油菜油体钙蛋白基因BnClol的克隆和表达

应用同源序列克隆法设计同源简并引物,结合RT-PCR和RACE-PCR技术.从甘蓝型油菜中分离克隆了编码28.1 kD油体钙蛋白(caleosin)的基因BnClol.其全长1 058 bp的BnClol mRNA(GenBank中序列号为AY966447)包含完整的开放阅读框和3'末端Poly(A)尾巴结构,染色体DNA结构上含6个外显子和5个内含子.Northern杂交结果表明,油菜中BnClol在种子形成中期开始丰富表达,在种子形成后期,即种子开始脱水成熟时期,高量稳定地表达.半定量PCR结果显示,BnClol在油菜种子吸水膨胀后前2d的茎中明显表达.证明在油菜种子发育期间,BnClol对mRNA的转录表达是由胚胎发育来调控的.具有显著的时空特性,并与油体的形成和积累密切有关.推测的caleosin蛋白为245个氨基酸残基(GenBank中序列号为AAY40837)组成的两性蛋白质,主要含3个结构域即由N末端1～16位氨基酸残基组成的a-螺旋和17-61位氨基酸残基组成的强亲水性的随机卷曲构成的N-末端亲水性结构域;由80-120位氨基酸残基组成的中间疏水性结构域和C-末端亲水性结构域.N-末端亲水性结构域包含一个潜在的结合Ca2 的EF-手结构.中间疏水性结构域包含一个潜在的脯氨酸-结(proline-knot)模体,在92～114位氨基酸残基组成的a-螺旋跨膜区域,推测在caleosin蛋白与单层磷脂层和油体锚定结合上及增加种子油体的稳定性上起着重要的作用.     

凤丹油体膜蛋白基因PoOLE17.5的克隆与表达特性分析

油体是油料作物种子储存脂肪的细胞器,油体膜蛋白是构成油体的重要组分,对维持油体大小和稳定起着重要作用。本研究以油体膜蛋白为对象,克隆了凤丹的油体膜蛋白基因(OLE),其cDNA全长783 bp,开放阅读框长507 bp,共编码169个氨基酸,蛋白的分子量为17.48 k D,故将该基因命名为PoOLE17.5。生物信息学分析显示,该基因编码的蛋白包含2个亲水性区域和1个疏水性区域,并具有1个保守结构域"Oleosin domain"。而后的表达模式显示,PoOLE17.5在凤丹种子发育过程中的表达水平逐渐上升,在种子接近成熟时表达水平最高,而后略有下降。这一结果有利于今后通过基因工程手段调控凤丹油体发育。     

甘蓝型油菜BnACP5基因克隆及表达分析

ACP5基因是参与植物体中脂肪酸代谢的重要基因之一,在甘蓝型油菜中该基因还未被深入研究。为了弄清楚BnACP5基因在甘蓝型油菜中的螺旋结构及功能,以湘油15为试材,采用同源克隆法得到一个与脂肪酸合成相关的基因BnACP5。BnACP5基因CDs序列长417 bp,共编码138个氨基酸。生物信息分析表明,BnACP5蛋白的相对分子质量15.25 ku,理论等电点(p I)为5.94,其不稳定参数为43.03,属于不稳定蛋白;其总疏水平均系数(GRAVY)为-0.148,是一种亲水性蛋白;不含跨膜结构,也不含信号肽序列;二级结构中含有59.42%的α螺旋、23.19%无规则卷曲、14.49%延伸链和2.90%β转角;其中18个可能是磷酸化位点;该蛋白位被定位在线粒体内;ACP氨基酸序列比对表明,植物ACP磷酸泛酰巯基乙胺结合位点周围的氨基酸序列高度保守;UPGMA聚类分析表明,BnACP5与白菜型油菜(XP_009111933.1)亲缘关系最近。荧光定量PCR结果显示,BnACP5基因的表达量在甘蓝型油菜种子发育的不同阶段呈一定规律变化,在授粉后15 d达到峰值,20 d呈下降趋势,25~35 d呈上升趋势,此后迅速下降,45 d表达量降到最低水平。由此分析得出,该基因可能与种子脂肪酸的合成有关。     

