向日葵盐胁迫相关基因的cDNA-AFLP差异表达

目的:研究向日葵盐胁迫前后基因表达的变化,分离并鉴定耐盐相关基因。方法:采用c DNA-AFLP技术分析盐胁迫产生的差异表达基因片段。结果:从256对引物组合中筛选到232对有差异表达的引物组合。用其进行选择性扩增,获得差异表达的上调TDFs 845条。经二次PCR扩增及反向Northern blot验证,获得42个阳性TDFs。对其中12个TDFs进行克隆及序列测定,得到10条TDFs核苷酸序列。经Blastx比对及功能分析,10个TDFs均与应答盐胁迫相关,涉及信号转导相关蛋白、胁迫相关功能蛋白、衰老相关蛋白以及与蛋白相互作用有关的蛋白。结论:利用c DNA-AFLP技术鉴定出一批盐胁迫应答基因,为揭示向日葵耐盐分子机制及指导向日葵耐盐分子育种实践奠定基础。     

花生抗青枯病分子标记研究

利用抗、感青枯病的花生品种为素本配制杂交组合中花5号×远杂9102,构建重组近交系,以其F6为研究材料,分析青枯痛抗性遗传规律,结果表明,花生青枯病抗性是由两时主效基因控制的遗传,并且主效基因的遗传力较高,为84%;同时采用AFLP技术和BSA分析方法,获得两个与花生青枯病抗性连锁的分子标记,标记与抗性间的遗传距离分别为8.12cM和11.46cM.利用获得的分子标记对抗、感青枯病的花生种质进行了分子鉴定,证实了标记P3M59与膏枯病抗性的符合率为70%,标记PIM58的符合率为50%,从而为花生青枯病抗性辅助选择育种提供理论基础.     

小麦抗黄矮病相关基因cDNA-AFLP差异表达片段的验证

cDNA-AFLP技术是研究基因差异表达的有利工具,广泛用于植物抗病、抗逆和生长发育等研究领域.本研究以cDNA-AFLP技术分离的抗黄矮病小麦与感黄矮病小麦间差异表达片段为对象,利用反向Northern方法,从cDNA-AFLP技术筛选的46个候选抗黄矮病防御基因差异表达片段中,筛选出抗黄矮病相关防御基因片段6个;采用对回收的差异片段进行再扩增(延伸引物再扩增),再与原选扩产物一起进行PAGE电泳分析法,并结合半定量RT-PCR分析法,又验证了候选抗黄矮病相关基因表达片段3个.反向Northern方法以及对回收的差异片段进行再扩增(PAGE再分析法),结合(半)定量RT-PCR分析方法,可以比较准确地验证cDNA-AFLP所筛选出的基因差异表达片段.     

花生抗青枯病种质脂肪酸组成的遗传多样性

通过对123份不同类型抗青枯病花生种质种子脂肪酸的鉴定测试,分析了抗青枯病花生种质在这些性状方面的遗传分化,并与6006份资源组成的花生基础收集品进行了比较。研究结果表明,我国抗青枯病花生资源的油酸含量平均为51.78%,显著高于基础收集品的对应值(45.64%);亚油酸含量平均为28.88%,显著低于基础收集品的对应值(34.36%);高油酸种质较多,油酸含量达61%以上的资源23份,所占比重为18.7%,显著高于基础品中的相应比重(2.65%)。标准差、变异系数以及遗传多样性指数的分析结果表明,抗青枯病资源在油酸和亚油酸含量方面的遗传分化程度高。     

