玉米opaque2突变体子粒表型及营养成分分析

通过回交转育构建o2突变型的近等基因系CAL58/o2,对其子粒进行扫描电镜和透射电镜观察,发现与CAL58相比,CAL58/o2中淀粉颗粒排列松散,形状更加规则,CAL58/o2子粒中蛋白体体积变小。分析子粒的营养成分,CAL58/o2赖氨酸含量极显著提高了55.88%,氨基酸总量显著减少9.79%,粗蛋白显著减少8.86%,粗脂肪显著增加30.96%,粗淀粉显著增加5.87%。同时,提取成熟子粒醇溶蛋白进行比较,CAL58/o2子粒的α、β-醇溶蛋白含量明显降低,γ-醇溶蛋白含量增加。     

玉米opaque2近等基因系中相关基因的表达量分析

Opaque2(O2)基因编码产生OPAQUE2蛋白,该蛋白为碱性亮氨酸拉链家族的一种转录因子,可作用于基因的启动区,调控基因的转录。辽2345/o2为本实验室构建的辽2345的opaque2(o2)基因回交导入系,蛋白组学分析表明,辽2345与辽2345/o2在授粉后18 d的胚乳细胞中表达蛋白的种类和数量存在很大差异,从中筛选出差异较明显的9种蛋白质。为了进一步验证O2与9种蛋白间的作用关系,应用相对荧光定量PCR的方法对9种蛋白进行了RNA水平表达量的检测。结果表明,在胚乳发育过程中Chfi很可能受到O2的抑制作用,O2很可能对Ubc、Lgl、Sdh、LOC541920、Shmt、Ppdk等基因存在某种促进或激活作用。     

郑58/opaque2近等基因系中opaque2基因突变位点分析与高效分子标记开发

opaque2突变体材料是最常用的高赖氨酸玉米供体。对已获得的郑58/o2近等基因系研究表明,胚乳发育不同时期22-kDα-醇溶蛋白的积累均明显低于郑58。荧光定量PCR结果表明,α-醇溶蛋白家族基因Z1A、Z1B、Z1C和Z1D的表达均显著低于郑58。郑58/o2胚乳发育不同时期Opaque2基因均正常表达。测序分析发现,郑58/o2中o2基因ATG后713 bp处缺失10个碱基,ORF预测Opaque2蛋白翻译提前终止。针对突变缺失位点开发基因内分子标记o2-indel-1,利用该标记进行回交转育,结果表明,o2-indel-1与o2突变表型完全连锁,错选率为0。opaque2基因突变位点的解析有助于高效分子标记的开发,有效降低错选率,提高优质蛋白鲜食玉米多基因聚合育种的选择效率。     

玉米胚乳突变体Wrk1基因定位及候选基因预测

Wrkl是在EMS诱导群体中分离得到的玉米胚乳显性突变基因.Wrk1突变体玉米胚乳严重皱缩,导致玉米子粒的千粒重降低30％～50％.研究利用B73为回交亲本构建BC1分离群体,通过9 800个单株把Wrk1基因定位在开发的SSR标记Mssr1824和Mssr2042之间,二者之间的物理距离约1.2 Mb.在此基础上,另外构建一个以M017为回交亲本的BC1群体,通过11280个单株把Wrk1基因定位在开发的SSR标记Mssr1916.2和Mssr1838之间,二者之间的物理距离约185 kb.通过基因预测和注释,发现在该区域内有7个候选基因,为Wrk1基因的最终克隆奠定了基础.     

野生二粒小麦粒重QTLs位点分析

为了挖掘野生二粒小麦的优异基因资源,对从以色列魏茨曼科学院引进的一套以普通小麦品种中国春为遗传背景的野生二粒小麦染色体臂渐渗系(Introgression lines,渐渗系)进行了一年两点的田间试验,考查了构成粒重的三个重要因子即粒长、粒宽和千粒重。结果表明,8个渐渗系(3AS、7AL、5AS、1BS、7AS、3BL、2AL、1BL)的籽粒显著宽于亲本,推断在这些染色体臂上至少有一个正效QTL控制野生二粒小麦的粒宽;同样可以推断,12个控制野生二粒小麦粒长的正效QTLs分别定位在2AS、7AL、1BL、4BS、3AS、7BS、4BL、7AS、5BS、2BS、3AL、6AS上;5个控制野生二粒小麦千粒重的正效QTLs分别定位在7AS、4BS、7AL、1BS、6AS上。各粒重构成因子关联性分析表明,粒长、粒宽和千粒重主要受遗传因素调控,粒长和粒宽都与千粒重显示出较高的遗传相关性。     

