salt tolerance ILs in the Ce258‐ILs and ZGX1‐ILs. Eighteen main‐effect QTLs were identified for the four traits in the two IL populations, and the IR75862 alleles at most loci showed increased and decreased salt tolerance in the ZGX1 and Ce258 backgrounds, suggesting overwhelming genetic background effects on QTL detection for salt tolerance. The qDSS11 simultaneously detected in the two backgrounds was validated in a F₂ population derived from a salt tolerance line and ZGX1. Our results indicated that salt tolerance‐enhancing allele could be identified in the elite susceptible breeding lines and that introgression of the favourable alleles could facilitate the development of superior lines with greater salt tolerance levels.     

Chilean potato germplasm with resistance to potato viruses Y and S

Summary Four clones of the Chilota Potato Collection from the germplasm bank of the Universidad Austral de Chile, with potential combined resistance to PVX, PVY and PVS, were mechanically inoculated with these viruses in order to demonstrate wether they were truly resistant or not. Virus coat protein in the inoculated clones was determined by means of NCM-ELISA, DAS-ELISA, Western Blot and NASH.Two of the four inoculated clones were resistant to both PVY and PVS. This information will allow the utilization of these two clones in plant breeding programs.Abbreviations NCM-ELISA Nitrocellulose membrane enzyme-linked immunosorbent assay - DAS-ELISA double antibody sandwich enzyme-linked immunosorbent assay - NASH nucleic acid spot hybridization     

Correlation between Potato Clones Grown in vivo and in vitro under Sodium Chloride Stress Conditions

Salinity is one of the most serious and widespread agricultural problems resulting in losses of yield and arable land. One strategy available to cope with saline soil is to choose salt-tolerant crops or to select salt-tolerant cultivars within a crop. Difficulties in breeding for salt tolerance arise from its polygenic control and from the variable salt composition and distribution in the soil. An in vitro approach for screening large amounts of parental material is suggested and the present results confirm previous work conducted on in vitro response of the potato to a high level of sodium chloride. Furthermore, a highly significant correlation was found between in vitro growth parameters and field performance of ten potato clones.