黄瓜抗黑星病相关基因的差异表达分析

以抗黑星病黄瓜材料HX1为试材,接种黑星病菌（Cladosporium cucumerinum）2h、8h、20h、32h和72h的叶片作为试验方（Tester）,相应的未接种叶片作为对照方（Driver）,利用SSH技术,构建了黑星病菌侵染初期的正向和反向cDNA-SSH文库。用巢式引物PCR检测插入片段,获得了200个阳性克隆,通过测序,除去重复序列,共得到105个Unique ESTs。与非冗余蛋白数据库进行BLASTx比对,结果显示,17条ESTs未找到同源序列,88条非重复序列和已知基因的同源性较高,占全部ESTs序列的83.8%,其中86条ESTs与非冗余蛋白数据库已知功能的蛋白具有高度的相似性。结合高密度点阵膜杂交差异筛选,阳性率为75.0%。经初步分析这些序列的功能,差异表达的ESTs功能涉及能量和基础代谢、信号转导、蛋白和核酸代谢、光合作用及逆境中特异表达的基因等方面。为研究黄瓜抗黑星病基因提供了依据。     

用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较

由Ralstonia solanacearum E.F.Smith引起的青枯病是若干亚洲和非洲国家花生生产的重要限制因子,利用抗病品种是防治这一病害最好的措施。虽然一大批抗青枯病花生种质资源材料已被鉴定出来,但对其遗传多样性没有足够的研究,限制了在育种中的有效利用。本研究以31份对青枯病具有不同抗性的栽培种花生种质为材料,通过简单序列重复(SSR)和扩增片段长度多态性(AFLP)技术分析了它们的遗传多样性。通过78对SSR引物和126对AFLP引物的鉴定,筛选出能显示抗青枯病种质多态性的SSR引物29对和AFLP引物32对。所选用的29对多态性SSR引物共扩增91条多态性带,平均每对引物扩增3.14条多态性带;32对多态性AFLP引物共扩增72条多态性带,平均扩增2.25条多态性带。在所筛选引物中,4对SSR引物(14H06,7G02,3A8,16C6)和1对AFLP引物(P1M62)检测花生多态性的效果优于其他引物。SSR分析获得的31个花生种质的遗传距离为0.12-0.94,平均为0.53,而AFLP分析获得的遗传距离为0.06～0.57,平均为0.25,基于SSR分析的遗传距离大于基于AFLP分析的遗传距离,疏枝亚种组的遗传分化相对大于密枝亚种组。基于两种分析方法所获得的聚类结果基本一致,但SSR数据聚类结果与栽培种花生的形态分类系统更为吻合。根据分析结果,对构建青枯病抗性遗传图谱群体的核心亲本和抗性育种策略提出了建议。     

番茄抗青枯病基因的AFLP分子标记

用番茄高抗青枯病品种“T51A”与高感青枯病品种“T9230”配制杂交组合,接种鉴定其正反交Ｆ₁代及Ｆ₂代分离群体的青枯病发生情况。结果表明,T51A对青枯病的抗性属于细胞质遗传,受1对杂合基因加性控制。用64个EcoRＩ/ＭseＩ引物组合对“T51A”、“T9230”两个亲本及其Ｆ₂代抗病和感病基因池进行AFLP分析,共扩增出约4200条可分辨的带,其中2条为稳定的差异。用“T51A”和“T9230”杂交产生的Ｆ₂代分离群体对2个特异条带与目的基因的遗传连锁性进行分析,发现特异条带AAG/CAT与暂定名为RRS-342的抗青枯病基因紧密连锁,二者之间的遗传距离为6.7 cM。将AAG/CAT片段回收、克隆和测序,成功地将其转化为SCAR标记,可以更加方便地用于对番茄青枯病基因的标记辅助选择。      

差异显示法分离水稻抗稻瘟病相关基因

采用mRNA差异显示技术,分析水稻稻瘟病抗源材料“地谷”叶片受稻瘟病菌侵染前后的基因的表达差异,获得87个差异片段。对这87个差异片段进行了回收、重扩增与克隆,并对其中的81个片段进行了杂交鉴定。斑点杂交结果证实其中6个片段受稻瘟病菌诱导表达。进一步克隆测序并进行数据库比对分析表明其中一个与水稻4号染色体中一推测的苹果酸合成酶高度同源,一个与水稻11号染色体上的RPR1基因高度同源,RPR1基因具有保守的NBS-LRR结构,并与水稻防卫反应的信号传导有关;另一个与水稻第6号染色体上一推测的硫氧还蛋白高度同源,其余3个为新的cDNA片段。     