Kernel amino acid composition and protein content of introgression lines from Zea mays ssp. mexicana into cultivated maize

Development of high yield maize germplasm with satisfying nutritional quality has been a major objective for maize breeders. Introgression lines from hybrids between Zea mays ssp. mexicana, a close wild relative of cultivated maize, and elite maize inbred line Ye515 were obtained. Ear-related traits of the lines showed wide range of variation compared to the maize parent. Kernel protein contents in the progeny lines ranged from 7.89% to 12.44%. Considerable variability of protein fractions and amino acid composition of mature endosperms was observed. Protein contents for some lines such as SD00362, SD00312 and SD00259 were significantly higher than that of Ye515, and Lys and some other essential amino acids were conspicuously improved. It was concluded that introgression from Z. mays ssp. mexicana into a maize background had great effects on protein content and composition, and that desirable inbred lines with enhanced protein contents and improved nutritional value were produced via alien introgression.     

玉米Opaque-2基因在毕赤酵母中的表达

从玉米自交系辽2345授粉后18 d的胚乳中提取总RNA,经反转录后得到第一链cDNA,利用Opaque-2基因的特异引物克隆出该基因的全编码区,将该编码区的全序列以正确的阅读框架利用Infusion同源重组的方法亚克隆到毕赤酵母表达载体pPIC9的α因子下游,并置于AOXⅠ启动子控制下,利用pPIC9表达载体上的特异引物进行PCR鉴定,鉴定后的重组质粒用Bgl Ⅱ酶切线性化后,电转化导入甲醇型毕赤酵母菌株GS₁15中,利用MM、MD和菌落PCR的方法筛选出Mut⁺表型,得到的重组子以甲醇诱导表达蛋白。经SDS-PAGE结果显示,Opaque-2蛋白得到表达,其相对分子质量（Mr）约为47 KD。     

荆州黑麦 NPR1同源基因 ScNPR1的克隆与特性分析

为了明确荆州黑麦ScNPR1基因的功能,对荆州黑麦 NPR1同源基因ScNPR1进行克隆并分析了其表达特性。利用同源序列法和RACE技术从荆州黑麦中克隆得到 NPR1同源基因的3 185 bp全长cDNA序列,该基因包含了一个编码507个氨基酸(1 524 bp)的开放阅读框、终止密码子TGA、5′端1 517 bp的非编码区和3′端144 bp的非编码区,命名为ScNPR1。利用生物信息学软件对其结构进行分析,ScNPR1编码的氨基酸序列与已知的小麦、水稻 NPR1基因编码的氨基酸序列具较高的同源性,分别达到92.4%和90.3%。荧光定量PCR发现,ScNPR1基因在小麦不同器官中均有表达,在叶、茎、根中表达较高;ScNPR1基因在植物抗病相关信号分子水杨酸、茉莉酸和乙烯处理后上调表达,在白粉病菌、纹枯病菌和赤霉病菌的诱导下,ScNPR1基因也上调表达。研究结果表明,ScNPR1基因与水杨酸和乙烯信号转导途径有关,参与寄主对病原菌侵染的防御反应。     

玉米对小斑病T小种抗性的遗传模型分析

用广义遗传模型方法,研究了玉米对小斑病T小种的抗性遗传特性.结果表明:玉米对小斑病T小种的核抗性主要受加性和显性效应控制,以显性效应为主.3个病害指标之间有极显著的正相关,相关性主要归因于加性效应,病斑数和病级之间还有显著的显性遗传相关.另外,根据遗传效应预测值对供试亲本作了评价。     

Ecophysiological traits in maize hybrids and their parental inbred lines: Phenotyping of responses to contrasting nitrogen supply levels

