抗旱基因在小麦抗旱基因工程中的应用进展

干旱严重影响小麦的生长发育及产量,小麦抗旱育种是保障小麦生产的重要措施,利用基因工程技术提高小麦抗旱性是优于传统育种的有效途径。抗旱基因主要包括调节基因（蛋白激酶、蛋白酶和转录因子基因）和功能基因。目前,已证实的可提高小麦抗旱性的基因主要为转录因子基因CBF/DREB1、MYB、NAC（NAM、ATAF1、ATAF2和CUC2）、HD-Zip和WRKY等和功能基因LEA蛋白基因、甜菜碱合成酶基因和海藻糖合成酶基因等。本文从转录因子基因和功能基因2个方面概述国内外利用基因工程技术提高小麦抗旱性的研究进展,并对目前存在的问题进行分析,以期为小麦抗旱遗传改良及育种提供参考。     

小麦叠氮化钠诱变群体重要功能基因的KASP标记检测

为了解沧麦6005叠氮化钠诱变群体中小麦重要功能基因的组成情况,利用高通量的KASP标记技术对小麦株高、抗病性、抗旱性、抗穗发芽、春化和品质等性状相关的基因进行了检测分析。结果表明:1)控制小麦株高的基因Rht-B1和Rht-D1在73份叠氮化钠诱变材料中出现6种组成类型,分别是Rht-B1a+197bp+Rht-D1a(1份)、Rht-B1a+197bp+Rht-D1b(39份)、Rht-B1a+Rht-D1a(1份)、Rht-B1a+Rht-D1b(13份)、Rht-B1b+Rht-D1a(13份)和Rht-B1b+Rht-D1b(3份),含有Rht-D1b的家系占全部突变家系的75.34%。2)在小麦抗病和抗逆性方面,73份诱变材料中发现含抗叶锈病基因Lr68的材料7份,含抗赤霉病基因Fhb1的材料5份,抗叶锈病基因Lr34在供试材料中未发现;在抗穗发芽方面,有3份材料含TaSdr-B1基因的TaSdr-B1a抗穗发芽单倍型,有53份材料含有PHS1基因的Rio Blanco type抗穗发芽单倍型,有16份材料含有TaMoc-A1基因的Hap-H抗穗发芽单倍型,在所有供试材料中含TaMFT-A1基因的Jagger-type和Zen/2174-type抗穗发芽单倍型的材料数分别为15份和22份;在抗旱性方面,有55份材料含有COMT-3B基因的3Ba单倍型,有28份材料含有Dreb-B1基因的TaDREB-B1a抗旱单倍型,有52份材料含有TaSST-4A基因的A2a抗旱单倍型;3)在小麦春化和早熟性方面均为一种单倍型,在春化方面,所有的诱变材料均为冬性类型。在早熟性方面,所有的供试材料在TaELF3-B1基因上均检测为晚开花的单倍型。4)在小麦品质方面,有45份材料含有高分子量麦谷蛋白亚基5+10,有18份材料含有Glu-A1基因的Ax1 or Ax2*强筋单倍型。综合表明,叠氮化钠诱变方法和KASP标记技术结合起来,可以作为一种小麦分子辅助育种的有效策略,能显著地提高小麦育种效率。     

Variance components and heritability of traits related to root: shoot biomass allocation and drought tolerance in wheat

Enhanced root growth in plants is fundamental to improve soil water exploration and drought tolerance. Understanding of the variance components and heritability of root biomass allocation is key to design suitable breeding strategies and to enhance the response to selection. This study aimed to determine variance components and heritability of biomass allocation and related traits in 99 genotypes of wheat (Triticum aestivum L.) and one triticale (X. Triticosecale Wittmack) under drought-stressed and non-stressed conditions in the field and greenhouse using a 10?×?10 alpha lattice design. Days to heading (DTH), days to maturity (DTM), number of tillers (NPT), plant height (PH), spike length (SL), shoot and root biomass (SB, RB), root to shoot ratio (RS), thousand kernel weight (TKW) and yield (GY) were recorded. Analyses of variance, variance components, heritability and genetic correlations were computed. Significant (p?<?0.05) genetic and environmental variation were observed for all the traits except for spike length. Drought stress decreased heritability of RS from 47 to 28% and GY from 55 to 17%. The correlations between RS with PH, NPT, SL, SB and GY were weaker under drought-stress (r?≤???0.50; p?<?0.05) compared to non-stressed conditions, suggesting that lower root biomass allocation under drought stress compromises wheat productivity. The negative association between GY and RS (r?=???0.41 and ??0.33; p?<?0.05), low heritability (<?42%) and high environmental variance (>?70%) for RS observed in this population constitute several bottlenecks for improving yield and root mass simultaneously. However, indirect selection for DTH, PH, RB, and TKW, could help optimize RS and simultaneously improve drought tolerance and yield under drought-stressed conditions.     