抗青枯病烟草种质资源在云南省的评价

筛选出抗性稳定的种质资源是选育抗病品种的重要基础。本文采用人工接种和病田自然发病方法鉴定了48份烟草种质的青枯病抗性表现。土壤盆栽接种鉴定表现为高抗的材料有CF207、岩烟97、TI448A、DB101、G80、RG17、GTH-1等7份材料,表现抗病的有MSK149、Oxford 2028、NC95、YN108、K346、K358、Enshu FC、Oxford 207、RG11等9份材料。苗期恒温水培接种鉴定结果表明,Oxford 207和岩烟97表现为高抗,Enshu FC表现抗病,抗病材料与云烟85和K326杂交F1的抗性表现为中感至抗病。田间自然发病鉴定结果表明,我国审定的中抗青枯病的品种RG17、RG11、K358和K346,在云南省田间抗性表现为中抗,产值较高。TI448A田间表现为抗青枯病,但易感黑胫病和空茎病。Oxford 2028和Oxford 207田间表现为抗病至高抗,产值较高。G3和岩烟97田间分别表现为高抗和抗病,产值较低。根据接种鉴定和田间病圃2年抗性鉴定,筛选出育种潜力较大的青枯病抗源Oxford 207、Enshu FC,岩烟97和TI448A。     

花生抗黄曲霉相关ARAhPR10基因克隆及其原核表达

花生黄曲霉污染已成为制约我国花生及花生制品出口贸易的关键因素.基于花生抗黄曲霉相关EST序列,RT-PCR法克隆花生ARAhPR10基因(gb|EU661964.1),开放读码框471 bp,编码157个氨基酸,分子量16.9 kD,等电点5.03.与已报导AhPR10(gb|AY726607)相似性为49.3%.推导氨基酸序列含有PR10家族的高度保守"P-loop"基序(G*GG*G)和Betv1保守疏水结构域"GVALP PTAEK ITFET KLVEG PNGGSIGKLT LKY",推测ARAhPR10是该花生PR10家族的新成员.其基因组扩增序列长度561 bp,两个外显子间存在一个长度为87 bp的内含子.原核表达ARAhPR10的融合蛋白约25 kD,Ni+-NTA树脂亲和纯化后获得电泳单一条带.纯化的ARAhPR10融合蛋白具有体外核酸酶活性,预测其具有抗黄曲霉活性.该基因的克隆及其原核表达融合蛋白的获取为ARAhPR10功能研究奠定了基础.     

Boron mobility in peanut (Arachis hypogaea L.)

In most plant families, boron (B) is phloem immobile. For plants such as peanut which bury their fruit, the mechanism for B delivery and the B source for fruit and seed growth remains enigmatic. Therefore, this study aimed to establish evidence of B retranslocation in peanut and to identify its importance in plant development. In a sand culture experiment, the increase in B contents in new organs after B withdrawal and the corresponding decline in B contents in older organs was evidence of B redistribution. In a foliar ¹⁰B experiment, the ¹⁰B abundance of treated-leaves decreased and ¹⁰B was detected in leaves and flowers formed after the application of foliar B. Application of ¹⁰B to the roots for a period also provided evidence for the retranslocation of ¹⁰B accumulated during the first growth period. The ¹⁰B abundance in older plant parts declined and ¹⁰B appeared in new organs (flowers, pegs, leaves) that had developed after the ¹⁰B supply had been replaced by ¹¹B. In the fourth experiment, foliar application of B reduced hollow heart, a symptom of B deficiency in seeds, in cv. TAG 24 from 39 to 8% and in Tainan 9 from 63 to 18%. These experiments all provide evidence for B retranslocation in peanut, but further work on the relative importance of the xylem and phloem pathways for B loading into the fruit is needed.     

Next‐generation sequencing identified genomic region and diagnostic markers for resistance to bacterial wilt on chromosome B02 in peanut (Arachis hypogaea L.)