Maize (Zea mays L.) breeding based primarily on final grain yield has been successful in improving this trait since the introduction of hybrids. Contrarily, understanding of the variation in ecophysiological processes responsible of this improvement is limited, especially between parental inbred lines and their hybrids. This limitation may hinder future progress in genetic gain, especially in environments where heritability estimation is reduced because grain yield is severely affected by abiotic stresses. The objective of this study was to analyze the genotypic variation between inbred lines and derived hybrids in the physiological determinants of maize grain yield at the crop level, and how differences among hybrids and parental inbreds may effect contrasting responses to N stress. Special emphasis was given to biomass production and partitioning during the critical period for kernel number determination. Phenotyping included the evaluation of 26 morpho-physiological attributes for 6 maize inbred lines and 12 derived hybrids, cropped in the field at contrasting N supply levels (N₀: no N added; N₄₀₀: 400 kg N ha⁻¹ applied as urea) during three growing seasons. Tested genotypes differed in the response to reduce N supply for most measured traits. Grain yield was always larger for hybrids than for inbreds, but N deficiency affected the former more than the latter (average reduction in grain yield of 40% for hybrids and of 24% for inbreds). We also found (i) a common pattern across genotypes and N levels for the response of kernel number per plant to plant growth rate during the critical period, (ii) a reduced apical ear reproductive capacity (i.e., kernel set per unit of ear growth rate) of inbreds as compared to hybrids, (iii) similar RUE during the critical period and N absorption at maturity at low N levels for both groups of genotypes, but enhanced RUE and N absorption of hybrids at high N supply levels, and (iv) an improved N utilization efficiency of hybrids across all levels of N supply. Results are indicative of a more efficient use of absorbed N by hybrids than by parental inbreds. Larger grain yield of hybrids than of inbreds at N₀ was associated to (i) enhanced dry matter accumulation due to improved light interception during the life cycle and (ii) enhanced biomass partitioning to the grain.     

opaque2对糯玉米子粒食味和营养品质的影响分析

玉米opaque2(o2)是胚乳发育的核心调控基因,对子粒发育至关重要。基于9组糯玉米o2近等基因系(o2-NILs)及对应轮回亲本,对鲜食期和成熟期子粒的种皮厚度、总淀粉含量、可溶性糖含量等性状进行分析。结果表明,黄糯2/o2o2和中63-2/o2o2鲜食期子粒的食味品质性状均有提高,其他o2-NILs子粒品质变化不一。鲜食期o2突变对种皮厚度影响较大,但与成熟期无直接相关性。o2突变使玉米子粒赖氨酸含量增加,营养价值提高,但多数近等基因系成熟期淀粉含量和百粒重降低,仅糯2/o2o2、淀粉含量和百粒重均不受影响。糯2/o2o2、SY1-2/o2o2、赵OP-6/o2o2中27 KD γ-zein表达量显著增加、子粒饱满度与轮回亲本相近,但均表现为粉质胚乳,因此认为,优质蛋白糯玉米硬质胚乳的恢复需多个o2胚乳修饰基因协同作用。     

Molecular Mapping of Sterility QTLs qSS-3, qSS-6a and qSS-7 as Single Mendelian Factors via NIL strategy

Hybrid sterility between Oryza glaberrima and O. sativa seriously hampers the introgression of favorable genes from each other. In order to further understand this issue, identification and isolation of hybrid sterility QTLs as single Mendelian factors are an effective strategy. A genetic map was constructed using a BC₁F₁ population derived from a cross between an O. sativa japonica cultivar and an O. glaberrima accession. Four main-effect QTLs for pollen sterility were detected in the BC₁F₁. Five BC₈F₁ advanced backcross populations were developed via successive backcrosses based on phenotype and molecular selections. The BC₈F₁ populations showed bimodal distribution for pollen fertility and could be classified into semi-sterile and fertile types, fitting single Mendilian factor inheritance ratios. Three QTLs detected in the BC₁F₁ corresponding to qSS-3, qSS-6a and qSS-7 were mapped on chromosomes 6, 3 and 7, respectively, as single Mendilian factors.     

小麦抗条锈基因Yr1和Yr2分子检测体系研究

为建立小麦抗条锈病基因分子检测体系,分别以Yr1和Yr2紧密连锁的标记为检测标记,以Sull-1、CYR29、CYR32、CYR33和V26/CM42为小麦条锈菌鉴别菌株,构建Yr1和Yr2分子检测体系,并对181份小麦高代系材料进行了抗病鉴定和Yr1和Yr2基因分析。结果表明,gwm372和gwm382可作为Yr1的检测标记;wmc364可作为Yr2的检测标记;5个鉴别菌株对Yr1和Yr2具有较好的区分能力。Yr1和Yr2基因在181份小麦高代系材料中比例较低,表明这两个基因在我国小麦抗病育种过程中丢失严重。     