DNA甲基化参与调控马铃薯响应干旱胁迫的关键基因挖掘

植物受到干旱胁迫时,会通过DNA甲基化做出快速反应以帮助其应对胁迫。为探究在干旱胁迫下,DNA甲基化是如何影响基因转录表达,本研究对甘露醇模拟干旱和5-azadC(去甲基化)处理下,抗旱性不同的2个马铃薯品种(抗旱型,青薯9号;干旱敏感型,大西洋)进行转录组学分析,以Fold-change>2和校正后P<0.01进行差异表达基因(DEG)的筛选。GO富集分析发现,2种处理都共同显著富集到氧化应激和碳水化合物代谢过程相关的GO term。说明不同耐旱性马铃薯在响应干旱胁迫时,与这些GO term相关的基因也受DNA去甲基化调控。对既响应干旱又响应DNA去甲基化的1345个DEG进行KEGG功能富集发现,与植物抗旱相关的通路有植物MAPK信号途径、植物激素信号转导途径、植物谷胱甘肽代谢通路、糖酵解与糖异生和磷酸肌醇代谢通路。说明这些通路相关基因在大西洋和青薯9号2个抗旱性不同的马铃薯品种中,响应干旱的敏感性受DNA甲基化调控。接着对DEG上游1500 bp启动子区域进行顺式作用原件和甲基化CpG岛分析发现,干旱胁迫下参与植物谷胱甘肽代谢的GST基因通过DNA去甲基化来降低启动子区ABRE和CAAT-box作用元件的甲基化水平,进而激活该基因的表达以应对干旱胁迫。因此,利用比较转录组学分析干旱和DNA去甲基化处理下的差异基因,可挖掘到DNA甲基化参与调控马铃薯响应干旱胁迫的相关基因,为研究马铃薯干旱胁迫响应的表观遗传学机理提供新的研究思路。     

小麦VQ家族全基因组鉴定与表达分析

近年来,VQ蛋白因发现与WRKY转录因子相互作用而引起广泛关注。目前,关于VQ基因家族在小麦中的研究鲜有报道。为了解小麦VQ基因的存在数量和结构等特征,本研究采用生物信息学技术在小麦基因组中鉴定了25个小麦VQ基因,并对其编码蛋白的结构、性质、亚细胞定位和进化关系进行了分析。结果表明,大多数小麦VQ蛋白序列较短,为中性或偏碱性蛋白,由单外显子基因编码;小麦VQ蛋白主要分布于细胞核中。25个VQ基因不均匀地分布在21条小麦染色体上,片段复制是小麦VQ基因家族的主要扩张方式。通过基因表达模式分析,发现部分小麦VQ基因响应干旱和高温的胁迫,并呈组织表达特异性。研究结果为进一步分析该家族成员,并深入探讨其在小麦中的生物学功能和进化模式奠定了基础。     

冬小麦水旱品种间杂交主要性状的遗传分析

利用水地和旱地两种生态型的品种 ,采用 4× 4不完全双列杂交试验 ,对水旱品种间杂交主要性状的配合力和遗传表现进行了研究。结果表明 ,水地亲本的一般配合力效应对杂种后代主要性状的影响 ,除千粒重外 ,均达到显著或极显著水平 ;旱地亲本的一般配合力效应主要对株高、穗下节长、单株穗数、收获指数等与抗旱性有关的性状影响明显 ,且除株高和收获指数外 ,其余性状P1的方差均大于P2 的方差。说明水地亲本对杂种后代产量性状影响大 ,旱地亲本主要影响抗旱性状 ,水旱两种生态型杂交 ,实现了抗旱与丰产的有机结合。株高、穗下节长、穗粒数、单株产量、单株生物产量和收获指数以加性效应为主 ,而显性基因对单株穗数、千粒重、单穗重的影响较大。旱地小麦产量性状遗传力均较低 ,而株高、穗下节长、收获指数等与抗旱性有关的性状遗传力较高     