Bacterial wilt, caused by Ralstonia solanacearum, is a devastating disease affecting over 350 plant species. A few peanut cultivars were found to possess stable and durable bacterial wilt resistance (BWR). Genomics‐assisted breeding can accelerate the process of developing resistant cultivars by using diagnostic markers. Here, we deployed sequencing‐based trait mapping approach, QTL‐seq, to discover genomic regions, candidate genes and diagnostic markers for BWR in a recombination inbred line population (195 progenies) of peanut. The QTL‐seq analysis identified one candidate genomic region on chromosome B02 significantly associated with BWR. Mapping of newly developed single nucleotide polymorphism (SNP) markers narrowed down the region to 2.07 Mb and confirmed its major effects and stable expressions across three environments. This candidate genomic region had 49 nonsynonymous SNPs affecting 19 putative candidate genes including seven putative resistance genes (R‐genes). Two diagnostic markers were successfully validated in diverse breeding lines and cultivars and could be deployed in genomics‐assisted breeding of varieties with enhanced BWR.     

Fungal populations in the rhizosphere of peanut (Arachis hypogaea L.)

Summary A comparison of fungal populations in the rhizospheres of eight varieties of peanut grown in a red lateritic soil amended with farmyard manure was made by the dilution-plate technique. There was a marked increase in fungi in the rhizospheres of TMV 2, TMV 4, Pollachi Red and EC 1698, the increase was smaller in Spanish Improved and RS 1 while very little rhizosphere effect was shown by TMV 3 and Pondicherry 8. Age of the plant had a significant influence on numbers of fungi in the rhizosphere. High R/S ratios were obtained when the plants were 30 days old, at which time attained maximum vegetative growth and started to flower. The ratios gradually decreased after that age until the plants were three months old when there was again a small increase. This later rise in fungal populations is interpreted to be due to an increase in microbial activity around dead or senescent roots. No correlation could be established between numbers of root nodules produced by a variety and its rhizosphere effect. Preferential stimulation of certain fungi in the rhizosphere of some of the varieties was noticed.     

Plant regeneration through somatic embryogenesis in peanut (Arachis hypogaea L.)

Somatic embryos were induced from immature cotyledons and immature embryonal axis ofArachis hypogaea L. on L-6 basal medium supplemented with NAA, picloram or 2,4-D at 5–50 mg 1^-1. Immature embryonal axis produced a higher number of somatic embryos in comparison with immature cotyledons. The highest number of responding cultures was produced on medium supplemented with NAA (50 mg 1^-1), while the highest average number of somatic embryos per culture was produced on medium with 2,4-D (10 or 20 mg 1^-1) and picloram (30 mg 1^-1) from cotyledons. The somatic embryos developed into plants on basal medium supplemented with activated charcoal and about 100 plants were successfully transferred to the field. Acknowledgement: The authors wish to thank Nuclear Agriculture Division, BARC for supplyingA. hypogaea seeds and Mr. R.M. Mudliar for photography.     

花生镉污染研究进展

花生既是世界主要的油料作物,又是重要的植物蛋白来源和食品加工原料．随着花生直接食用和食品加工的不断增加,国际上对花生籽粒Cd含量问题越来越关注．我国是世界上重要的花生生产国和出口国．近年来,花生Cd含量偏高已经成为制约我国出口贸易的重要因素．本文从花生籽粒Cd富集能力、花生Cd含量的种内差异、籽粒中Cd的分布规律、影响花生籽粒Cd积累的机制和降低花生籽粒Cd含量技术等方面,对花生Cd污染研究的现状与问题进行了论述．指出在花生cd污染控制方面有2种策略可以考虑,一是降低花生对土壤Cd的吸收;二是控制Cd向籽粒的迁移富集．为此需要从3个方面加强对花生籽粒Cd积累机制的研究,即花生根系活性特征参数及其与籽粒Cd积累的关系;花生果荚Cd吸收机制及其对籽粒Cd含量的贡献;花生植株体内Cd迁移机制及其与籽粒Cd含量的关系．     

Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.)

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - SDS Lauryl sulfate