小麦TaWRKY2基因组分析及在进化和育种材料中的检测

小麦WRKY转录因子在抗旱抗逆等方面有重要作用。为小麦抗旱抗逆分子模块组装育种提供参考,本研究在已有小麦TaWRKY2基因cDNA序列（GenBank登录号：EU665425）的基础上,从小麦品种晋麦79号中克隆获得TaWRKY2基因全长序列,并对TaWRKY2的基因结构、基因组信息进行研究,同时检测其在部分小麦亲缘种、黄淮麦区水旱地代表品种、本课题组选育的高代品系及部分水旱地杂交组合F₁代中的分布。结果表明,克隆所得到的TaWRKY2基因全长DNA序列包含4个外显子和3个内含子,其中,3个内含子长度变化范围为111~172 bp;外显子和内含子的交接处序列符合GT-AG原则;与乌拉尔图小麦（Triticum urartu,AA）和粗山羊草（Aegilops tauschii,DD）中的片段序列同源性分别为93%和99%,与中国春小麦（AABBDD）1AS、1BS和1DS染色体的片段序列同源性分别为93%、88%和99%。除野生一粒小麦外,在小麦进化祖先种、黄淮海水旱地推广的代表品种、课题组选育的高代品系及F₁代共78份材料中,都能够检测出该基因的普遍存在,说明该基因可应用于小麦抗旱抗逆分子设计育种中。     

Endo-β-1,4-xylanase inhibitors in leaves and roots of germinated maize

Extracts of both leaves and roots of germinated maize were found to contain endo–β–1,4–xylanase inhibitors, previously reported only from whole maize meal. The inhibitors seem to be of the xylanase inhibitor protein (XIP) type, since they inhibit endoxylanases of families 10 and 11 and also show some other characteristics similar to XIP inhibitors described in other cereals. Inhibitors from leaves and roots appeared to be similar. A novel property of the inhibitors described in this work is their unusual thermostability. The half-life of inhibitors at pH 4.5 and 100 °C is greater than 10 h. However, the inhibitors are less thermostable at higher pH levels. Because they did not inhibit a plant endoxylanase, the inhibitors may play a role in maize defense against phytopathogens.     

小麦品种东农冬麦2号根中TaEXPA7部分同源基因的克隆及表达特性分析

膨胀素(expansin)是植物生长发育过程中诱导细胞壁松弛和伸展的蛋白,包括EXPA、EXPB、EXLA、EXLB四个基因家族,对植物根系的形成及快速发育具有重要作用。本试验以寒地冬小麦品种东农冬麦2号两叶一心期的根为材料,克隆了TaEXPA7基因的3个CDS全长,其氨基酸序列同源性较高,仅信号肽处有两个氨基酸不同,其核酸序列长度均为777bp,编码258个氨基酸,分别命名为TaEXPA7-A、TaEXPA7-B、TaEXPA7-D,且分别定位于2AL、2BL、2DL染色体。蛋白均含有DPBB-1和Pollen-allerg-1两个保守结构域,分子量为27 670.74Da,等电点为8.09,均为疏水性蛋白;与节节麦、二穗短柄草的相似性分别为99%和92%。采用两叶一心期的根进行qRT-PCR分析发现,TaEXPA7-A/B/D三个基因在根尖中的表达量均较高,伸长区和成熟区次之;对冬小麦根进行低温、干旱和激素处理后,这三个基因均下调表达,并且TaEXPA7-B基因的相对表达量较高,TaEXPA7-D的相对表达量较低。由此可见,多倍体小麦不同染色体上的基因在应对不同环境胁迫时,表达以及应答模式存在差异,并且在不同的环境胁迫下,发挥主导作用的染色体也存在差异;此外,该基因在根中的表达可能与低温、干旱以及外源激素的胁迫有一定的关系,可能是促进根系生长的重要基因。     

华山新麦草穗发芽抗性相关基因AIP2的克隆及其序列分析

为了挖掘和利用华山新麦草(Psathyrostachys huashanica,2n=14,NsNs)的优良基因,拓宽小麦穗发芽抗性基因资源,以华山新麦草为材料,采用同源克隆的方法克隆AIP2基因,利用生物信息学软件分析其基因结构及功能。结果表明,从华山新麦草中成功克隆AIP2基因,该基因的开放阅读框为840bp,编码279个氨基酸残基,结构域预测结果显示AIP2蛋白含有完整的Ring保守域,属于锌指环家族成员。AIP2的gDNA含5个外显子,4个内含子,华山新麦草AIP2氨基酸序列与感穗发芽小麦品种中优9507的AIP2氨基酸序列的相似性为84%,与中优9507相比缺失了44个氨基酸,与抗穗发芽的乌拉尔图小麦的AIP2氨基酸序列的相似性为80%,华山新麦草和乌拉尔图小麦都有氨基酸缺失,推测这些缺失的氨基酸可能与华山新麦草穗发芽抗性强有很大关系。本研究为小麦穗发芽抗性改良提供了新的候选基因。     