利用cDNA-AFLP技术分析小麦应答低磷胁迫的特异表达基因

以磷高效小麦品种石新828为材料,采用cDNA-AFLP技术,鉴定了短期(1~6 h)、中期(12~48 h)和长期(72~144 h)低磷胁迫根系特异上、下调表达基因的表达序列标签(EST)。共有非重复的上调ESTs 142个,下调ESTs 94个。胁迫下的前者分别含短、中和长期23、53和66个;后者分别含短、中和长期17、39和38个。对其功能比对发现,上调ESTs在功能上归属于信号转导、转录调控、代谢、逆境响应、发育、物质运输、脂类代谢和功能未知等类别,下调EST除上述类别外,还含有蛋白质合成和降解等类别。部分转录因子基因(如水稻OsPTF1和拟南芥ZAT10高度同源的转录因子基因)、促分裂原激酶基因MAPK1a、钙依赖蛋白激酶基因CPK1A和蛋白激酶基因(如serine/threonine kinase)、高亲和磷转运蛋白基因(PHT3和PT2)、过氧化物酶基因(如peroxidase 73)和谷胱甘肽-S-转移酶基因(glutathione S-transferase),受到低磷胁迫的特异增强诱导,在改善小麦植株对低磷胁迫的适应能力中可能具有重要作用。研究表明,小麦对低磷胁迫的响应,在分子水平上存在着植株感受低磷胁迫信号和信号转导、进一步在生理生化方面对胁迫信号产生应答等复杂的过程。     

小麦转录因子基因TaNAC67参与调控穗长和每穗小穗数

NAC转录因子是植物特有的一类转录因子,在植物生长发育和逆境胁迫应答反应中发挥着重要作用。前期研究表明, TaNAC67参与对多种逆境胁迫的应答,过量表达能增强拟南芥的抗逆性。为进一步揭示其在调控小麦主要农艺性状发育方面的作用,本研究以36份普通小麦组成的高多态性群体为材料,测序分析了TaNAC67-6A、TaNAC67-6B、TaNAC67-6D序列多态性,发现TaNAC67-6A启动子区–1516 nt有1个A/G转换SNP,在–873～–748 nt处有1个126 bp的InDel; TaNAC67-6B启动子区–2014和–1916 nt处各有1个C/T转换SNP; TaNAC67-6D启动子区–1795 nt有1个T/G颠换SNP,编码区357 nt处有1个C/T转换SNP。根据多态性分别开发了功能分子标记,扫描由282份普通小麦构成的自然群体,并将基因型和表型性状检测结果进行关联分析,TaNAC67-6A、TaNAC67-6B的标记与表型性状无显著关联,而TaNAC67-6D的2个标记SNP-D-1和SNP-D-2分别与小麦穗长和每穗小穗数显著相关。单倍型分析发现,自然群体中存在3种主要单倍型,其中Hap-6D-3是增加穗长和每穗小穗数的最优单倍型,在我国小麦育种历史中受到了正向选择。转基因水稻表型分析发现,TaNAC67过表达能显著增加水稻主穗穗长、穗分枝和穗粒数,验证了小麦关联分析结果。因此,TaNAC67-6D可用于改良农作物穗部性状,其分子标记可用于小麦分子标记辅助选择育种。     