油菜开花时间调控基因SVP的克隆与表达特性分析

应用同源克隆法和RT-PCR技术分别从异源四倍体油菜湘油15号和四川黄籽花序组织中克隆了Short vegetative phase(SV)基因的同源基因,分别命名为BnSVP-1、BnSVP-2、BnSVP-3、BnSVP-4、BnSVP-5和BjSVP-1、BjSVP-2、BjSVP-3,GenBank登录号分别为JQ906717、JQ906718、JQ906719、JQ906720、JQ316471和JQ906715、JQ906716、JQ316472.序列分析结果表明,这些SVP同源基因编码区长726 bp,编码241个氨基酸残基,具有典型的MIKC结构域,是一类MADS-box调控基因.表达结果分析表明,光周期和GA3处理会使SVP基因的表达模式发生改变,而春化处理后的表达模式与对照相似,其表达模式的改变会导致开花时间提早;在开花前,SVP基因在根、茎、叶组织中均有表达,花期,在花器官的雄蕊、雌蕊和萼片中均有表达,在花瓣中无表达;幼角果的角果皮中该基因的表达水平要高于幼嫩种子.     

芸芥中DnaJ同源基因的克隆与表达分析

为研究芸芥自交亲和机理,采用mRNA差异显示反转录PCR(DDRT-PCR)分析技术,从芸芥自交亲和系开花前柱头中获得1条差异片段。测序及DNA序列的生物信息学分析表明,该差异片段与拟南芥DnaJ蛋白同源基因At J3有较高同源性,暂命名为esAtJ3基因。该序列含有一个长度为297 bp的完整开放阅读框,编码98个氨基酸残基,含有一个DnaJ-C保守结构域,属于植物DnaJ超家族。esAtJ3基因编码的蛋白无信号肽,是一个亲水性蛋白,该蛋白主要由不规则卷曲和α螺旋组成。实时荧光定量PCR分析表明,esAtJ3基因在芸芥SC系开花前柱头中表达量较高,初步推测该基因可能参与芸芥亲和性调控。     

甘薯IbNPR1全长cDNA序列的分离与表达特性分析

在植物系统获得性抗性(SAR)中,NPR1蛋白是水杨酸介导的基因表达中关键调控因子。本研究以青农2号为试验材料,利用同源序列法和RACE技术分离甘薯SAR 途径的主要抗病信号元件NPR1 (none expresser of PR gene)的全长cDNA序列。序列分析表明,IbNPR1基因全长2 353 bp,包含一个编码586个氨基酸残基的开放阅读框,包含有类似拟南芥NPR1蛋白中的BTB/POZ和锚蛋白重复氨基酸序列结构域。聚类分析显示IbNPR1与来源于番茄的NPR1基因关系最近。Southern杂交及半定量RT-PCR分析表明,甘薯NPR1基因属于低拷贝基因家族,表达模式为组成型表达,并且SA能提高其表达水平。由该结果推测,IbNPR1可能在甘薯抵御病原物的侵染中起重要的作用。     