转小麦 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥植株的主要抗旱性研究

核氧还蛋白（nucleoredoxin,NRX）可通过还原目标蛋白的二硫键来调控其生物活性,在植物的生长发育和抗逆境胁迫中发挥着重要作用。蛋白质二硫键异构酶（protein disulfide isomerase,PDI）、h型硫氧还蛋白（h-type thioredoxin,TRXh）和蛋白磷酸酶2A催化亚基（protein phosphatase 2A catalytic subunit,PP2Ac）是小麦核氧还蛋白TaNRX1的互作蛋白。为了明确TaNRX1互作蛋白的抗旱性功能,本研究在拟南芥中过表达了小麦 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因,对野生型和转基因拟南芥的表型和抗旱相关生理指标进行了鉴定。结果表明,干旱胁迫处理后,转 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥的根长、存活率、脯氨酸含量均大于野生型,离体叶片失水率、丙二醛（MAD）含量均小于野生型。二氨基联苯胺（diaminobenzidine,DAB）对H₂O₂组织定位染色结果表明,干旱胁迫处理后,转 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥的H₂O₂含量均低于野生型。上述结果说明,TaNRX1的互作蛋白基因 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D增强了拟南芥对于干旱胁迫的抵抗能力。本研究可为小麦抗旱育种提供候选基因和理论基础。     

小麦地方品种黄方柱中赤霉病主效抗性位点 Fhb1的精细定位

位于小麦3BS染色体上的赤霉病抗性主效基因Fhb1的效应较大而且比较稳定,是培育抗赤霉病品种最常用和最可靠的位点。我国小麦地方品种黄方柱（HFZ）高抗赤霉病且在3BS染色体上携带Fhb1基因。本研究对HFZ/Wheaton重组自交系（RIL）群体的Fhb1位点进行分子标记检测,筛选到两个杂合子株系,并对这两个杂合子株系连续自交3代,衍生出2个次级分离群体（L30和L112）,将这两个次级分离群体在扬州大学农学院试验田种植,并在扬花期进行单花滴注接种鉴定赤霉病,调查这两个群体中各株系的病小穗数、病小穗率、病粒数和病粒率。结果表明,L30和L112两个群体的病小穗数和病小穗率均显著正相关,病粒数和病粒率均显著正相关。同时依据小麦3B染色体参考序列开发了7个新的多态性标记,分别是 X1550、X2214、X2300、X2357、X2455、X2460和X2467。用这些多态性分子标记进行基因型检测,在目标位点发现5个交换株系,根据基因型和病小穗率,进一步将Fhb1定位在分子标记 X2214与 X2357之间,在ctg0954b上这两个分子标记之间的物理距离约为143 kb,NCBI预测该区间有3个候选基因,但是FGENESH在相同区段预测了29个基因,说明目标区段所具有的基因数目很可能多于3个,但少于29个。     

青稞法尼基转移酶β亚基编码基因HbERA1的克隆及表达分析

ERA1(Enhanced response to ABA)基因编码法尼基转移酶(Farnesyl transferase)β亚基,该酶在干旱胁迫下对ABA信号负向调控因子的修饰起着关键作用。本研究以青稞(Hordeum vulgare subsp.vulgare)抗旱品种喜马拉雅10号为材料,利用RT-PCR技术克隆获得了ERA1基因全长cDNA序列,命名为HbERA1(登录号:KJ699392)。生物信息学分析表明,该基因全长1 401bp,可编码466个氨基酸序列,蛋白分子量为51.14kD,等电点为5.00。Prosite Scan分析结果表明,HbERA1含有多个干旱胁迫响应蛋白的作用位点,如酪蛋白激酶Ⅱ磷酸化位点、N-糖基化位点、蛋白激酶C磷酸化位点及N-豆蔻酰化位点。利用实时定量PCR方法研究了HbERA1在干旱胁迫条件下及复水后不同时间点的表达情况,发现在水分过剩处理下(土壤绝对含水量15.5%),HbERA1在土壤绝对含水量为33.4%时表达量最高,并随着土壤绝对含水量的下降而下调表达;进行干旱胁迫后(15.5%)基因表达量也明显下调表达;复水后表达逐渐恢复,复水8h时超过正常表达水平,表明HbERA1基因可能参与调控水涝和干旱胁迫双重信号传导。