河北省小麦重要农艺性状的KASP标记检测

为了将分子标记技术尽快应用到小麦育种工作中,利用高通量KASP (Kompetitive allele specific PCR)标记检测了河北省153份审定小麦品种的光周期、春化、株高、粒重、穗发芽、抗旱和抗病相关基因。结果表明在Ppd-B1和Ppd-D1光周期位点分别检测到24个和5个光周期不敏感品种。Vrn-D1b春化基因占比45.1%,Vrn-A1位点3个标记检测春化基因占比分别是0.7%,6.5%和6.5%。株高基因中Rht-B1位点2个标记检测矮杆基因占比分别是41.2%和7.2%,Rht-D1b矮杆基因占比35.9%。TaSus2-2B、TaGs3-D1、TaCKX-D1、TaGASR7-A1、TaCwi-4A、TaCwi-5D、TaMoc-A1和TaTGW6是与粒重有关的8个基因,优异等位变异占比分别是22.9%、58.8%、57.5%、7.2%、66.0%、100%、29.4%和89.5%。控制穗发芽基因TaPHS1、TaMFT-A1和TaVp1B1各开发2个KASP标记,检测抗穗发芽基因占比分别是54.9%和53.6%、64.7%和89.5%、56.2%和2.6%,控制穗发芽基因TaSdr-B1位点优异等位变异占比25.5%。两个与抗旱相关的基因TaDREB-B1和Ta1-feh w3优异等位变异占比分别是49.0%和39.9%。抗叶锈病基因Lr14a和Lr68优异等位变异占比分别是41.8%和26.8%;Lr34基因开发了2个KASP标记,仅一个标记检测Lr34抗性基因,占比0.7%;Lr15抗条锈病基因占比2.0%;Fhb抗赤霉病基因占比3.9%;抗小麦黄斑叶枯病基因Tsn1占比63.4%。综上所述,KASP标记高效检测小麦重要农艺性状的优异等位变异,在河北省小麦重要农艺性状改良方面将有良好的应用前景。     

干旱胁迫下水稻转录因子表达变化

本研究是探索在干旱应激下水稻转录因子的变化及可能的调控机制。本研究整合GEO数据库中关于水稻耐旱研究的基因芯片数据，通过TAC软件对基因芯片数据分析筛选出差异基因，利用在线数据库DAVID 6.8 和Plant TF DB筛选出相应的转录因子。敏感品种有9个转录因子表达方向一致（同时上调表达的有7个转录因子，下调的有2个转录因子），耐旱品种有34个转录因子表达方向一致（同时上调表达的有23个转录因子，下调表达的有11个转录因子）。敏感品种和耐旱品种间有8个表达方向一致的重叠转录因子。干旱胁迫会显著影响转录因子的表达，不同水稻品种中，这些转录因子的表达既有较强的品种特异性，也有部分相似性。水稻可通过诱导和抑制转录因子的表达，来提高自身抗旱能力。     

211份小麦种质资源抗旱性的评价

为了筛选更多抗旱小麦种质资源供育种利用,选用来自我国17个省的211份小麦品种资源,连续2年分别在旱地和水地种植,调查其株高、穗长、每穗小穗数、主穗粒数、单株产量和千粒重6个产量相关性状,利用综合抗旱系数、抗旱指数和隶属函数综合评价值3种方法对试验材料进行抗旱性评价。结果表明,在干旱胁迫下,这些产量相关性状均受到了不同程度的抑制,单株产量对干旱胁迫的敏感程度最大,综合3种抗旱评价方法共鉴定出8份高度抗旱种质：藁城8901、蒙科3号C、海宁75-39、济南13、烟农15、有芒红18号、郑麦9023和中优16,其中藁城8901与蒙科3号C是利用3种方法同时鉴定为高度抗旱类型的优良种质资源。     

Chromosomal locations and genetic relationships of tiller and spike characters in wheat

Number of tillers per plant, plant growth habit in seedling and adult stages, and spike and spikelet characters are agronomically important features of the gross morphology of wheat. To localize to wheat chromosomes the genes for these traits, we scored them in a set of wheat recombinant-inbred mapping lines already well genotyped with molecular markers. Quantitative-trait analysis revealed a region near Gli-A2 (Xpsr10) on the short arm of chromosome 6A strongly affecting tiller number and the correlated trait of seedling growth habit. Genes with opposing effects on adult plant type were localized on the short arms of chromosomes2A and 3A, while genes affecting spike development were assigned to several A- and B-genome chromosomes. None of these genes showed synteny with counterpart QTLs reported to affect the same traits in rice. In the chromosome 2D region containing the photoperiod-insensitivity gene Ppd-D1, the major determinant of heading date in these autumn-sown lines, earliness alleles reduced tiller and spikelet numbers and increased erect seedling growth habit, but showed no influence on adult plant type or spike length. Though several of these morphological traits are generally considered to be associated with winter hardiness and their phenotypic intercorrelations were consistent with the genetic mapping evidence, no association was found between newly identified loci and known vernalization-response or frost-resistance loci. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic systems regulating flowering response in wheat