棉花中一个钝叶醇14α-脱甲基酶基因同源基因（GhCYP51G1）的克隆、序列特征和表达分析

为研究植物固醇在棉花纤维细胞生长发育中的作用和信号传导机制,通过筛选棉花EST数据库并对目标EST序列进行整合和分析,从陆地棉栽培品种徐州142正在发育的纤维中克隆了植物固醇合成途径的重要酶基因——钝叶醇14α-脱甲基酶基因的同源基因,命名为GhCYP51G1 (GenBank登录号为EU727154)。该基因编码486个氨基酸残基,其分子量和等电点分别为55.2 kD和8.87。推导的氨基酸序列与烟草、马铃薯和葡萄等物种中CYP51家族成员有较高的同源性。而且具有钝叶醇14α-脱甲基酶序列中的典型保守结构域,如多个底物结合位点和血红素结合域。说明该克隆基因是钝叶醇14α-脱甲基酶基因的同源基因。实时定量RT-PCR的结果表明GhCYP51G1基因在快速伸长期的纤维中具有较高的表达水平,而在子叶、雌蕊和雄蕊以及开花后6 d胚珠、开花后0 d和2 d的胚珠纤维中表达水平较低。在开花后8 d的纤维细胞中GhCYP51G1的表达水平最高,这些结果说明该基因在纤维细胞的伸长生长中具有重要作用。同时在纤维生长过程中,由于生长素能够下调GhCYP51G1基因的表达,暗示植物固醇在植物激素,特别是油菜素类固醇物质和生长素的相互作用中具有一定作用。     

柚FIE同源基因的克隆及表达分析

FIE基因在受精前抑制胚乳发育并在受精后通过特异位点阻碍胚发育相关基因的转录。在拟南芥中,FIE突变最终导致种子败育。本研究从马家柚中克隆到1个FIE同源基因,开放阅读框(ORF)长为1 110 bp,编码369个氨基酸,命名为CmFIE (GenBank:IDMG680456),包含一个保守的WD40 motif结构域。推测其蛋白分子质量为41.443 3 k D,等电点为6.02,属于不稳定亲水性蛋白,并以折叠结构和环状结构为主。系统进化树分析表明,与甜橙(Citrus sinensis) CsFIE亲缘关系最近。qRT-PCR分析显示,CmFIE基因在马家柚种子和幼苗根、茎、叶中均有表达,但在种子中的表达量相对更高。CmFIE在正常种子中的发育过程持续表达,并在授粉后4～7周(胚乳细胞化之前和开始时)表达量下降,另外胚乳退化型败育种子中CmFIE表达水平被发现从授粉2周后开始一直高于正常种子。结果表明CmFIE对种子发育起到重要的调控作用,这与模式植物拟南芥FIE基因的功能一致。通过探究柚FIE基因生物信息学特性及在种子中的表达情况,可以为FIE基因在种子发育过程中的功能研究提供理论基础。     

苋菜AmPCS基因的克隆及表达载体构建

合成植物络合素(PCs)是高等植物减轻重金属毒害的重要机制。通过对镉超积累苋菜品种天星米植物络合素合成酶基因(PCS)的克隆及表达载体构建,旨在为植物修复镉污染土壤奠定基础。依据同源克隆原理,通过RT-PCR和RACE方法克隆苋菜PCS基因。序列分析表明:苋菜PCS基因cDNA全长1 770 bp,包含完整的阅读框,编码485个氨基酸;苋菜PCs合成酶与拟南芥、遏蓝菜、芥菜及油菜PCs合成酶相似性为91.96%～95.67%,其氨基酸序列N端有1个Pfam:Phytochelatin结构域(功能为催化合成植物络合素)、C端有1个Pfam:Phytochelatin_C结构域(功能为重金属离子感应器)。因此,推断苋菜PCS基因编码蛋白是PCS-like家族的新成员,具有催化合成植物络合素的功能,将它在GenBank注册,序列号为:JN979370,命名为AmPCS。在AmPCS基因的编码区加入BamH I和Sac I酶切位点,双酶切植物表达载体pBI-121和带有酶切位点的PCR产物,成功获得苋菜PCS基因正义表达载体pBI-PCS。     