Genetic systems regulating bread wheat ontogenesis have been studied at Ukraine's Plant Breeding and Genetics Institute, for more than two decades. The influence of Vrn genes is the most obvious; dominant alleles of Vrn genes inhibit the vernalisation requirement. The Vrn genotypes of more than 1000 cultivars were determined and the peculiarities of gene geography were shown. Dominant Vrn1 or Vrn2 seemed to be replaced by Vrn3 in regions closer to the equator. In the developed sets of near-isogenic (congenic) lines, the value of different genes was characterised for certain environments (favourable – phytotron, natural – early or late drought) based on their effects. Methods of Vrn gene manipulation were elaborated, including methods for winter genotype selection from spring x spring crosses. The possibility of alien homoeologous Vrn loci introgression was shown. In the introgressed lines, the new genes were identified and found to be nonallelic to known Vrn genes in wheat. In studying congenic lines for three Ppd genes, differences were observed in duration and intensity of photoperiodic response, vernalisation requirement and effects on agronomic traits. For typical winter wheats, two loci were identified that influenced the duration of the vernalisation requirement. One system, controlling intrinsic earliness, might be responsible for the differences in reaction to light intensity, as selection of earlier genotypes is supposed to be more effective at lower light intensity. This revised version was published online in August 2006 with corrections to the Cover Date.     

TaMyb2-Ⅱ基因在普通小麦(Triticum aestivum L.)及其近缘种中的单核苷酸多态性分析

以39份抗旱性不同的普通小麦、5份A基因组材料、4份拟斯卑尔脱山羊草（Aegilops speltoides）、6份粗山羊草（Aegilops tauschii）和2份四倍体小麦,分析TaMyb2基因的核苷酸序列长度多态性和单核苷酸多态性,及其与抗旱性的关系。结果发现,TaMyb2在A基因组材料中无目标片段扩增,在其他材料中检测到Ⅰ、Ⅱ、Ⅲ 3种类型序列。经详细分析,TaMyb2-Ⅱ序列长1 606 bp,在供试材料77 088 bp的核苷酸序列中包括34个单核苷酸变异,其中26个SNP,8个InDel,二者出现的频率分别为1/2 965 bp和1/9 636 bp,编码区π值（0.00055）小于非编码区的π值（0.00185）, 说明编码区的遗传变异小于非编码区的遗传变异。从SNP水平上分析,发现普通小麦与其D基因组供体种粗山羊草及四倍体小麦的亲缘关系较近,与B基因组供体种拟斯卑尔脱山羊草的亲缘关系较远。48份材料的TaMyb2-Ⅱ序列共分为18个单倍型（haplotype）,其中haplotype 2、3、5、6、8、9均为旱地栽培的普通小麦品种,说明普通小麦TaMyb2-Ⅱ的这几个haplotype结构可能与抗旱性有关。     

Selection for Yield and its Components in a Winter Wheat Population under Different Environmental Conditions in Albania

Wheat breeding programmes in Albania depend upon the use of diverse genetic material of local origin and on special environmental conditions; the present study being conducted under drought stress conditions. Grain yield, yield components, plant height and test weight were estimated in an F₅ generation of a winter wheat population at Kamez in 1989—90 and 1990—91 and at Lushnje in 1989—90. The highest values of heritability were observed for plant height, kernel weight and spike length. Due to drought stress the heritability of kernels per spike was low. None of the correlated responses was higher than the direct response for yield. Maximum genetic gain was expected when yield, plant height, spike length, kernels/spike and kernel weight were included in the index.     