玉米籽粒发育相关基因ZmMADS-RIN的克隆及表达分析

玉米籽粒发育及产量是玉米重要的经济性状。本研究以玉米骨干自交系郑58为试材,采用同源克隆法得到一个与玉米籽粒发育相关的基因ZmMADS-RIN。该基因的cDNA全长859 bp,开放阅读框为768 bp,编码255个氨基酸,与玉米B73中所对应的cDNA的编码区序列相比,共有6个SNPs位点的差异,3个氨基酸残基发生了变化。生物信息学分析表明,ZmMADS-RIN蛋白的相对分子量为29.23 kD,理论等电点(pI)为8.84,是一种亲水性蛋白,不含信号肽序列,也不含跨膜结构;具有5个磷酸化位点;二级结构中含有50.20%的α-螺旋(alpha helix)、27.45%的无规则卷曲(random coil)、14.51%的延伸链(extended strand)和7.84%的β-转角(beta turn);该蛋白位于细胞核内;其氨基酸序列中含有高度保守的MADS结构域、相对保守的K结构域、低保守的I结构域和最不稳定的C结构域,是典型的MIKC型MADS-box蛋白;系统进化树分析表明,ZmMADS-RIN蛋白属于AGL6分枝,与水稻基因OsMADS6的相似性最高,为89%。ZmMADS-RIN基因在玉米自交系郑58的籽粒中特异表达,在根及不同时期的叶片中没有检测到表达信号。荧光定量PCR结果显示,ZmMADS-RIN基因的表达量在玉米籽粒发育的不同阶段呈一定规律的变化,在授粉后0~20 d,呈上调表达趋势,授粉后25 d表达量迅速下降至较低水平,而在授粉后30~40 d则检测不到其表达。推测该基因可能与玉米籽粒的发育有关。     

花生溶血磷脂酸酰基转移酶基因的克隆与表达分析

溶血磷脂酰基转移酶(LPAT)是植物油脂合成途径的一个关键酶,在植物油脂品质改良和提高种子含油量方面具有重要的应用价值。本研究通过构建花生种子全长cDNA文库,结合大规模EST测序和功能注释,从花生中克隆了溶血磷脂酸酰基转移酶基因,命名为AhLPAT。该基因cDNA全长1 629 bp,对应的基因组序列5 531 bp,由11个外显子和10个内含子组成,内含子剪接方式符合GT-AG剪接规则。根据编码区预测AhLPAT编码一条387个氨基酸组成的多肽,预测分子量为43.2 kD,等电点为9.42。AhLPAT蛋白含有一个典型的酰基转移酶保守功能结构域以及溶血磷脂酰基转移酶相似的保守区域。该蛋白的氨基酸序列与已报道的其他物种LPAT蛋白序列有较高的一致性。AhLPAT与旱金莲、油菜、海甘蓝、蓖麻和拟南芥的LPAT蛋白氨基酸相似性依次为90%、89%、89%、88%和87%。系统进化分析表明,AhLPAT与拟南芥AtLPAT2亲缘关系较近,且同属于内质网型LPAT蛋白。RT-PCR分析表明,AhLPAT基因在花生根、茎、叶、花、果针和种子中均有表达,在花生开花后50~60 d,果针和种子中的表达量最高,且AhLPAT的表达量与花生种子含油量积累速率变化一致,二者显著相关(r=0.63,P<0.05)。推测AhLPAT基因在花生种子油脂合成中起重要作用。     

大白菜DREB类转录因子cDNA的克隆及植物表达载体的构建

以干旱处理的大白菜自交系85-1为材料,利用RT-PCR技术获得了大白菜DREB类转录因子基因的cDNA编码序列,命名为BcDREB1(GenBank登录号为:EU924266).序列分析表明,BcDREB1核苷酸序列长663 bp,编码214个氨基酸,含有一个典型的AP2结构域,具有DREB转录因子的典型特征.将该基因的cDNA编码序列连接到植物表达载体pCAMBIA2301中,成功构建了大白菜BcDREB1基因的植物表达载体pCAM-BcDREB1,为进一步通过转基因技术研究该基因的功能奠定了基础.     

Effect of Nitrogen Fertilization and Foliar Application of Plant Growth Retardants and Zinc on Cottonseed, Protein and Oil Yields and Oil Properties of Cotton

Field experiments were conducted in two successive seasons at the Agricultural Research Centre, Giza, Egypt (on a clay loam soil), to determine the effects of N fertilization (added at rates of 107 and 161 kg N ha^–1) and foliar application of plant growth retardants (Pix, Cycocel and Alar; each applied once at 300 p.p.m., 75 days after planting) and zinc (applied at 0.0 and 50 p.p.m., two times, 80 and 95 days after planting) on cottonseed, protein and oil yields and oil properties of the Egyptian cotton cultivar Giza 75. The higher N rate, as well as the application of all growth retardants and zinc, resulted in an increase in cottonseed yield ha^–1, seed index, seed protein content, oil and protein yields ha^–1, seed oil refractive index, unsaponifiable matter and total unsaturated fatty acid content (oleic and linoleic). However, those treatments decreased the oil acid value, saponification value, and total saturated fatty acid content. The seed oil content tended to decrease when the high N rate was applied, but tended to increase with the application of all growth retardants and zinc. There were some differences between Pix, Cycocel and Alar regarding their effects on the studied characters. The highest increase in seed yield ha^–1, seed index, and oil and protein yields ha^–1 was found with Pix, followed by Cycocel. The Cycocel treatment gave the lowest total saturated fatty acid oil content, followed by Alar.     

甘蓝型油菜oleosin基因片段的克隆及序列分析

以油菜（Brassica napus）基因组DNA为模板,通过设计一对特异性引物,利用多聚酶链式反应（PCR）扩增获得oleosin基因片段。将其克隆到E.coli质粒上进行序列分析。结果表明,该基因由878个碱基组成,含一个内含子,编码193个氨基酸。不计附加的18 bp凝血酶切割序列,与已报道的序列相比较,核苷酸序列及推导出的氨基酸序列的同源性分别为79.20%和91.21%。推测的氨基酸序列构成的蛋白质具有oleosin的基本结构特点,包含3个结构区域：两性N末端区（an amphipathic N-terminal region）,中心疏水区（a central hydrophobic domain）,两性C-末端区（an amphipathic C-terminal domain）。     

西藏青稞LEA3蛋白新抗旱基因的克隆与序列分析

以强抗旱青稞冬青8号和弱抗旱青稞品比14为材料,将幼苗经干旱诱导处理12 h提取总RNA,RT-PCR同源克隆到2个有差异的LEA3蛋白抗旱基因的全编码框序列。冬青8号、品比14的LEA3蛋白基因序列全长分别为661 bp和694 bp,其中,来源于品比14的LEA3蛋白基因编码框全长639 bp,编码含213个氨基酸残基的蛋白质,该多肽含有9个重复的、由11个氨基酸残基组成的保守基元序列。而来源冬青8号的LEA3蛋白基因与之相比较除缺少33 bp核苷酸外,另有6个碱基的差异,所编码的蛋白质也相应地缺少第4个保守基元序列,由8个重复的保守基元序列组成。二者在DNA与氨基酸序列上的同源性分别为94.38%和92.02%。结果证实抗旱性不同的青稞品种之间LEA3抗旱蛋白保守基元拷贝数有差异,推测抗旱蛋白结构的差异可能对植物的抗旱性有影响。     

Identification of quantitative trait loci underlying seed protein and oil contents of soybean across multi‐genetic backgrounds and environments

Seed protein and oil contents are important quantitative traits in soybean. Previously, quantitative trait loci (QTL) associated with seed protein and oil were mostly identified in single genetic background. The objective of this work was to identify QTL and their epistatic effects underlying seed protein and oil contents in three recombinant inbred line populations (two of them used one common female parent) across eight environments by composite interval mapping. Forty QTL underlying protein content and 35 QTL underlying oil content were identified. Among them, nine were universal QTL underlying protein content and four were universal QTL underlying oil content. Epistatic interactions between QTL underlying seed protein/oil and different genetic backgrounds were detected. Different pairs of epistatic interactions were observed in diverse genetic backgrounds across multi‐environments. Common marker intervals were observed to simultaneously underlie seed protein and oil contents with different epistatic interactions. The results in this study suggested that a specific genotype with high oil content and low protein content might significantly affect the selection of soybean lines for high seed protein.     