干旱胁迫下棉花SSH文库构建及其抗旱相关基因分析

以耐旱自交系邯郸177为材料,利用抑制性差减杂交技术(SSH),构建棉花苗期叶片的正向差减文库。挑取300个阳性克隆进行PCR验证,并对验证后的单克隆进行测序和分析,共获得284个有效序列。聚类后得到202条uniESTs序列,其中174条singlets,28条contigs。经过BlastN分析,156个unigene可以在GenBank中找到同源序列,46个unigene未能找到同源匹配。经BlastX分析,40个unigene与未知功能蛋白或假定蛋白有较高相似性,116条unigene与已知功能蛋白有较高同源性。用KOBAS系统将33个unigene定位到55个Pathways中,其中P值小于0.5的Pathway有23条。初步分析发现, 丙酮酸盐代谢(pyruvate metabolism)途径、乙醛酸和二羧酸代谢(glyoxylate and dicarboxylate metabolism)途径与棉花抗旱相关性较大。这些unigene基因涉及信号传导、能量代谢、蛋白质代谢、核酸代谢、光合作用及膜运输等代谢过程。发现了苹果酸合成酶基因(Ms1, 001_B03; Ms2, 003_E04)、苹果酸脱氢酶基因(Md1, 001_C12; Md2, 002_F01);NAC(001_C08)、锌指蛋白(zfp, 003_C06)、BZR1/BES1(003_G04)等转录调节因子,以及翻译控制肿瘤蛋白基因(TCTP,002_C04)等耐旱相关基因。     

Identification of QTL associated with durable adult plant resistance to stem rust race Ug99 in wheat cultivar ‘Pavon 76’

Stem rust of wheat, caused by Puccinia graminis f. sp. tritici, was under control worldwide for over 30 years by utilizing genetic resistance. The emergence of stem rust in 1998 in eastern Africa in form of race Ug99 and its evolving variants with virulence to many resistance genes were recognized as potential threats to wheat production. In this study we identified genomic regions contributing to putatively durable, adult plant resistance (APR) to wheat stem rust. A recombinant inbred line (RIL) population of 298 lines was previously developed at CIMMYT from a cross between ‘Avocet S’ and ‘Pavon 76’. Pavon 76 has been described to carry APR to stem rust. Avocet S carries the race-specific resistance gene Sr26. A subset of RILs without Sr26 segregated for APR to stem rust race Ug99 when evaluated in Kenya for three years. Single year and joint year analysis by inclusive composite interval mapping using 450 DArT markers identified five quantitative trait loci (QTL) that contributed to the resistance of wheat to stem rust race Ug99. Three of these, including QSr.cim-3B, which probably represents the Sr2 gene, were contributed by Pavon 76 whereas the remaining two QTL were contributed by Avocet S. QSr.cim-3B, or putatively Sr2, on chromosome arm 3BS explained 32 % of the phenotypic variation while the additional QTL in Pavon contributed 24 and 20 %, respectively. Two QTL from Avocet S explained 8 and 6 % of phenotypic variance, respectively. A combination of APR QTL from the two parents resulted in transgressive segregants expressing higher levels of resistance than Pavon 76. Our results indicate that it is possible to accumulate several minor resistance genes each with a small to intermediate effect resulting in a variety that exhibits negligible disease levels even under high stem rust pressure.     

Upland rice breeding in Brazil: a simultaneous genotypic evaluation of stability, adaptability and grain yield

A durum wheat recombinant inbred line population developed from PDW 233 × Bhalegaon 4 cross was analyzed in five environments to understand the genetic network responsible for test weight (TW), thousand kernel weight (TKW), grain yield (YLD), spike length (SL), spikelets per spike (SPS), kernels per spike (KER) and kernel weight per spike (KWS). Genotype, environment and their interactions were main sources of variance for all the traits. TW and TKW were influenced by 11 main effect QTL and 6 digenic epistatic interactions detected on chromosomes 2A, 2B, 4B and 7A. Grain yield was influenced by three epistatic interactions and five main effect QTL, of which two on chromosome 2A were most consistent. A major QTL for spike length was observed on chromosome 3B. QTL for spike characters were distributed over 9 chromosomes. All the traits showed significant influence of digenic epistasis (QQ) and, to a certain extent, QTL × environment interactions (QQE). Therefore, while breeding for complex traits like kernel characters and grain yield components, these interactions should also be considered important. The consistent QTL on chromosome 2A between the marker interval Xgwm71.2–Xubc835.4 with pleiotropic effect on TW and TKW, may be utilized in early generation selection to improve TW and TKW and thereby the milling potential of the durum wheat.