显性黄籽油菜种子特性研究

本文以甘蓝型显性黄籽油菜为材料,研究了黄籽杂交种和亲本种子特性,结果表明:(1)该黄籽与遗传背景相同的黑籽相比,皮壳率低2.68个百分点、含油量高2.01个百分点、蛋白质高1.60个百分点;黄籽杂交种的含油量比黄籽父本具有明显的杂种优势.对皮壳和胚的分析表明,黄籽皮壳中的含油量和蛋白质含量明显高于黑籽,而胚中增加较少.表明黄籽的含油量和蛋白质含量比黑籽高,主要是因为:黄籽的胚的比例增加,并且黄籽皮壳和胚中的油分含量和蛋白质含量均比黑籽高.(2)在黄籽油中,饱和脂肪酸的含量减少,不饱和脂肪酸的含量增加,其中,亚麻酸的含量增加最多.(3)黄籽与黑籽的色泽差异,主要是由于花色素和黑色素含量的差异引起,黑籽的花色素和黑色素含量分别是黄籽的4.86倍和4.24倍.     

Unconditional and conditional QTL underlying the genetic interrelationships between soybean seed isoflavone,and protein or oil contents

Selection for soybean (Glycine max L. Merr.) rich in isoflavones, protein and oil has been difficult due to negative genetic interrelationships. In this study, genetic interrelationships among seed isoflavones and protein and oil contents were evaluated using both unconditional and conditional QTL mapping. Daidzein (DZ), genistein (GT), glycitein (GC) and total isoflavone (TI) contents were analysed in F_5:6, F_5:7 and F_5:8 recombinant inbred lines (RILs) derived from a cross between ‘Zhongdou 27’(TI 3791 μg/g; protein content 42.84%; oil content 18.73%) and ‘Jiunong 20’ (TI 2061 μg/g; protein content 34.05%; oil content 21.42%). When DZ, GT, GC and TI were analysed for their genetic relationships with protein or oil contents, eight conditional QTL were detected, which included DZ|pro, GC|pro, GT|pro, TI|pro, DZ|oil, GC|oil, GT|oil and TI|oil. Seventeen QTL that had significant genetic associations between seed isoflavone, and seed protein or oil contents were found, including two for DZ conditioned on protein (qDZ|proK‐1, qDZ|proF‐2); one for GC conditioned on protein (qGC|proM‐1); three for GT conditioned on protein (qGT|proM‐1, qGT|proA2‐1, qGT|proL‐1); three for TI conditioned on protein (qTI|proM‐1, qTI|proA2‐1, qTI|proF‐2); one for DZ conditioned on oil (qDZ∣oil K_1); one for GC conditioned on oil (qGC∣oilI_1); four for GT conditioned on oil (qGT∣oil A2_1, qGT∣oil F_1, qGTF_2, qGT∣oilD2_1); three for TI conditioned on oil (qTI∣oilA2‐1, qTI∣oilE‐1, qTI∣oilL‐1). Few epistatic interactions among loci were detected. These loci may be valuable for improving seed isoflavone, protein and oil contents.     

Recurrent selection for increased protein content in yellow mustard (Sinapis alba L.)

Increasing protein content is an important objective in breeding high protein oilseed yellow mustard (Sinapis alba L). The objectives of this research were to increase meal protein content, study population variation during three cycles of selection for increased meal protein content, and quantify the relationships of protein with oil and 1000‐seed weight. Recurrent selection was employed with half‐sib family evaluation in replicated field trials. Meal protein content increased by an average of 1 % per cycle. The correlation between meal protein and seed oil content was negative (r= ‐0.49 to ‐0.58). The population shifts, with selection, reflected successful increase of average meal protein content, and an increased frequency of genotypes with high meal protein content. Furthermore, simultaneous selection for meal protein and seed oil content was possible. The correlation between meal protein content and seed weight was positive (r= 0.29‐0.39) and thus selecting for increased meal protein content posed no risk of decreasing seed weight in this yellow mustard germplasm.